http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=20&target=Hartlmueller2010.igem.org - User contributions [en]2024-03-28T21:02:20ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:TU_Munich/PartsTeam:TU Munich/Parts2010-11-20T19:37:54Z<p>Hartlmueller: </p>
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BioBricks<br />
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=Submitted Parts=<br />
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<groupparts align="center" width="100%">igem2010 TU_Munich</groupparts><br />
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==Malachite Green Binding Aptamer - <partinfo>BBa_K494000</partinfo>==<br />
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Methods to visualize nucleic acids via fluorescence are rare, partly due to the size of fluorescent reporters. Thus, we present the malachite green-binding aptamer to the partsregistry. By adding only 38 bp, fluorescent determination of specific nucleic acids becomes possible allowing evalutation of PoPS-based devices via in vitro transcription.<sup>[[Team:TU_Munich/Parts#ref1|&#91;1&#93;]]</sup> Binding of triphenyl dye malachite green to the aptamer increases fluorescence by 2360-fold. This leads to an significant increase and a shift in absorbance from 618 to 630 nm. With an emission maximum at 652 nm, aptamer-bound malachite green fluoresces at longer wavelength than most dyes and does not interfere with those.<sup>[[Team:TU_Munich/Parts#ref2|&#91;2&#93;]]</sup> We provide this part for efficient ''in vitro'' evaluation of PoPS-based devices in general and switches based on our concept in particular. <br />
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The malachite green binding aptamer has been successfully used in screening systems being both robust and easy to produce. Aptamers provide specifities in the range of antibodies and can be evolved to target small molecules and proteins.<sup>[[Team:TU_Munich/Parts#ref3|&#91;3&#93;]]</sup> <br><br />
Since malachite green is a membrane permeable dye, its uses are not limited to ''in vitro'' measurements. The malachite green aptamer can be used to tag and follow any RNA, including messengar and small RNAs to study questions about their metabolism and biological functions.<sup>[[Team:TU_Munich/Parts#ref2|&#91;2&#93;]]</sup> Aside from the application as a mere reporter, the malachite green binding aptamer has already been utilized to build up modular sensors which can together with another RNA-binding domain sense and report small molecules like ATP for example. This new detection method seems to provide promising future applications and sensors.<sup>[[Team:TU_Munich/Parts#ref4|&#91;4&#93;]]</sup> Since the principle of modularizing fits well into our concept of building networks, we like to provide this part to allow further engineering considering ''in vitro'' sensing systems. <br />
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==Plasmids==<br />
In general we want to provide a new principle of gene regulation which can be further developed, tested and optimized by everybody. Therefore we focus on providing the parts needed for verification and testing of new individual switches. We provide a plasmid backbone which can be used for further cloning, a positive control to test the general functionality and measurements setups and the constructs we characterized for comparison. <br />
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===Screening system: Backbone <partinfo>BBa_K494001</partinfo>===<br />
Backbone <partinfo>BBa_K494001</partinfo> is a new backbone which can be utilized not only for evaluation of terminator-based switches but also for testing basically all PoPS devices. In principle, <partinfo>BBa_K494001</partinfo> is a modified version of <partinfo>pSB1A10</partinfo>, which allows more flexibility in regard of the reporter protein and suits very well for testing and screening of switches based on bioLOGIC principles ''in vivo'' and with an ''in vitro'' translation assay. As an internal control, eGFP is integrated into the backbone to evaluate measurement setups and induction quality. eGFP is under the control of an pBAD promoter, therefore the whole pBAD promoter cassette being also located on <partinfo>BBa_K494001</partinfo>. In comparison to <partinfo>pSB1A10</partinfo> a new cloning site was created right after the eGFP protein which allows the insertion of every DNA encoded BioBrick reporter. Reporter proteins may be exchanged to fit the challenge and can be easily exchanged if not well-functioning. <br />
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|[[Image:TUM2010 BBa K494001.png|300px|left]]<br />
|For efficient usage of this part, several cloning steps must occur. First of all, the second reporter protein needs to be chosen. This can be directly introduced into the backbone using the standard BioBrick restriction enzymes EcoRI and PstI and it was done in the case of <partinfo>BBa_K494002</partinfo>.<br />
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The improved screening system is optimized for the evaluation of PoPS-based devices in fluorescence measurements. RFP which was known to contain an RNase restriction site was replaced by mCherry which combines good expression yield, short maturation times and an acceptable and well-characterized quantum yield. A unique challenge for the characterization of our switches is the expression of a corresponding signal independent of the input/output measurement. Thus, we moved the BioBrick cloning site resulting in the fluorescent reporter being inside the cloning site and giving the possibility to clone independent parts behind the reporter protein. <br />
To fully function our screening plasmid need the arabinose inducible promoter BBa_I13453 in front of the PoPS-based device to screen. Using a second arabinose inducible promoter, we were able to keep eGFP as an internal standard for the tunable input via the Pbad arabinose-inducible induction system. The two identical promoters ensure the same rate of induction for eGFP and the tested PoPS-based device. Thus, obtaining comparable screening results is easy. Unfortunately, this design implicates a minor disadvantage. Two cloning steps are needed to gain an functional construct for testing any PoPS-based device.--><br />
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===Screening System: Positive Control <partinfo>BBa_K494002</partinfo>===<br />
BBa_K494002 is the backbone <partinfo>BBa_K494001</partinfo> with an mCherry inserted into the standard BioBrick cloning site using EcoRI and PstI. mCherry was further equipped with an arabinose inducible promoter (pBAD) for good expression yield and can be used as a further reference and internal standard to compare measurements and setups. With the mCherry fluorescence in the red, a fast maturation time and a satisfying quantum yield, mCherry is the perfect complement to eGFP if fluroscence is used as an output. mCherry is expressed together with eGFP after induction with arabinose without delaying elements in between both proteins. <br><br />
With mCherry inserted as a reporter protein, further cloning can proceed. This is another advantage of the <partinfo>BBa_K494001</partinfo> backbone: Despite the nessity of more than one cloning step, it does not cost you more time than conventional cloning strategies since a positive is generated on the way. Since the reporter protein was inserted according to BioBrick standards, additional cloning can be done using the restriction sites for EcoRI and XhoI upstream of the promoter protein and SpeI and PstI downstream of the promoter protein. This was done in the case of <partinfo>BBa_K494003</partinfo>.<br />
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Here we present a ready to use composite part consisting of our screening system <partinfo>pSB1A10</partinfo>mod <partinfo>BBa_K494001</partinfo>, the PBad Promotor <partinfo>BBa_I13453</partinfo> and the reporter protein mCherry <partinfo>BBa_J06702</partinfo> including RBS and double Terminator <partinfo>BBa_B0015</partinfo>. This part belongs to the screening system and is intended as positive control.<br><br />
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[[Image:TUM2010_PicPosControl3cInd.png|70px|left|]]<br />
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[[Image:TUM2010_graphPosControl3cInd.png|400px|thumb|center|Emission spectra of induced pSB1A10mod positive control BBa_K494002 ; A: eGFP fluorescence ex: 501 nm, B: mCherry fluorescence ex: 587 nm]]<br />
[[Image:TUM2010 BBa K494002.png|left|500px]] <br><br><br><br><br><br><br><br><br />
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===Screening System: <partinfo>BBa_K494003</partinfo> - exemplary insert into <partinfo>BBa_K494001</partinfo>===<br />
With the reporter protein inserted, EcoRI and XhoI upstream of the chosen reporter protein and SpeI and PstI downstream the reporter protein are now free for insertion of further proteins. In the case of <partinfo>BBa_K494003</partinfo>, <partinfo>BBa_K494001</partinfo> was utilized to evaluate a terminator for both termination efficiency and evaluation of antitermination. To test the termination efficiency, a terminator was inserted upstream of the reporter protein mCherry, in this case a Rho-independent terminator based on the regulative hairpin in front of the ''his-operon'' from ''Salmonella enterica''. With this strong terminator right before mCherry, no mCherry fluorescence can be detected anymore while the eGFP fluorescences grows steadily over time. Another example of the same principle is shown in <partinfo>BBa_K494005</partinfo>. A further part can be inserted following the reporter protein using SpeI and PstI, this was done in the case of <partinfo>BBa_K494004</partinfo>. <br />
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Here we present a ready to use composite part consisting of our screening system <partinfo>pSB1A10</partinfo>mod <partinfo>BBa_K494001</partinfo>, the PBad Promotor BBa_I13453, the His Terminator and the reporter protein mCherry BBa_J06702 including RBS and double Terminator BBa_B0015. This part belongs to the screening system and is intended as negative control.<br />
[[Image:TUM2010_HisTermInd.png|400px|thumb|center|Emission spectra of induced <partinfo>pSB1A10</partinfo>mod HisTerm <partinfo>BBa_K494004</partinfo> ; A: eGFP fluorescence ex: 501 nm, B: mCherry fluorescence ex: 587 nm]]<br />
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===Screening System: <partinfo>BBa_K494004</partinfo> - exemplary insert into <partinfo>BBa_K494001</partinfo>===<br />
BBa_K494004 was constructed using <partinfo>BBa_K494001</partinfo> as a backbone and the cloning steps described before. Starting from <partinfo>BBa_K494003</partinfo>, a signal sequence was inserted downstream of the reporter protein mCherry. The main goal of our project is to construct and evaluate switchable elements based on terminators, our so called BioLOGICS in response of a short RNA signal strand. Therefore the signal needs to be incorporated in the plasmid. The inserted signal follows an IPTG inducible promoter, for tight regulation of the transcription. In theory every promoter can be used and any other DNA encoded signal or addition sequence can be cloned downstream of the reporter protein. Upon induction with IPTG the signal is transcribed and can in theory bind to the terminator stem loop, leading to antitermination and therefore detectable mCherry fluorescence. Another example of the same principle is shown in <partinfo>BBa_K494006</partinfo>. Again, it is no time is lost due to more than one cloning step, since like in the case of <partinfo>BBa_K494002</partinfo>, a negative control for measurements based on <partinfo>BBa_K494004</partinfo> is given by <partinfo>BBa_K494003</partinfo>. <br />
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[[Image:TUM2010_graphTrpTermInd.png|400px|thumb|center|Emission spectra of induced <partinfo>pSB1A10</partinfo>mod TrpTerm <partinfo>BBa_K494004</partinfo> ; A: eGFP fluorescence ex: 501 nm, B: mCherry fluorescence ex: 587 nm]]<br />
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=== Screening System: <partinfo>BBa_K494005</partinfo> ===<br />
BBa_K494005 is constructed following the same principles like <partinfo>BBa_K494003</partinfo>. Instead of the ''his-operon'' attenuation sequence, a Rho-independent terminator from ''E. coli'' which regulates the transcription of the ''trp-operon'' was chosen. The Trp-terminator does not inhibit transcription as efficiently as the His-terminator, mCherry fluorescence is still detectable in the measurements. <partinfo>BBa_K494005</partinfo> was used for further cloning of <partinfo>BBa_K494006</partinfo>.<br />
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[[Image:TUM2010_graphTrpTermInd.png|400px|thumb|center|Emission spectra of induced <partinfo>pSB1A10</partinfo>mod TrpTerm <partinfo>BBa_K494004</partinfo> ; A: eGFP fluorescence ex: 501 nm, B: mCherry fluorescence ex: 587 nm]]<br />
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=== Screening System: <partinfo>BBa_K494006</partinfo> ===<br />
BBa_K494006 again is similiar to <partinfo>BBa_K494004</partinfo>. Like in the case of <partinfo>BBa_K494004</partinfo>, a signal sequence which is constructed to bind into the hairpin-structure of the Trp-Terminator and therefore resolving it, is inserted downstream of the reporter protein. Since the Trp-terminator does not terminate as efficiently, the noise level is higher which must be concerned upon measurement evaluation. <br />
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[[Image:TUM2010_graphTrpTermInd.png|400px|thumb|center|Emission spectra of induced <partinfo>pSB1A10</partinfo>mod TrpTerm <partinfo>BBa_K494004</partinfo> ; A: eGFP fluorescence ex: 501 nm, B: mCherry fluorescence ex: 587 nm]]<br />
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=Parts Falsification and Verification=<br />
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{| class="bordertable" width=700 align="center" style="text-align: center; font-size:80%"<br />
<br />
|- align=”center” ß Textausrichtung in den Spalten<br />
<br />
! style="color:#0065bd;font-size:110%" | Identifier <br />
<br />
! style="color:#0065bd;font-size:110%" | Name<br />
<br />
! style="color:#0065bd;font-size:110%" | Part Type<br />
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! style="color:#0065bd;font-size:110%" | Description<br />
<br />
! style="color:#0065bd;font-size:110%" | Validation<br />
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! style="color:#0065bd;font-size:110%" | Availability<br />
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|- align=”center” <br />
| <partinfo>BBa_J04450</partinfo> <br />
| RFP Coding Device <br />
| Reporter <br />
| Basic BioBrick Insert for usage as cloning reporter <br />
| style="background:#4e9d20"|worked <br />
| existing<br />
<br />
|- align=”center”<br />
| <partinfo>BBa_R0011</partinfo> <br />
| Promoter (lacI regulated, lambda pL hybrid) <br />
| Regulatory<br />
| IPTG inducable promoter<br />
| style="background:#4e9d20"|worked <br />
| existing<br />
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|- align=”center”<br />
| <partinfo>BBa_I13453</partinfo> <br />
| Pbad promoter<br />
| Regulatory<br />
| Arabinose inducable promoter<br />
| style="background:#4e9d20"|worked <br />
| existing<br />
<br />
|- align=”center”<br />
| <partinfo>BBa_J06702</partinfo> <br />
| mCherry with RBS<br />
| Reporter<br />
| Fluorescent protein generator: mCherry<br />
| style="background:#4e9d20"|worked <br />
| existing<br />
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|- align=”center” <br />
| <partinfo>pSB1A2</partinfo> <br />
| pSB1A2<br />
| Plasmid Backbone<br />
| Basic plasmid backbone with ampicillin resistance<br />
| style="background:#4e9d20"|worked <br />
| existing<br />
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|- align=”center” <br />
| <partinfo>pSB1A3</partinfo> <br />
| pSB1A3<br />
| Plasmid Backbone<br />
| Basic plasmid backbone with ampicillin resistance<br />
| style="background:#4e9d20"|worked <br />
| existing<br />
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|- align=”center” <br />
| <partinfo>pSB1K3</partinfo> <br />
| High copy BioBrick assembly plasmid <br />
| Plasmid Backbone<br />
| Basic plasmid backbone with kanamycin resistance<br />
| style="background:#4e9d20"|worked <br />
| existing<br />
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|- align=”center” <br />
| <partinfo>pSB1A10</partinfo> <br />
| BioBrick backbone for measuring termination efficiency<br />
| Plasmid Backbone<br />
| Plasmid backbone with ampicillin resistance (based on pSB1A2)<br />
| style="background:red"|failed<br>[[Team:TU_Munich/Parts#Falsification_of_pSB1A10 | more details]]<br />
| existing<br />
|}<br />
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==Falsification of pSB1A10==<br />
The Screening Plasmid pSB1A10 is intended for the characterization of an Input/Output for a PoPS based device on the basis of two fluorescent proteins. While the first fluorescent protein, eGFP quantifies the input from an arabinose induced promoter, the second reporter, RFP detects the output. <br />
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However, our experiments designed for testing switches revealed the part to be non-functional. In the current available form, RFP fluorescence is not induced even when the PoPS device does not interfere with the Polymerase.<br />
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{|<br />
|- align="center"<br />
|[[Image:invivo1.png|center|500px| pSB1A10 screening plasmid in its originally form available at parts registry]] <br />
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[[Image:TUM2010_pSB1A10falsification.png|300px|thumb|left|Kinetic measurement of pSB1A10(induced & uninduced). GFP fluorescence ex: 501 nm, RFP fluorescence ex: 584 nm]] Although induction with arabinose worked fine indicated by GFP fluorescence, we could not detect any RFP output. The kinetic measurment on the left shows pSB1A10 postive control with a nonsense sequence inserted into the BioBrick site between the two fluorescent proteins GFP and RFP. Nevertheless induction does not trigger any RFP expression. Therefore no output signal is created in our experimantal setup. This fact renders the part as it is unsuitable for testing PoPS-based devices. We first assumed degradation of RFP-mRNA which is known to have an RNase site might be responsible. But comparison with our new screening system reveals a transcriptional problem to be most likely as replacing RFP did not improve the construct. Though, introducing a second arabinose promoter finally resulted in the desired output signal. <br />
<br><br />
In order to create a functional screening device for measurements we suggest cloning an additional PBad promoter <partinfo>BBa_I13453</partinfo> in front of the part to be tested. Thus, eGFP is still able to function as an internal standard for expression rate via the Pbad arabinose-inducible induction. Therefore comparable results for PoPS-based devices are achievable.<br />
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=References=<br />
<br />
<html><a name="ref1"></a></html>[1] Grate, D. and C. Wilson, Laser-mediated, site-specific inactivation of RNA transcripts. Proceedings of the National Academy of Sciences of the United States of America, 1999. 96(11): p. 6131.<br />
<br />
<html><a name="ref2"></a></html>[2] Babendure, J.R., S.R. Adams, and R.Y. Tsien, Aptamers switch on fluorescence of triphenylmethane dyes. J. Am. Chem. Soc, 2003. 125(48): p. 14716-14717.<br />
<br />
<html><a name="ref3"></a></html>[3] Rowe, W., M. Platt, and P.J.R. Day, Advances and perspectives in aptamer arrays. Integrative Biology, 2009. 1(1): p. 53-58.<br />
<br />
<html><a name="ref4"></a></html>[4] Stojanovic, M.N. and D.M. Kolpashchikov, Modular aptameric sensors. J. Am. Chem. Soc, 2004. 126(30): p. 9266-9270.<br />
<html><a name="ref6"></a></html>[5] http://en.wikipedia.org/wiki/Logic_gate#Symbols<br />
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{{:Team:TU_Munich/Templates/End}}</div>Hartlmuellerhttp://2010.igem.org/Team:TU_Munich/LabTeam:TU Munich/Lab2010-10-28T03:59:24Z<p>Hartlmueller: </p>
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=Experiment Design=<br />
In this section we do not only want to present the experiments and results we gained but also to encourage you to evaluate your own switch based on the protocols and general procedure on how to evaluate basic parameters of a switch. In theory, every terminator can be turned into a switch with minor modifications and the right signals which are based on individual applications. While the principle of how to turn a terminator into a switch is explained in detail [https://2010.igem.org/Team:TU_Munich/Project here], experimental setups and protocols are provided in the following. Due to time and equipment limitations we could not perform all the experiments we planned but next to the hope that another iGEM team might proceed with our project we would also like to encourage you to design and test some basic switches on which you can base a complete, tightly regulated network. <br />
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The complexity of our experimental setups vary, since we planned to characterize an individual switch with one exemplary signal on all relevant levels: Starting from the most general, complicated but also relevant level, ''in vivo'' measurements we approached to testing different switches on each smaller scale: We developed setups for ''in vitro'' translation which can be done without much effort following the ''in vitro'' measurements and also provide detailed description of ''in vitro'' transcription verification providing an inside to the molecular functionality of our basic idea. We do not see the methods we used here as the gold standard for bioLOGICS evaluation and encourage you to include your own ideas as well as check in our outlook section where we suggest experiments we could not do during the limited iGEM 2010 time. Together with our [https://2010.igem.org/Team:TU_Munich/Parts Biobrick submissions] this year, we offer a complete set for switch evaluation on all cellular levels.<br />
<br><br />
Most measurements are based on fluorescence reporters which provide easy handling, fast output and are well studied. Next to the fluorescent proteins GFP and mCherry we used ''in vivo'', a malachite green binding aptamer serves as a reporter ''in vitro'' providing a reliable fluorescent output upon antitermination. <br />
<br><br />
Most setups up to now were only used to evaluate switches with an default state "off" which are applied for AND/OR devices. In principle the same methods can be used for NOT devices which are based on a switch with an default state "off". Again, time limitation circumvented further tested from our team but we hope that further studies can be done in the future. <br />
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<!-- Our initial idea to prove our concept of antitermination was to use fluorescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks ''in vivo'' as well as ''in vitro'', the latter using a translation kit based on <i>E. coli</i> lysate. Later on, we decided to develop an experiment, that relies only on transcription. In this set-up, we used a fluorescent dye, malachite green, that binds a specific RNA aptamer and thus makes it possible to detect transcription activity. --><br />
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==''In vivo'' Measurements==<br />
''In vivo'' measurements have the highest complexity compared to any other experimental set-up. Different parameters and circumstances deriving from both the cellular environment as well as technical considerations like scatter have to be taken into account. Nevertheless, the measurements are essential, as our switches should finally work inside cells to fulfill our vision of an intracellular logic network. This year's submitted Biobricks provide you with a basic kit of plasmids which allow a quick beginning of the measurements. <br />
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===Design===<br />
For the measurements ''in vivo'' we decided to use an expression cassette consisting of Green Fluorescent Protein (GFP) coding sequence upstream of the switch and another fluorescent protein coding sequence downstream of it. Both protein coding sequence share the same ribosome binding site allowing the usage of the GFP as an internal control in measurements. Since the spectra should not overlap and to avoid FRET as well as an pure overlap of the spectra, we settled on the usage of red fluorescent protein variants, namely mRFP1 in the first try and mCherry in an modified variant of the pSB1A10 vector. <br />
<br><br />
While the GFP fluorescence can be used to normalize the measurements, the RFP fluorescence serves as a reporter to detect and evaluate termination and antitermination. To stimulate the expression of the fluorescent proteins, we took advantage of the pBAD promoter family (sensitive towards arabinose). The signal upon which the antitermination events and therefore switching relies on were under the control of an IPTG inducible promoter. We went with this well-established pair of controllable promoters to deliver an easy setup in the beginning, like described [https://2010.igem.org/Team:TU_Munich/Software here], every sort of input may later be combined with our basic switching units. <br />
<br />
<br><br />
[[Image:invivo3.png|500px|thumb|center|general measurement principle]]<br />
<br><br />
<br />
The GFP internal control carries the advantage that errors in the measurement set can be detected easily. Lack of arabinose or promoter insensitivity can be recognized as well as problems with the fluorescence measurement itself. Plus, it allows normalizing measurements to compare different preparations in relation to each other. <br />
<br><br />
Upon binding of a signal to the terminator switch, termination is circumvented and the reporter protein behind the switch can be translated. In the experimental setup presented here, this will result in an RFP expression, but again, every protein or DNA-encoded element in general may be used as an output. Since the RFP fluorescence spectra do not overlap with GFP it offers an easy possibility to evaluate the effect of signal induction. Next to GFP fluorescence, RFP fluorescence will show up.<br />
<br><br />
[[Image:TUM2010_Expected_emission_spextra.png|375px|thumb|center|schematic estimated fluorescence spectra]]<br />
<br><br />
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===Construction and Cloning===<br />
<br />
In a first try we cloned a measuring construct based on pSB1A10. The resulting plasmid, nicknamed pMonsterplasmid due to its size was tested in the fluorescent measurements described [https://2010.igem.org/Team:TU_Munich/Lab#Switch_evaluation_in_vivo below]. Unfortunately after two months of cloning we had to recognize that the plasmid in use did not work for us (see also [[Team:TU_Munich/Parts#Falsified_Parts|pSB1A10 Falsification]]). <br><br />
So after the first unsuccessful attempts we decided to reclone the system, substituting RFP to mCherry, a dsRED derivative with a spectrum in the far red, and adding arabinose inducible promoters in front of both fluorescent proteins to guarantee stable and comparable expression of both proteins<br />
{|<br />
|[[Image:TUM2010 Plasmid1flo.png|350px|right|first measuring construct]]<br />
|[[Image:TUM2010 Plasmid2flo.png|350px|left|BBa_K494006 cloned in BBa_K494001]]<br />
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<br><br />
To control the expression of the switch, the particular DNA sequence itself is under the control of an IPTG dependent promoter. In the future we want our networks to be able to respond to a variety of external signals like small metabolites, ions or whatever can be found in the parts registry. For basic switch evaluation, an established and well-working system like the ''lac''-operon was chosen to avoid side-effects of less well-characterized promoters.<br />
<br><br />
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===Measurements based on submitted Biobricks===<br />
The Biobricks BBa_K494001-BBa_K494006 are constructed for easy design of a switch-evaluation system. Detailed information can be found [https://2010.igem.org/Team:TU_Munich/Parts#Plasmids here].<br />
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===Switch evaluation ''in vivo''===<br />
To evaluate the switching efficiency, output with and without signal needs to be monitored. In this case, GFP fluorescence (internal control) will always appear upon arabinose induction, while RFP/mCherry fluorescence is only present upon binding of a signal and occurring antitermination. <br><br><br />
Upon induction with arabinose a rise of GFP expression can be seen. To monitor changes in gene expression we used a fluorimeter and measured fluorescence of whole living cells. While this approach provides easy handling and monitoring, too much scattering has to be carefully avoided: the cell density should not exceed an OD<sub>600</sub> of 0.05. RFP/mCherry emission should be visible only in case of a working switch or inefficient termination.<br />
<br><br><br />
For evaluation of the measuring plasmid itself we incorporated a positive control in every measurement. A random sequence in between GFP and RFP/mCherry was chosen in a corresponding length instead of a terminator. An increase in both GFP and mCherry was detectable in comparable amounts after quantum yield correction, showing the measuring plasmid to beworking nicely. While the positive control may be the same for all evaluated devices, the negative control has to be specific for every switch or terminator, respectively. <br />
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[[image:TUM2010_bacteriaAll.JPG|thumb|375px|center|Bacterial cultures after incubation of 16h]]<br />
<br><br />
The negative control contained the evaluated switch without any possibility for induction of the corresponding signal. Thereby the switch's function is limited to termination, leading to no detectable RFP/mCherry fluorescence. By definition every switch type has to be tested using a negative control without a corresponding signal, since termination efficiency may vary depending on the terminator itself, cell strain and general growth conditions. We recommend to chose your terminator of choice and evaluate it using the provided plasmids. <br><br />
In our experimental part we evaluated terminators based on the regulatory unit of the tryptophan (Trp-Term) and histidine (His-Term) operons. Those synthetic operons are regulated based on the principle of attenuation, a terminator in front of genes involved in amino acid biosynthesis avoids transcription until environmental stimulis suggest a lack of those amino acids. Since both sequences are known to be regulated by changes in secondary structure, those two attenuators became the basis for our designed switches. <br><br />
The terminators we tested can be found in the Parts Registry. With the construction of the backbone [http://partsregistry.org/Part:BBa_K494001 BBa_K494001], potential switches and signals can be easily subcloned in two steps and tested. [http://partsregistry.org/Part:BBa_K494002 BBa_K494002] was constructed as a positive control, producing maximal mCherry fluorescence which may be used to characterize terminator and switch efficiency. [http://partsregistry.org/Part:BBa_K494003 BBa_K494003] and [http://partsregistry.org/Part:BBa_K494004 BBa_K494004] carry the His-Terminator with and without the corresponding signal, [http://partsregistry.org/Part:BBa_K494005 BBa_K494005] and [http://partsregistry.org/Part:BBa_K494006 BBa_K494006] being the same for Trp-Terminator. <br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==''In vitro'' Translation==<br />
To go more into detail, the next complexity level is to study the effect of switches on a translational level. ''In vitro'' measurements with ''E. coli'' lysate make the fluorescence signals independent of cell growth and physical or biological factors like cell density or growth stadium.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
===Design===<br />
Since the same constructs can be used both for ''in vivo'' and ''in vitro'' translation, no additional cloning effort is needed. This implements, that the Biobricks we [https://2010.igem.org/Team:TU_Munich/Plasmids provided this year], can again be used as the groundwork for constructing vectors for measurements. <br><br />
Reporter proteins GFP and mCherry are well expressed ''in vitro'', the limiting factors are mostly the capacity of a kit versus the maturation time of fluorescent proteins. Since we used a fast-folding GFP variant and mCherry, which was characterized with a maturation time of 15 minutes by Tsien and Coworkers, the problem should be minimized. Alternative tags may be considered, a major advantage of measuring translation ''in vitro'' may be the use of non-cell permeable tags for switch evaluation.<br />
<br />
===Measurements===<br />
We used the cell-free ''E. coli'' S30 extract system for circular DNA provided by Promega<sup>[[Team:TU_Munich/Lab#ref1|&#91;1&#93;]]</sup>, which is prepared by modifications of the Method Zubay ''et al.''<sup>[[Team:TU_Munich/Lab#ref2|&#91;2&#93;]]</sup>. The characterization of the kit can be obtained from the [http://partsregistry.org/Cell-free_chassis/Commercial_E._coli_S30 Parts Registry]. <br><br />
Experiments were performed at 37°C with an amount of approximately 1 µg plasmid in a reaction volume of 50 µL. Fluorescence was followed over time in a jasco fluorolog with wavelength corresponding to those used ''in vivo''. <br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==''In vitro'' Transcription==<br />
To monitor transcription termination and antitermination on a the molecular level, ''in vitro'' transcription of individual switches and their response to signals offer an elegant way for fast and easy prove of principle. Most side effects occurring in a complex environment given in a cell or a cell lysate do not arise here. Another major advantage of ''in vitro'' transcription experiments is the possibility to test many signals for one switch to optimize antitermination efficiency and binding specificity without much cloning work. Data gained by ''in vitro'' transcription experiments can be used to improve switches and signals for ''in vivo'' usage. <br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Since we are working on a totally new principle of transcriptional control, we used this approach beside the above mentioned advantages for easy variation of different variables like the length of the core unit and the switch to signal ratio. <br><br />
To study the switches on a transcriptional level offers the advantage, to reduce interference and possible artifacts to a minimum. Since we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems, ''in vitro'' transcription was also used as a minumum system from which more complexity can derive.<br />
<br><br />
Working with in vitro systems also has the advantage that an input is not needed anymore and the output can also be generated easily. We used '''two readouts''' with '''two different transcription systems''' to check and investigate our devices: First, we used an [https://2010.igem.org/Team:TU_Munich/Parts malachitegreen-binding aptamer] for an fluorescence output and second, we simply put our reaction educts on an denaturing acrylamide-gel to check for RNA varying in length. As for two different transcription systems we used on the one hand ''E. coli''-RNA Polymerase (RPO) based transcription since the aim is to apply the so gained results ''in vivo'' and on the other hand T7 based transcription which is well established through literature and delivers good RNA yields.<br />
<br><br />
=== T7 RNA polymerase ===<br />
<br />
<div align="justify"><br />
<br />
The T7 RNA polymerase is known for satisfying RNA yields together with easy handling. In our approach we had PCR amplified, double stranded switches with an malachitegreen binding aptamer following after a T7 terminator which was constructed to function as a switch. Different signals were tested varying in length of the specificity site and the triggering unit. <br />
<br> <br />
For in vitro expression the T7 RNA Polymerase requires a double stranded promotor region at the beginning of the DNA template but is otherwise capable of handling single stranded DNA, so a sense strain corresponding to the T7 promoter region was added. Transcription is more effective with double stranded DNA as template. Apart from that, no more requirements are needed in theory which makes the evaluation of many signals especially easy. Since we ordered the signal sequences we tested we chose the cheaper way in the beginning by using single stranded signals with corresponding sense T7 pieces and switched to double stranded constructs after narrowing down the most promising switch/signal pairs. Later on we also used double stranded signals and switches since transcription rates are higher with those. <br />
<br><br />
As a positive control, the malachite green binding aptamer right behind the T7 promoter was used. Transcription proceeds without termination and the maximal fluorescence intensity should be gained. <br />
<br> Transcription termination can also be estimated by measuring just the switch without interfering signals. Since upon transcription of a signal sequence, less RNA Polymerase is available, the transcription rate of the switch and therefore the fluorescence output is reduced by merely adding the signal. Therefore randomly chosen short sequences in the range of the tested signals were added to the negative control. <br />
<br />
</div><br />
=== ''E. coli'' RNA polymerase ===<br />
In comparison to the T7 RNA Polymerase the ''E. coli'' RNA Polymerase requires slightly more sophisticated proceedings when it comes to the design of switches and handling of the enzyme. The biggest in our case was to store it properly since the only -80°C fridge was in another building, so make sure you have a big supply of dry ice ready if you encounter the same problem. <br><br />
E. coli RPO was ordered saturated with σ70-factor.<br />
<br />
=== Denaturing Polyacrylamide gel electrophoresis ===<br />
<br />
<div align="justify"><br />
Polyacrylamide gel electrophoresis (PAGE) was used for evaluation of termination and switching efficiency. Gels containing 15 % acrylamide and 6 M urea were used for separation of terminated and readthrough RNAs. The same constructs as designed for the malachite green binding aptamer were used. <br />
<br><br />
Polyacrylamide gels separate RNA and DNA according to their size in an electric field. Since the negative charge equals the size of nucleotides in the RNA/DNA, the number of base pairs can be compared between two samples often with one base pair resolution. Since RNA forms three-dimensional structures, the samples are preheated and run in 6 or 7 M urea. The polyacrylamide gel is stained in SybrGold afterwards which binds to both single and double stranded DNA and RNA. A Dnase digestion was applied before running the samples to avoid confusion caused by DNA templates.<br />
<br><br />
Denaturing PAGE is a simple yet elegant way to check for transcription efficiency and termination rates. Since it is a very direct way and it provides a simple yet clear readout, we used it as another method beside the more sophisticated malachitegreen binding assay to evaluate and characterize our switch. Equipment for denaturing PAGE can be found in nearly every biochemical lab, so this method also applies for an easy controlexperiment. <br />
</div><br />
<br />
=== Malachite green assay ===<br />
<br />
<div align="justify"><br />
<br />
[[Image:TUM2010_Malachit_emission.png|200px|thumb|left|Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA]]<br />
In this year's DNA submission we contribute the [[Team:TU_Munich/Parts#Malachite_Green_Binding_Aptamer_-_BBa_K494000 | malachite green binding aptamer]] which can be used as a transcription reporter in ''in vitro'' transcription experiments. <br><br />
Malachite green is a dye with a negligible fluorescence in solution but undergoes a dramatic increase about 3000 times if bound by the RNA aptamer making it an exceptional good marker. Since the binding is very specific, transcription in dependence of a signal can be monitored by measuring the fluorescence of malachite-green over time if the aptamer is located behind the switch. Transcription of the aptamer will only take place after anti-termination by a signal. An increase should be visible over time. Other triphenyldyes are also recognized with weaker effects on the fluorescence but may also serve as reporters if the emission or excitation of malachite green does not fit the experimental setup. <br />
<br><br><br><br />
[[Image:TUM2010_Malachitgruen-2.png|600px|center|thumb|Description of the malachit green assay. Antitermination allows the polymerase to produce the malachite green aptamer ]]<br />
Malachite green binding can be used to follow RNA transcription over time, a rise in the fluorescence is then detectable. Fluorescence marker of specific RNA structures are still rare, so the malachite green binding aptamer provides one of the only possibilities to continuously monitor transcription reactions. In comparison to PAGE, kinetics can be taken, while with PAGE only end point estimations can be made. This makes the malachite green binding aptamer a valuable tool to study ''in vitro'' transcription in general and the principles underlying our switch in principle. <br />
<br />
<br />
<!--For the T7-based measurements we ordered single stranded signals for a first attempt and added matching single strands complementary to the T7 promoter region. The switch was amplified using PCR and consisted of the following elements: Primer-binding site - T7 promotor - switch - malachitegreen binding aptamer. Upon binding of a correct signal to the switch, the stem loop dissolves and transcription is possible. <br />
<br />
<br />
<br><br><br />
<br />
OLD: A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramatically (about 3000 times) upon binding of a specific RNA-aptamer. The RNA-aptamer<br />
<br><br><br />
---concept to be described, as well as literature---<br />
<ref>refs</ref><br />
<br><br><br />
<br><br />
<br />
We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.<br />
<br><br />
Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level. --><br />
</div> <br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
=Lab Book=<br />
<br />
==Explanations==<br />
In the following we present an overview regarding our work in the lab. For easier understanding we summarized the work of each week using colored boxes. To get a better overview we used the following color code for the boxes:<br />
{|<br />
|-<br />
| {{:Team:TU_Munich/Templates/RedBox | text=&nbsp;}} The red box represents general cloning steps that were required for our measurements. See the [[Team:TU_Munich/Lab#Molecular_Biology | protocol section]] for further details.<br />
|-<br />
| {{:Team:TU_Munich/Templates/BlueBox | text=&nbsp;}} The blue box indicates <i>in vivo</i> measurements which are described [[Team:TU_Munich/Lab#In vivo Measurements | here]].<br />
|-<br />
| {{:Team:TU_Munich/Templates/GreenBox | text=&nbsp;}} The green box indicates <i>in vitro</i> measurements relying on <i>in vitro</i> transcription and malachite green measurements. Details can be found [[Team:TU_Munich/Lab#In vitro Transcription | here]].<br />
|-<br />
| {{:Team:TU_Munich/Templates/YellowBox | text=&nbsp;}} The yellow box represents measurements done with an <i>in vitro</i> translation kit and is described in more details [[Team:TU_Munich/Lab#In vitro Translation | here]].<br />
|}<br />
<br><br />
To learn more about the work and results of a specific week, just click on the according week number. You will find detailed notes on our daily lab work. We present these notes in an unedited form as a record of our work, for for processed results please check the [[Team:TU_Munich/Project#Results | results section on our project page]].<br />
<br />
==Chronological Lab Book==<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week01{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===08.04.2010===<br />
==Chronological Lab Book==<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week01{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===08.04.2010===<br />
Flo & Philipp<br />
<br />
'''PCR''' <br />
*samples:<br />
** R0011_His<br />
** R0011_Trp<br />
** Control<br />
*protocol: [[Team:TU_Munich/Lab#Molecular_Biology| protocol]]<br />
**templates: purified PCR products from 5.2.2010<br />
**primer G1004/1005<br />
**polymerase: Taq<br />
**programm: igempcr<br />
<br />
<br />
'''Purification of PCR products with QIAquick PCR purification Kit '''<br />
*protocol followed. exceptions: DNA-binding/unbinding with 3min 6000rpm followed by 60sec full speed<br />
<br />
<br />
'''2% Agarose Gel'''<br />
*in 1x TAE.<br />
<br />
===09.04.2010===<br />
Philipp & Flo<br />
<br />
----<br />
'''Gel of PCR products from 08.04.2010'''<br />
*loaded: 10 µL sample+2 µL 6x GLD, 4/2 µL LMW standard<br />
* 110 V, 90 min<br />
*stained with Sybrgold, 20 min, 1:10.000 dilution in TAE<br />
*Standard - Control - R0011_His - R0011_Trp - Standard(=low molecular weight (see [[Team:TU_Munich/Lab#Molecular_Biology Lab_Protocols]]))<br />
[[Image:TUM2010_100409.JPG]]<br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week02{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
<br />
===15.04.2010===<br />
Philipp & Flo<br />
<br><br>[http://web.e14.physik.tu-muenchen.de/igem/index.php/Team:TU_Munich/Lab#Molecular_Biology PCR PCR] of B0014 and R0011<br />
<br />
===16.04.2010===<br />
Philipp & Flo<br><br><br />
<br />
*'''Purification''' of PCR products from [[15.04.2010]] using [[Team:TU_Munich/Lab#Molecular_Biology QIAquick_purification_Kit|QIA kit]] <br><br><br />
<br />
*'''Concentrations''' measured with nanodrop:<br><br><br />
<br />
{| width="200" cellspacing="1" cellpadding="1" border="1" align="center"<br />
|-<br />
| B0014 <br />
| 2.5 ng/µL<br><br />
|-<br />
| R0011<br> <br />
| 27.5 ng/µL<br><br />
|}<br />
<center><br> --&gt; worked for R0011, not for B0014 <br> </center> <br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|PCR]] '''of B0014<br><br />
**Purification with the [[Team:TU_Munich/Lab#Molecular_Biology ZYMO_RESEARCH_DNA_Clean.26Concentration_Kit|Zymo Kit]], Elution in 20 µL H2O<br />
**Concentration measured with nanodrop, 17.5 ng/µL --> worked<br><br><br />
<br />
*'''Digestions''' of:<br><br><br />
<br />
{| width="618" cellspacing="1" cellpadding="1" border="1" align="center" style=""<br />
|-<br />
| '''template'''<br> <br />
| '''restriction enzymes (biobrick assembly)'''<br><br />
|-<br />
| B0014 (from Christoph, verified PCR products, 21 ng/uL)<br> <br />
| EcoRI, PstI<br><br />
|-<br />
| R0011 (from PCR [15042010], 27.5 ng/µL<br> <br />
| SpeI<br><br />
|-<br />
| HisSig (1:100 dilution)<br> <br />
| XbaI<br><br />
|-<br />
| TrpSig (1:100 dilution)<br> <br />
| XbaI<br><br />
|-<br />
| psB1K3 (with RFP insert, from HiWiPhilipp, 81 ng/µL) <br />
| EcoRI, PstI<br />
|}<br />
<br />
<br> <br />
<center>5 µL template used for each setup. [[Team:TU_Munich/Lab#Molecular_Biology Restriction|protocol]] followed</center> <br />
<br> <br />
<br />
*'''Gel''' for purification of the cleaved plasmid <br />
**2% Agarose in 1x TAE <br />
**120 V, 90 min <br />
**[[Team:TU_Munich/Lab#Molecular_Biology stain|stained]] with SybrGold<br />
**digestion, digestion, [[Team:TU_Munich/Lab#Molecular_Biology standards|1 kb ladder]]<br />
***Digestion worked (partly). band at 2000 bp (backbone) cut<br><br><br />
[[Image:TUM2010_100416.png]]<br><br />
<br />
<br><br><br />
*'''Purification of DNA from Gel'''<br />
**using the [[Team:TU_Munich/Lab#Molecular_Biology ZYMO_RESEARCH_DNA_Clean.26Concentration_Kit|Zymo Kit]]<br />
<br />
<br><br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' of HisSig/TrpSig with R0011in 2 reactions<br><br><br />
<br />
{| width="617" cellspacing="1" cellpadding="1" border="1" align="center" style=""<br />
|-<br />
| <br> <br />
| '''used Volume'''<br> <br />
| '''approx. concentration*'''<br><br />
|-<br />
| HisSig<br> <br />
| 6 µL<br> <br />
| 7 ng/µL<br><br />
|-<br />
| TrpSig<br> <br />
| 6 µL<br> <br />
| 5 ng/µL<br><br />
|-<br />
| R0011<br> <br />
| 3 µL<br> <br />
| 6 ng/µL<br><br />
|}<br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
* approximated from the amount used in the digestion before<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week03{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===19.04.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|PCR]] '''of R0011-TrpSig and R0011-HisSig<br><br />
**Purification with the [[Team:TU_Munich/Lab#Molecular_Biology ZYMO_RESEARCH_DNA_Clean.26Concentration_Kit|Zymo Kit]], Elution in 30 uL H2O<br />
**Concentration measured with nanodrop: c(R0011-TrpSig)=20 ng/µL, c(R0011-HisSig)=12.5 ng/µl --> worked<br><br><br />
<br />
*'''Gel''' for analysis of ligation and PCR <br />
**2% Agarose in 1x TAE <br />
**110 V, 90 min <br />
**[[Team:TU_Munich/Lab#Molecular_Biology stain|stained]] with SybrGold 1:10000 20 min<br />
**pure R0011 PCR product used as control<br />
<br>[[Image:TUM2010_GEL_20100419beschriftet.png]] <br><br />
{| width="617" cellspacing="1" cellpadding="1" border="1" align="center" style=""<br />
|-<br />
| '''LMW'''<br> <br />
| 4 µl<br> <br />
|-<br />
| '''R0011-TrpSig'''<br> <br />
| 5 µL<br> <br />
|-<br />
| '''R0011-HisSig'''<br><br />
| 5 µL<br> <br />
|-<br />
| '''R0011'''<br><br />
| 5 µL<br> <br />
|-<br />
<br />
|}<br />
<br />
<br><br />
Samples seem to have run further than the buffer/dye-Front! But: Ligation Products show bands at shorter lengths than R0011 alone --> Ligation didn't work ?!?<br />
<br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' of HisSig/TrpSig with R0011in 2 reactions<br><br><br />
<br />
{| width="617" cellspacing="1" cellpadding="1" border="1" align="center" style=""<br />
|-<br />
| <br> <br />
| '''used Volume'''<br> <br />
| '''concentration'''<br><br />
|-<br />
| pSB1K3<br> <br />
| 5 µL<br> <br />
| 10 ng/µL (nanodrop)<br><br />
|-<br />
| B0014<br> <br />
| 3 µL<br> <br />
| 5 ng/µL approx.*<br><br />
<br />
|}<br />
<br />
<br />
<br />
* approximated from the amount used in the digestion before<br />
===20.04.2010===<br />
*'''Gel''' for analysis of ligation and PCR (repeat of [[19.04.2010|yesterday's gel]])<br />
**2% Agarose in 1x TAE <br />
**130 V, 75 min <br />
**[[Team:TU_Munich/Lab#Molecular_Biology stain|stained]] with SybrGold 1:10000 60 min<br />
**pure R0011 PCR product used as control<br />
**Excision and purification of marked bands at 200 bp using QIA Kit, elution in 30 µl H2O<br><br />
[[Image:TUM2010_Gel100420marked.png ]]<br />
<br />
<br />
<br><br><br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|PCR]] '''of excised and purified bands of R0011-TrpSig and R0011-HisSig<br><br />
**complete samples (30 µl) used as templates<br />
**Purification with the [[Team:TU_Munich/Lab#Molecular_Biology ZYMO_RESEARCH_DNA_Clean.26Concentration_Kit|Zymo Kit]], Elution in 30 uL H2O<br />
**Concentrations of PCR-products: 0.5-1 ng/µl --> Gel excision or PCR didn't work<br />
<br />
<br><br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]] ''' (Woehlke-Lab)<br />
**8 µl of ligation product pSB1K3-B0014 to 50 µl XL-10 competent cells<br />
**200 µl plated on a Kanamycin-containing Plate<br />
**remaining 800 µl stored @4°C in S1-lab<br />
<br />
===21.04.2010===<br />
*'''Gel''' for analysis of ligation and PCR (repeat of yesterday's gel) <br />
**2% Agarose in 1x TAE <br />
**110 V, 90 min <br />
**[[Team:TU_Munich/Lab#Molecular_Biology stain|stained]] with SybrGold 1:10000 80 min <br />
**pure R0011 PCR product used as control <br />
**Excision and purification of marked bands at 200 bp using Zymo 5 Kit, elution in 20 µl H2O<br><br />
<br />
[[Image:TUM2010_100421beschriftet.gif??]] <br />
<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|PCR]] '''of excised and purified bands of R0011-TrpSig and R0011-HisSig<br> <br />
**complete samples (20 µl) used as templates <br />
**Purification with the [[Team:TU_Munich/Lab#Molecular_Biology ZYMO_RESEARCH_DNA_Clean.26Concentration_Kit|Zymo Kit]], Elution in 25 uL H2O <br />
**Concentrations of PCR-products: <br />
*** R0011-TrpSig: 22.5 ng/µl<br />
*** R0011-HisSig: 9.5 ng/µl<br />
--> worked!!!!!<br />
<br><br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]] '''<br />
**7 Colonies picked and resuspended in 20 µl LB+Kana (each)<br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified)<br />
**15 µl of each sample mixed with 3 µl GLPn and loaded to Gel<br />
<br />
<br />
[[Image:TUM2010_100421colony.png]] <br />
<br><br><br />
*Overnight cultures: <br />
**remaining 18 µl of samples 1, 3, 6, and 7 added to 5 ml LB + kanamycin<br />
**37°C on Shaker<br />
<br />
===22.04.2010===<br />
*'''Gel''' for purification of PCR products R0011-TrpSig and R0011-HisSig ([[21.04.2010|yesterday's result]]) <br />
**2% Agarose in 1x TAE <br />
**110 V, 90 min <br />
**[[Team:TU_Munich/Lab#Molecular_Biology stain|stained]] with SybrGold 1:10000 30 min <br />
**pure R0011 PCR product used as control <br />
**Excision and purification of marked bands at 200bp using Zymo 5 Kit, elution in 20 µl H2O<br><br />
<br />
[[Image:TUM2010_100422beschriftet.png]]<br />
<br />
<br><br><br />
*Miniprep<br />
**Result: about 4 µg Plasmid<br />
<br />
===23.04.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Restriction|Digestion]]''' of:<br><br><br />
<br />
{| width="618" cellspacing="1" cellpadding="1" border="1" align="center" style=""<br />
|-<br />
| '''template'''<br> <br />
| '''template volume'''<br> <br />
| '''restriction enzymes'''<br><br />
| '''Buffer'''<br> <br />
|-<br />
| HisSig (1:100 dilution)<br> <br />
| 5 µl<br><br />
| EcoRI, SpeI<br><br />
| NEB4<br><br />
|-<br />
| TrpSig (1:100 dilution)<br> <br />
| 5 µl<br><br />
| EcoRI, SpeI<br><br />
| NEB4<br><br />
|-<br />
| psB1K3-B0014 from Miniprep (No 7, 35 ng/µl) <br />
| 5 µl<br><br />
| EcoRI, XbaI<br />
| NEB4<br><br />
|}<br />
<br />
<br> <br />
Incubated 90 min @ 37°C<br />
<br><br />
<br />
*'''Gel''' for purification of the cleaved plasmid <br />
**2% Agarose in 1x TAE (leftover from yesterday)<br />
**140 V, 90 min <br />
**[[Team:TU_Munich/Lab#Molecular_Biology stain|stained]] with SybrGold 40 min<br />
**4 µl [[Team:TU_Munich/Lab#Molecular_Biology standards|1 kb ladder]], 10 µl purified digestion + 2 µl GLPn, 10 µl purified digestion + 2 µl GLPn<br />
***Digestion worked (partly). band at 2400 bp cut out<br><br><br />
[[Image:TUM2010_100423beschriftet.png]]<br><br />
<br />
<br><br><br />
*'''Purification of DNA from Gel'''<br />
**using the [[Team:TU_Munich/Lab#Molecular_Biology ZYMO_RESEARCH_Gel_DNA_Recovery_Kit|Zymo Kit]]<br />
**elution in 25 µl H2O<br />
* A260/A230 and A260/A280 values were strange (see labbook)<br />
<br><br><br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week04{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
<br />
===26.04.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of pSB1K3-B0014 with EcorI and XbaI<br />
** 10 µl template (No1, 50 ng/µl)<br />
** 5 µl BSA, 5 µl Buffer NEB#4<br />
** 1 µl EcoRI, 1 µl XbaI<br />
** 28 µl H2O<br />
** 1.5 h @ 37°C<br />
**Purification with Zymo5 Kit, elution in 20 µl H2O<br />
<br><br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' of Signals and PSB1K3-B0014<br />
**3 µl of each sample, end volume 20 µl<br />
<br><br />
*Preparation of Measurement Plasmid from Folder, Transformation<br />
**Plate 1022, Spots 1E, 1G, 2A: pSB1A10 with different Inserts, all inserts are Zinc-finger constructs with about 1.6 kb<br />
<br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]] ''' of XL10 with Ligation Products (8 µl each) and pSB1A10 (2 µl each)<br />
<br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Preparation of BioBricks from distribution 2008|Preparation]]''' of Measurement Plasmid from Folder, Transformation<br />
**Plate 1022, Spots 1E, 1G, 2A: pSB1A10 with different Inserts, all inserts are Zinc-finger constructs with about 1.6 kb<br />
<br><br />
*growing over night cultures of remaining PSB1K3-B0014-transformed cells<br />
**2x 5 ml, 2x 1 ml<br />
<br />
===27.04.2010===<br />
*Plenty of cultures on both HisSig and TrpSig Ligation plates, but nothing on pSB1A10 plates! --> repeat DNA extraction, ask Prof. Simmel for new Distribution<br />
<br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
**7 Colonies picked and resuspended in 20 µl LB+Kana (each)<br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified)<br />
**10 µl of each sample mixed with 2 µl GLPn and loaded to Gel<br />
<br />
[[Image:TUM2010_100427beschriftet.png]] <br />
<br />
Many colonies with pSB1K3-B0014, not one with pSB1K3-Sig-B0014<br />
<br />
*Miniprep of PSB1K3-B0014<br />
**Samples I and II: 5 ml overnight cultures, centrifuged 10 min @ 3200 g, resuspended in 600 µl of the same culture<br />
**Samples III and IV: 600 µl overnight cultures<br />
**all samples mixed with 100 µl lysis buffer, Miniprep with Zyppy kit, each sample eluted in 50 µl H2O <br />
**Concentration measured (Nanodrop, LP=1mm, Factor 10, 4 µl sample)<br />
***cI=61.5 ng/µl<br />
***cII=33.5 ng/µl<br />
***cIII=103 ng/µl<br />
***cIV=108 ng/µl<br />
<br><br />
--> Better results for 600 µl cultures without centrifuging!!!<br />
<br><br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Preparation of BioBricks from distribution 2008|Preparation]]''' of Measurement Plasmid from Folder, Transformation<br />
**Plate 1022, Spots 1E, 1F, 1G, 1H, 2A: pSB1A10 with different Inserts, all inserts are Zinc-finger constructs with about 1.6 kb<br />
<br><br />
<br />
===28.04.2010===<br />
*No colonies on plates from Yesterday's transformations, but on the older plates (from monday) some colonies appeared<br />
**7 Colonies picked and resuspended in 20 µl LB0 (each)<br />
**1&2 from plate "1E", 3&4 from plate "1G", 5,6&7 from plate "2A", 8 LB0<br />
<br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified)<br />
**15 µl of each sample mixed with 3 µl GLPn and loaded to Gel<br />
**1% Agarose in 1xTAE, 95 V, after 50 minutes changed to 110 V<br />
<br />
[[Image:TUM2010_100428beschriftet.png]] <br />
<br />
<br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of pSB1K3-B0014 with EcorI and XbaI<br />
** 10 µl template (No1, 50 ng/µl)<br />
** 5 µl BSA, 5 µl Buffer NEB#4<br />
** 1 µl EcoRI, 1 µl XbaI<br />
** 28 µl H2O<br />
** 1.5 h @ 37°C<br />
** heat inactivation 5min @60°C <br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Dephosphorylation|Dephosphorylation]]''' of restricted vector<br />
**Purification with Zymo5 Kit, elution in 20 µl H2O<br />
**loaded on gel (with 4 µl GLPn) (Gel shown above)<br />
*Gel excision with Zymo Kit<br />
**<br />
**<br />
<br><br />
<br />
<br />
===29.04.2010===<br />
<br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of pSB1K3-B0014 with EcorI and XbaI<br />
** 10 µl template (NoIV, 108 ng/µl)<br />
** 5 µl BSA, 5 µl Buffer NEB#4<br />
** 1 µl EcoRI, 1 µl XbaI<br />
** 28 µl H2O<br />
** 1.5 h @ 37°C<br />
** heat inactivation 5min @60°C <br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Dephosphorylation|Dephosphorylation]]''' of restricted vector<br />
**Purification with Zymo5 Kit, elution in 20 µl H2O<br />
**loaded on gel (with 4 µl GLPn)<br />
[[Image:x]] <br />
*Gel excision with Zymo Kit<br />
**<br />
**<br />
<br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of R0011 with SpeI<br />
** 10 µl template (R0011, X ng/µl)<br />
** 5 µl BSA, 5 µl Buffer NEB#4<br />
** 1 µl SpeI<br />
** 29 µl H2O<br />
** 1.5 h @ 37°C<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' <br />
** 5 µl R0011 (S-digested) with 12 µl TrpSig or HisSig, respectively (X-digested)<br />
** complete ligation (20 µl) loaded on Gel (with 4 µl GLPn)<br />
[[Image:TUM2010_100429beschriftet.png]] <br />
** Gel excision with Zymo Kit, eluted in 42 µl H2O<br />
<br><br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]]''' <br />
**50 µl XL-10 transformed with 2 µl of pSB1A10 prepared from IGem 2009 Distribution (13 µl left in pink Box @-20°C)<br />
*<br />
<br><br />
<br />
===30.04.2010===<br />
PCR R0011-HisSig and R0011-TrpSig --> 13 ng/µl x 20 µl<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week05{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===04.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of pSB1K3-B0014 with EcorI and XbaI<br />
** 10 µl template (NoIII, 103 ng/µl)<br />
** 5 µl BSA, 5 µl Buffer NEB#4<br />
** 1 µl EcoRI, 1 µl XbaI<br />
** 28 µl H2O<br />
** 1.5 h @ 37°C<br />
**Purification with Zymo5 Kit, elution in 15 µl H2O<br />
**loaded on gel (with 3 µl GLPn) <br />
[[Image:TUM2010_100504beschriftet.png]] <br />
*Gel excision with Zymo Kit<br />
**<br />
**<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of R0011-HisSig and R0011-TrpSig with EcorI and SpeI<br />
** 10 µl template (PCR-product)<br />
** 5 µl BSA, 5 µl Buffer NEB#4<br />
** 1 µl EcoRI, 1 µl SpeI<br />
** 28 µl H2O<br />
** 1.5 h @ 37°C<br />
**Purification with Zymo5 Kit, elution in 20 µl H2O<br />
<br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' <br />
** 4 µl R0011-Signal (E/S-digested) with 4 µl pSB1K3-B0014 (E/X-digested)<br />
**15 min @ RT, 20 min heat inactivation @ 65°C<br />
<br><br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]]''' <br />
**50 µl XL-10 transformed with 10 µl of Ligation mix<br />
<br><br />
<br />
*Overrnight liquid cultures of pSB1A10-RFP made from<br />
**1 and 2: picked clones from original plates from *[[29.04.2010|Do 29.04.2010]]<br />
**3a: picked clone from copy plate from *[[30.04.2010|Fr 30.04.2010]]<br />
**3b: resuspended clone N° 3 from *[[30.04.2010|Fr 30.04.2010]]<br />
--> each in 600 µl LB+Carbenicillin (=Ampicillin) @37°C<br />
===05.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Miniprep|Miniprep]]''' of pSB1A10; 4 samples (1, 2; 3a; 3b)<br />
**eluted in 50 µl H2O each<br />
**Concentrations:<br />
*** c1=37.5 ng/µl<br />
*** c2=56.5 ng/µl<br />
*** c3a=46.5 ng/µl<br />
*** c3b=30 ng/µl<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of pSB1A10 with EcorI and PstI, 4 samples (1, 2; 3a; 3b)<br />
** 15 µl template <br />
** 5 µl BSA, 5 µl Buffer NEB#3<br />
** 1 µl EcoRI, 1 µl PstI<br />
** 23 µl H2O<br />
** 1.5 h @ 37°C<br />
** heat inactivation 5min @60°C <br />
**Purification with Zymo5 Kit, elution in 15 µl H2O<br />
**loaded on gel (with 3 µl GLPn)<br />
[[Image:TUM2010_100505beschriftet.png]]<br />
<br> <br />
Insert @ 1 kb as expected, but vector @ 2 kb and not @ 5 kb as expected!!!!<br />
--> Wrong Plasmid! Comparison to the [http://partsregistry.org/cgi/assembly/plate_egel.cgi?id=615 Gel in the registry] shows: The Distribution contains the wrong plasmid!<br />
<br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of HisTerm and TrpTerm with EcorI and PstI<br />
** 5 µl template <br />
** 5 µl BSA, 5 µl Buffer NEB#3<br />
** 1 µl EcoRI, 1 µl PstI<br />
** 33 µl H2O<br />
** 1.5 h @ 37°C<br />
<br><br><br />
*Clones picked: 7 from each Plate (pSB1K3-R0011-TrpSig-Boo14 and pSB1K3-R0011-HisSig-Boo14)<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR)<br />
**15 µl of each sample mixed with 3 µl GLPn and loaded to Gel<br />
**2% Agarose in 1xTAE, 130 V, 90 min<br />
<br />
[[Image:TUM2010_100505bbeschriftet.png]] <br />
<br />
<br><br />
<br />
===06.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of pSB1K3 with EcorI and XbaI<br />
** 20 µl template (sample III, 103 ng/µl)<br />
** 5 µl BSA, 5 µl Buffer NEB#3<br />
** 1 µl EcoRI, 1 µl XbaI<br />
** 18 µl H2O<br />
** 1.5 h @ 37°C<br />
** heat inactivation 5min @60°C <br />
**loaded on gel (with 10 µl GLPn) in 4 lanes<br />
[[Image:TUM2010_100506beschriftet.png]] <br />
*Gel excision with Zymo Kit (lanes 1&2) and with Qiaquick Kit (lanes 3&4)<br />
** c1=4.5 ng/µl<br />
** c2=3.5 ng/µl<br />
** c3=2 ng/µl<br />
** c1=7 ng/µl<br />
* A260/A230 and A260/A280 values were strange (see labbook)<br />
<br><br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' <br />
** 4 µl R0011-Signal (E/S-digested) with 10 µl pSB1K3-B0014 (E/X-digested, from [[23.04.2010|23.04.]])<br />
**15 min @ RT, 20 min heat inactivation @ 65°C<br />
<br><br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]]''' <br />
**50 µl XL-10 transformed with 7 µl of Ligation mix<br />
<br><br />
<br />
===07.05.2010===<br />
<br><br />
*Clones picked: 7 from each Plate (pSB1K3-R0011-TrpSig-Boo14 and pSB1K3-R0011-HisSig-Boo14)<br />
<br> <br />
---Too damn stupid to do a PCR!!!---<br> <br> <br />
<br><br />
* replated picked clones on new plates, incubated at RT<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week06{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
<br />
===10.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]''' of picked clones from [[07.05.2010|Fr 07.05.2010]]<br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified)<br />
**15 µl of each sample mixed with 3 µl GLPn and loaded to Gel<br />
**2% Agarose in 1xTAE, 120 V, 110 min<br />
**stained in SybrSafe 50 min<br />
[[Image:TUM2010_100510beschriftet.png]] <br />
<br />
<br><br />
Interpretation: <br />
Colonies contain an Insert with Prefix and Suffix, length is 200 bp. This is too short for the desired R0011-Signal-B0014 (245 or 247 bp) construct, but longer than B0014 (136 bp) which was the Insert in the digested vector. <br />
Possible explanation: Ligation worked, but not with R0011-Signal-construct but with R0011 '''or''' Signal. Which?<br />
<br />
<br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| fragment<br />
| length without Prefix/Suffix <br />
| length with Prefix/Suffix<br />
|-<br />
| R0011<br />
| 55 bp <br />
| 96 bp<br />
|-<br />
| B0014<br />
| 95 bp<br />
| 136 bp<br />
|-<br />
| TrpSig/HisSig<br />
| 34 bp/32 bp<br />
| 75 bp/73 bp<br />
|-<br />
| TrpSig-B0014/HisSig-B0014<br />
| 135 bp/ 133 bp<br />
| 176 bp/ 174 bp<br />
|-<br />
| R0011-TrpSig-B0014/R0011-HisSig-B0014<br />
| 206 bp/ 204 bp<br />
| 247 bp/ 245 bp<br />
|-<br />
| R0011-B0014<br />
| 156 bp<br />
| 197 bp<br />
|-<br />
| R0011-TrpSig/R0011-HisSig<br />
| 95 bp/93 bp<br />
| 136 bp/134 bp<br />
|-<br />
|}<br />
<br />
Prefix: 20 bp; Suffix: 21 bp; X-S-scar: 6 bp<br />
<br />
--> it looks as if R0011 is ligated to B0014, which makes the whole construct wothless. The R0011-HisSig control looks more like R0011 alone as well.<br />
<br />
===11.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' <br />
** 4 µl Signal (E/S-digested; from ) with 5 µl pSB1K3-B0014 (E/X-digested; from)<br />
**15 min @ RT<br />
<br><br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]]''' <br />
**50 µl XL-10 transformed with 7 µl of Ligation mix<br />
<br><br />
<br />
===12.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]''' of picked clones from [[11.05.2010|Tu 12.05.2010]]<br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified)<br />
**15 µl of each sample mixed with 3 µl GLPn and loaded to Gel<br />
**2% Agarose in 1xTAE, 120 V, 110 min<br />
**stained in SybrSafe 60 min<br />
[[Image:TUM2010_100512beschriftet.png]] <br />
<br />
<br><br />
<br />
<br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| fragment<br />
| length without Prefix/Suffix <br />
| length with Prefix/Suffix<br />
| length after PCR<br />
|-<br />
| R0011<br />
| 55 bp <br />
| 96 bp <br />
| 104 bp<br />
|-<br />
| B0014<br />
| 95 bp<br />
| 136 bp <br />
| 154 bp<br />
|-<br />
| TrpSig/HisSig<br />
| 34 bp/32 bp<br />
| 75 bp/73 bp <br />
| 93 bp/91 bp<br />
|-<br />
| TrpSig-B0014/HisSig-B0014<br />
| 135 bp/ 133 bp<br />
| 176 bp/ 174 bp <br />
| 194 bp/ 192 bp<br />
|-<br />
| R0011-TrpSig-B0014/<br>R0011-HisSig-B0014<br />
| 206 bp/ 204 bp<br />
| 247 bp/ 245 bp <br />
| 265 bp/ 263 bp<br />
|-<br />
| R0011-B0014<br />
| 156 bp<br />
| 197 bp <br />
| 215 bp<br />
|-<br />
| R0011-TrpSig/R0011-HisSig<br />
| 95 bp/93 bp<br />
| 136 bp/134 bp <br />
| 154 bp/152 bp<br />
|-<br />
|}<br />
<br><br />
Prefix: 20 bp/29 bp after PCR; Suffix: 21 bp/30 bp after PCR; X-S-scar: 6 bp<br />
===14.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of pSB1K3 with EcorI and XbaI<br />
** 10 µl template (sample III, 103 ng/µl)<br />
** 2 µl BSA, 2 µl Buffer NEB#4<br />
** 1 µl EcoRI, 1 µl XbaI<br />
** 4 µl H2O<br />
** 1 h @ 37°C<br />
**loaded on gel (with 4 µl GLPn) in 1 lane<br />
[[Image:TUM2010_100514beschriftet.png]] <br />
*Gel excision with Zymo Kit <br />
<br />
<br><br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of HisSig and TrpSig with EcorI and SpeI<br />
** 10 µl template ("1:100")<br />
** 2 µl BSA, 2 µl Buffer NEB#3<br />
** 1 µl EcoRI, 1 µl SpeI<br />
** 4 µl H2O<br />
** 1.5 h @ 37°C<br />
** Purification with [[Team:TU_Munich/Lab#Molecular_Biology ZYMO RESEARCH DNA Clean&amp;Concentration Kit|Zymo 5 ]]<br />
** or heat inactivated (20 min @ 80°C)<br />
<br><br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' <br />
**<br />
<br />
<br><br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]]''' <br />
**50 µl XL-10 transformed with 10 µl of Ligation mix<br />
**50 µl untransformed cells plated on Kana-plate as control<br />
<br><br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week07{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===17.05.2010===<br />
*Plates from Friday:<br />
**plenty colonies on control plate --> XL10 cells are impure!<br />
**use DH5a from now on!!!!!<br />
<br />
<br><br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]]''' <br />
**50 µl DH5a transformed with 10 µl of Friday's Ligation mix<br />
**plated on Kana-Plates; Overnight @ 37°C<br />
<br><br />
<br />
*DNA Isolation from BioBrick Distribution 2010<br />
** 10 µl H2O added to Well 1A of plate 1 containing pSB1A10 with RFP-insert<br />
** 2 µl used for [[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]] of 50 µl DH5a-cells<br />
** plated on Carbenicillin (=Amp-analogon)-plates, Overnight @ 37°C<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Preparation of Gels|Polyacrylamide Gel]]''' prepared for tomorrow<br />
** 1 big denaturing Gel with 20 pockets<br />
<br />
===18.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]''' of picked clones <br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR)<br />
**10 µl of each sample mixed with 10 µl Formamide loading buffer and loaded to Polyacrylamide Gel<br />
<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Running of Gels|Polyacrylamide Gel]]'''<br />
Samples: <br />
LMW|R0011|HisSig|TrpSig|HisSig E/S-Dig|TrpSig E/S-Dig|B0014|LMW2|Colony PCR His1|His2|Trp1|Trp2|control|HisTerm|TrpTerm|HisTerm E/P-Dig|TrpTerm E/P-Dig<br />
<br />
*5 µl of each samples mixed with 5 µl formamide loading dye and loaded to gel(except Ladder and colonyPCR)<br />
**LMW: 3 µl LMW (Korbinian) + 3 µl Formamide loading Dye<br />
**LMW2: 5 µl LMW Quickload (with GLP) + 10 µl Formamide loading Dye<br />
*stained in SybrSafe<br />
<br />
<font color=red>Important Mistake! See below Gel! </font><br />
[[Image:TUM2010_100518beschriftet.png|600px?]]<br />
<br><br />
IMPORTANT MISTAKE: DENATURING GELS NOT USEFUL FOR dsDNA!!!<br />
REPEAT WITH NATIVE GEL, IGNORE INTERPRETATION!!!<br />
<br />
<br />
<br />
(*R0011 and B0014 look normal<br />
*ColonyPCR: bands that look like B0014 in all clones (and in control) --> Religation?<br />
*Signals at the wrong size: should be about 75 bp, look like 200 bp!!!<br />
*terminators completely strange: should be around 100 bp!<br />
<br />
--> are all of our sequences just wrong???<br />
What are we going to do? Order everything new?)<br />
===19.05.2010===<br />
* Gel from Korbinian<br />
*5 µl of each samples mixed with 5 µl formamide loading dye and loaded to gel(except Ladder and colonyPCR)<br />
**LMW: 3 µl LMW (Korbinian) + 3 µl Formamide loading Dye<br />
**colony PCR: from Tuesday, 8 µl sample with 8 µl Formamide loading Dye<br />
*stained in SybrSafe 20 min<br />
[[Image:TUM2010_100519paabeschriftet.png]]<br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
** 4 colonies picked from each Plate (Ligations from yesterday; Signal-B0014)<br />
**15 µl of each Sample mixed with 3 µl GLPn and loaded to Gel:<br />
** 3% Agarose in 1x TBE, 130 V<br />
[[Image:TUM2010_100519beschriftet.png600px]]<br />
<br />
===20.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' <br />
** template<br />
** 2 µl BSA<br />
** 2 µl Buffer <br />
** 1 µl of each enzyme<br />
** water to reach 20 µl<br />
<br><br />
{| width="618" cellspacing="1" cellpadding="1" border="1" align="center" style=""<br />
|-<br />
| '''Template'''<br> <br />
| '''Enzymes'''<br><br />
| '''NEB Buffer #'''<br />
|-<br />
| HisTerm & TrpTerm (10 µl)<br><br />
| EcoRI, PstI<br><br />
| 3<br />
|-<br />
| HisSig & TrpSig (10 µl)<br> <br />
| EcoRI, SpeI<br><br />
| 4<br><br />
|-<br />
| B0014 (5 µl)<br> <br />
| XbaI, PstI<br><br />
| 3<br><br />
|-<br />
| pSB1A10_RFP (14 µl)<br><br />
| EcoRI, PstI<br><br />
| 3<br />
|-<br />
| pSB1K3_RFP (14 µl)<br><br />
| EcoRI, PstI<br><br />
| 3<br />
|-<br />
| pSB1K3_B0014 N° 4 (14 µl)<br><br />
| EcoRI, XbaI<br><br />
| 4<br />
|}<br />
<br><br />
** incubated @37°C for 1.5 h<br />
**digested inserts heat inactivated (20 min @ 80°C)<br />
**digested plasmids loaded on gel (with 4 µl GLPn) in 1 lane<br />
[[Image:TUM2010_100520beschriftet.png]] <br />
*Gel excision with Zymo Kit <br />
**c(pSB1A10, I)=4.5 ng/µl<br />
**c(pSB1A10, II)=1.5 ng/µl ?!?!?!<br />
**c(pSB1K3 E/P)=7 ng/µl<br />
**c(pSB1K3_B0014 E/X)=2.5 ng/µl<br />
<br><br><br />
<br />
*Gel of PCR products<br />
**3% Agarose in 1x TBE; 2h @130 V<br />
[[Image:TUM2010_100520bbeschriftet.png]]<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' <br />
** templates<br />
**2 µl T4-buffer 10x<br />
**1 µl T4-Ligase<br />
**Water to reach 20 µl<br />
<br />
{| width="618" cellspacing="1" cellpadding="1" border="1" align="center" style=""<br />
|-<br />
| '''Vector'''<br> <br />
| '''Insert'''<br><br />
|-<br />
| psB1A10 (E/P; sample I) (10 µl)<br><br />
| TrpTerm (E/P) (4 µl)<br><br />
|-<br />
| psB1A10 (E/P; sample I) (10 µl)<br><br />
| HisTerm (E/P) (4 µl)<br><br />
|-<br />
| psB1K3_B0014 (E/X) (12 µl)<br><br />
| HisSig (E/S) (2 µl)<br><br />
|-<br />
| psB1K3_B0014 (E/X) (12 µl)<br><br />
| TrpSig (E/S) (2 µl)<br><br />
|-<br />
| psB1K3 (E/P) (8 µl)<br><br />
| HisSig (E/S)(2 µl) + B0014 (X/P)(1.5 µl)<br><br />
|-<br />
| psB1K3 (E/P) (8 µl)<br><br />
| TrpSig (E/S)(2 µl) + B0014 (X/P)(1.5 µl)<br><br />
|}<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]]''' <br />
** 50 µl DH5a transformed with 10 µl of Ligation mix<br />
** 50 µl DH5a transformed with 2 µl of pSB1K3_B0014<br />
** 50 µl DH5a transformed with 2 µl of pSB1K3_RFP<br />
** 50 µl DH5a transformed with 2 µl of pSB1A10_RFP<br />
===21.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
** 4 colonies picked from each Plate (pSB1K3_HisSig_B0014, pSB1K3_TrpSig_B0014, pSB1K3_HisSig_B0014 double ligation, pSB1K3_TrpSig_B0014 double ligation)<br />
** each clone resuspended in 20 µl LB0, 3 µl used as template for PCR <br />
** 15 µl of each Sample mixed with 3 µl GLPn and loaded to Gel:<br />
** 3% Agarose in 1x TBE, 130 V<br />
<br />
[[Image:TUM2010_100521beschriftet.png]]<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week08{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
<br />
===25.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
** 4 colonies picked from each Plate <br />
***pSB1K3_HisSig_B0014<br />
***pSB1K3_TrpSig_B0014<br />
***pSB1K3_HisSig_B0014 double ligation<br />
***pSB1K3_TrpSig_B0014 double ligation<br />
***pSB1A10_TrpTerm<br />
***pSB1A10_HisTerm<br />
** each clone resuspended in 20 µl LB0, 2 µl used as template for PCR <br />
** 15 µl of each Sample mixed with 3 µl GLPn and loaded to Gel:<br />
** 3% Agarose in 1x TBE, 220 V (double Gel, 35 cm)<br />
** stained in SybrSafe<br />
<br><br />
[[Image:TUM2010_100525beschriftet.|600px]]<br><br />
[[Image:TUM2010_100525bbeschriftet.png]]<br><br />
*overnight cultures made of<br />
**HisSig 3, DL1, DL4<br />
**TrpSig DL2, DL4<br />
**HisTerm/TrpTerm 1,2,3<br />
<br />
===26.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology #ZYMO RESEARCH DNA Clean&Concentration Kit|Miniprep]]''' of cultures set up [[25.05.2010]]<br />
**HisSig 3, DL1, DL4<br />
**TrpSig DL2, DL4<br />
**HisTerm/TrpTerm 1,2,3<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Restriction|Restriction]]'''<br />
**analytical: E/P HisSig(3, DL2); TrpSig(DL2, DL4): 1.5 h 37 °C<br />
**prep: E/X HisSig(3, DL2); TrpSig(DL2, DL4): 1.5 h 37 °C<br />
**prep: E/S R0011: 1.5 h 37 °C, inactivation 20 min @ 80 °C<br />
***total volume each 20 µl, 10 µL template<br />
*'''Gel''': 1% Agarose, TAE - 1,5 h 110 V<br />
**[[Team:TU_Munich/Lab#Molecular_Biology #ZYMO RESEARCH Gel DNA Recovery Kit|bands excised]]: all E/X cleaved vectors<br />
[[Image:TUM2010_100526beschriftet.png]]<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''':<br />
**HisSig_3 (E/X) with R0011 (E/S)<br />
**HisSig_DL2 (E/X) with R0011 (E/S)<br />
**TrpSig_DL2 (E/X) with R0011 (E/S)<br />
**TrpSig_DL4 (E/X) with R0011 (E/S)<br />
***'''batches'''<br />
***total volume 20 µL<br />
***2 µL R0011 (E/S)<br />
***2 µL T4 buffer<br />
***1 µL T4 Ligase<br />
***15 µL vector<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]]''' of ligations<br />
** DH5a<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|PCR]]'''<br />
**His and TrpSig<br />
**His and TrpTerm<br />
**R0011<br />
**B0014<br />
***50 µL total volume<br><br />
***1 µL template<br><br />
***1 µl G1004<br><br />
***1 µl G1005<br><br />
***0.2 µL Taq<br><br />
***5 µl Taq standard buffer<br><br />
***rest water<br />
<br />
===27.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
** 3 colonies picked from each Plate <br />
***pSB1K3_R0011_HisSig_B0014 (N° 3 from yesterday)<br />
***pSB1K3_R0011_HisSig_B0014 (N° DL1 from yesterday)<br />
***pSB1K3_R0011_TrpSig_B0014 (N° DL2 from yesterday)<br />
***pSB1K3_R0011_TrpSig_B0014 (N° DL4 from yesterday)<br />
***"N6"<br />
***"N15"<br />
** each clone resuspended in 20 µl LB0, 2 µl used as template for PCR <br />
** 15 µl of each Sample mixed with 3 µl GLPn and loaded to Gel:<br />
** 3% Agarose in 1x TBE, 220 V (double Gel, 35 cm)<br />
** stained in SybrSafe<br />
<br><br />
[[Image:TUM2010_100527beschriftet.png]]<br><br />
<br><br />
<br />
<br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| fragment<br />
| length without Prefix/Suffix <br />
| length after PCR<br />
|-<br />
| R0011<br />
| 55 bp <br />
| 104 bp<br />
|-<br />
| B0014<br />
| 95 bp<br />
| 154 bp<br />
|-<br />
| TrpSig/HisSig<br />
| 34 bp/32 bp<br />
| 93 bp/91 bp<br />
|-<br />
| TrpSig-B0014/HisSig-B0014<br />
| 135 bp/ 133 bp<br />
| 194 bp/ 192 bp<br />
|-<br />
| <font color=red>R0011-TrpSig-B0014/<br>R0011-HisSig-B0014<br />
| 206 bp/ 204 bp <br />
| <font color=red>265 bp/ 263 bp</font><br />
|-<br />
| <br />
| <br />
| <br />
|-<br />
| I712074 ("N6")<br />
| 46 bp<br />
| 105 bp<br />
|-<br />
| I719005 ("N15")<br />
| 23 bp <br />
| 82 bp<br />
|-<br />
|}<br />
<br><br />
Prefix: 29 bp after PCR; Suffix: 30 bp after PCR; X-S-scar: 6 bp<br />
<br />
*overnight cultures made of<br />
**HisSig 3_1, DL1_3<br />
**TrpSig DL4_1, DL4_3<br />
**N15 1&2<br />
**HisTerm/TrpTerm (picked colonies from yesterdays plates #2 each)<br />
<br />
<br />
*Purification of yesterday's PCR<br />
**elution in 50 µl H2O<br />
*** c(HisSig)=5.5 ng/µl<br />
*** c(TrpSig)=10 ng/µl<br />
*** c(HisTerm)=6.5 ng/µl<br />
*** c(TrpTerm)=6.5 ng/µl<br />
*** c(R0011)=13 ng/µl<br />
*** c(B0014)=12.5 ng/µl<br />
** 5 µl loaded on gel with 1 µl GLPn; 3% Agarose in 1x TBE, 220 V (double Gel, 35 cm)<br />
<br />
[[Image:TUM2010_100527bbeschriftet.png]]<br><br />
===28.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology ZYMO_RESEARCH_DNA_Clean.26Concentration_Kit|Miniprep]]''' of cultures from [[27.05.2010]], Elution in 50 uL nuclease free water <br />
** (1)HisSig 3_1 <br />
** (2)HisSig DL1_3 <br />
** (3)TrpSig DL4_1 <br />
** (4)TrpSig DL4_3 <br />
** (7)HisTerm#1 <br />
** (8)HisTerm#2 (7 ml culture) <br />
** (9)TrpTerm#1 <br />
** (10)TrpTerm#2 (7ml culture) <br />
** (5)N15-1 (=BBa_I719005) <br />
** (6)N15-2 (=BBa_I719005)<br />
*** ()=numbers on gel<br />
<br><br />
{| cellspacing="1" cellpadding="1" border="1" style="width: 367px; height: 271px;"<br />
|-<br />
| sample<br> <br />
| DNA concentration (ng/uL)<br><br />
|-<br />
| HisSig 3_1 <br> <br />
| 6<br><br />
|-<br />
| HisSig DL1_3<br> <br />
| 11<br><br />
|-<br />
| TrpSig DL4_1 <br> <br />
| 16<br><br />
|-<br />
| TrpSig DL4_3 <br> <br />
| 21.5<br><br />
|-<br />
| HisTerm#1<br> <br />
| 27<br><br />
|-<br />
| HisTerm#2<br> <br />
| 66<br><br />
|-<br />
| TrpTerm#1<br> <br />
| 34<br><br />
|-<br />
| TrpTerm#2<br> <br />
| 29.5<br><br />
|-<br />
| N15-1 (=BBa_I719005)<br> <br />
| 19.5<br><br />
|-<br />
| N15-2 (=BBa_I719005)<br> <br />
| 18.5<br><br />
|}<br />
<br />
<br> <br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Restriction|analytical digest]]''' (E/P)<br><br />
**of all samples. total volume 20 uL, 5 uL template used for Term-constructs, 10 uL teplate for all others<br />
<br />
*'''Agarose Gel''' <br />
**3% broad range agarose in TBE. Run in TBE, 140 V, 1.50 h <br />
**stained with SybrGold, 45 min<br />
**signals look fine<br />
**terminators also (without pre/suffix 97/104 bp)<br />
**T7 promoter without pre/suffix has a length of 23 bp + cut pre/szffix ca at 60 bp -->buffer?<br />
[[Image:TUM2010_100528_beschriftet.png]]<br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week09{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===31.05.2010===<br />
* Digestions<br />
**15N-1 (BBa_I719005, 19.5 ng/uL) with S/P<br />
**HisSig/TrpSig (6.5/10 ng/uL) with X/P<br />
::2 h 37 °C<br />
:heat inactivation of insert-digestions<br />
<br />
*Gel: 1% Agarose in 1x TAE<br />
:1 h 25 min, 115 V<br />
:stained with SybrGold, 40 min<br />
:[[Image:TUM2010_100531.png]]<br />
<br />
:Band at ~2100b cut und purified using the [[Team:TU_Munich/Lab#Molecular_Biology ZYMO RESEARCH Gel DNA Recovery Kit |zymo kit]]<br />
<br />
* [[Team:TU_Munich/Lab#Molecular_Biology Ligation|ligation]]<br />
** HisSig (4 µL of digest) with purified plasmid (with BBa_I719005)<br />
** TrpSig (2 µL of digest) with purified plasmid -=-<br />
::reason: concentration of His Sig before digest was 1/2 of TrpSig<br />
<br />
* [[Team:TU_Munich/Lab#Molecular_Biology Transformation|transformation]]<br />
: of DH5a with Ligation batches, HisSig1-3, HisSig3-1, TrpSig DL4-1, TrpSig 4-3<br />
===01.06.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
<br />
:of Ligations transformed into DH5a yesterday <br />
:4 Colonies of each Ligation<br />
<br />
**T7-HisSig <br />
**T7-TrpSig<br />
<br />
<br> <br />
<br />
*'''Gel: 3% broad range Agarose in 1xTBE'''<br />
<br />
:1.5 h 140 V <br />
:stained with SybrGold<br />
<br />
[[Image:TUM2010_100601.png]] <br />
<br />
:calculation for the expected size of the fragments<br />
<br />
:{| cellspacing="1" cellpadding="1" border="1" width="200"<br />
|-<br />
| part<br />
| size (bp)<br />
|-<br />
| HisSig<br />
| 32<br />
|-<br />
| TrpSig<br />
| 34<br />
|-<br />
| T7 promoter<br />
| 23<br />
|-<br />
| prefix<br />
| 20<br />
|-<br />
| suffix<br />
| 21<br />
|-<br />
| X/S scar<br />
| 6<br />
|}<br />
<br />
:in PCR we get additional bp due to the primers - +9 at pre/suffix=+18 bp<br> <br />
<br />
:overall size of the fragments expected to come out of the PCR: T7_HisSig: 120 bp, T7_TrpSig: 122 bp<br />
<br />
*'''ON cultures'''<br />
<br />
:5 ml cultures of pSB1K3_R0011_HisSig_B0014 (1_3 &amp; 3_1) and pSB1K3_R0011_TrpSig_B0014 (DL4_1 &amp; DL4_3) <br />
:1 ml cultures of each colony monitored in Colony PCR<br />
<br />
===02.06.2010===<br />
*Miniprep of yesterdays cultures using Zymokit, elution by nuclease-free water <br />
*Concentration determination <br />
*analytic digestion <br />
*results on gel:<br />
<br />
<br> <br />
<br />
*Sequencing <br><br />
<br />
JobNr. Barcode Last change Date/Time Last message / Files 6549287 AE2739 02.06.2010 / 13:51:12 HisSig 1-3-forward G1004 <br />
<br />
We just received your order. Many thanks.<br />
<br />
6549288 AE2738 02.06.2010 / 13:51:12 HisSig 3-1-forward G1004 <br />
<br />
We just received your order. Many thanks.<br />
<br />
6549289 AE2737 02.06.2010 / 13:51:12 TrpSig DL4-1-forward G1004 <br />
<br />
We just received your order. Many thanks.<br />
<br />
6549290 AE2736 02.06.2010 / 13:51:12 TrpSig DL4-3-forward G1004 <br />
<br />
We just received your order. Many thanks.<br />
<br />
6549291 AE2735 02.06.2010 / 13:51:12 HisTerm-forward G1004 <br />
<br />
We just received your order. Many thanks.<br />
<br />
6549292 AE2734 02.06.2010 / 13:51:12 TrpTerm-forward G1004 <br />
<br />
We just received your order. Many thanks.<br />
<br />
*Gel 3% broad range agarose in 1x TBE<br />
: [[Image:TUM2010_100602_beschr.png]]<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week10{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===07.06.2010===<br />
*'''Sequenbcing results from GATC''' <br />
**HisSig DL1-3 is ok <br />
**HisSig 3-1 is ok <br />
**TrpSig DL4-1 is ok <br />
**TrpSig DL4-3 is ok <br />
**TrpTerm + HisTerm bad runs... --&gt; new sequencing order with Primer 100 bp upstream (within GFP)<br />
<br />
:Files can be found stored in our [[Zugangsdaten für GATC|GATC account]]<br />
<br />
<br><br />
<br />
*'''Sequencing@GATC:''' both Term-constructs with primer pGFP-FP provided by GATC<br />
<br />
<br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Restriction|Restrictions]] '''<br />
**psB1A10_TrpTerm/HisTerm with Nsi1, Aat2 <br />
**pSB1K3_R0011_HisSig/TrpSig_B0014 with Pst1, Aat2 <br />
**T7 bb with Spe1, Pst1 <br />
**PCRProducts: HisSig/TrpSig with Pst1, Xba1, 2 h @37°C<br />
<br />
:all plasmid digests done''' sequential''' as enzymes do not have 100% activity in the same buffer, each reaction 1.5 h@37°C <br />
:psB1A10_TrpTerm/HisTerm and T7 bb dephosphorylated the last 30 min <br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]] ''' <br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]] '''<br />
<br />
<br> liquid culture (10 ml) of pSB1K3_R0011_HisSig/TrpSig_B0014<br />
===08.06.2010===<br />
*'''Sequencing results from GATC''': His/TrpTerm with pGFP-FP primer<br />
**HisTerm worked<br />
**TrpTerm worked<br />
: checked with blast2seq<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
: 8 colonies from each plate of T7His, T7Trp, MonsterHis, MonsterTrp as ligations resulted in many colonies<br />
<br />
*'''3% Agarose Gel in 1xTBE'''<br />
: for all colony PCR reactions<br />
[[Image:TUM2010_100608_beschriftet.png|600px]]<br />
[[Image:TUM2010_100608-2_beschriftet.png|600px]]<br />
<br />
<br> Interpretation/Info: The R0011_Sig_B0014 construct was cut with PstI, but not ligated into a PstI site but instead into the NsiI site --> even if sticky end are compatible, the bases in the 3` direction are different --> primer lags 7 bp compared to standard procedure --> we didn´t expect to find the signal construct by colony PCR --> control digestion tomorrow<br />
: T7-Signal constructs seem to have worked, expected size was 23 bp (T7)+ 32 (HisSig)/34 (TrpSig) bp + 30+29 (PCRPre+Suf)=114/116 bp<br />
*over night cultures<br />
<br />
===09.06.2010===<br />
* Miniprep of 4 Monster_His, 4 Monster_Trp, 3 T7_His and 3 T7_Trp cultures<br />
* Analytical digestion of plasmids mentioned above<br />
* gel of Monster_Plasmid digestion<br />
[[Image:TUM2010_100609_Monster_inverse.jpg|600px]]<br />
<br><br />
gel didn´t work at all --> even after > 2 h, bands were not separated correctly, even the 1kb ladder was "stacked" in the gel-pockets, the 100 bp ladder should show EQUAL distances between the lines [http://www.neb.com/nebecomm/productfiles/778/images/N3231_fig1_v1_000034.gif see here], it looks like the gel was "more dense" at the pockets---> no idea what happened --> repeat Monster-digestion tomorrow?<br />
<br><br />
* gel of T7_Plasmid digestion<br />
[[Image:TUM2010_100609_T7_sig_invers.jpg|600px]]<br />
<br><br />
T7_Trp E + S digestion 107 bp and T7_His 105 bp --> worked for all picked colonies. (regard that there is an excess of plasmid DNA-basepairs of factor >30 --> thats why the inserts are much weaker than the plasmid signals.<br />
<br><br />
occured trouble: <br />
** Ladders and loading dye´s empty --> i used those of eike, BUT: eikes 1 kb ladder is different --> compare [http://www.neb.com/nebecomm/products/productN3272.asp here] and his loading dye was much more diluted, even if there was also 6x Sac GLP written on it -> i hope this won´t cause any trouble<br />
<br />
===10.06.2010===<br />
*'''ordered'''<br />
:Promega E.coli S30 in vitro transcription/translation kit<br />
:Spe1, Aat2 from NEB, 500 U each<br />
<br><br />
* '''[[Team:TU_Munich/Lab#Molecular_Biology Restriction|analytical Digestion]]<br />
:of Ligation colonies from MonsterHis/trp 1-3 and T7His/Trp 1,2,5/1,2,3<br />
:2h digestion<br />
:: Monster: 6 µL DNA template with Aat2/Spe1 in Buffer 4/Bsa<br />
:: T7-Signal: 6 µL DNA template with E/P in Buffer 3<br />
<br><br />
* '''Agarose Gels'''<br />
:used standards: lmw, 2-log [[Team:TU_Munich/Lab#Molecular_Biology standards|click here]]<br />
: Gel1: 1% Agarose in 1xTBE for Digestions of Monsterplasmid<br />
:: run in big chamber @ 200 V for 1 h 20 min<br />
:[[Image:TUM2010_100610_t7sig.png]]<br />
:Gel2: 3% Agarose (broad range) in 1xTBE for Digestions of T7-Signal<br />
:: run in small chamber @140 V for 1 h 35 min<br />
:[[Image:TUM2010_100610_monster.png]]<br />
<br><br><br />
:Conclusions:<br />
# all T7-Signal ligations loaded on the gel worked<br />
# monsterplasmid didn't work? bands at 800 bp, 900 bp, 1.3 kbp, 2.2 kbp, 3 kbp, we SHOULD expect to see our Insert, wich is Prefix+R0011_Signal_B0014_small Suffix, which should run around 300-400 bp...<br />
<br />
===11.06.2010===<br />
*'''Gel''': large 1% Agarose in TAE. Load: The rest of N/A cut Messplasmids from [[07.06.2010]]. Run @220 V for 3.5 h<br />
: fragments expected are 5087 and 176. original size of plasmid is 5263. This is a Try to differ between 5087 and 5263 bp<br />
:[[Image:TUM2010_100611.png]]<br />
: Band @ 5000 bp of Trp_Term purified, obviously digestion was 100%. Bad point is that HisTerm includes an Nsi1 cleavage site...<br />
<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week11{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }}{{:Team:TU_Munich/Templates/ClearBox }}{{:Team:TU_Munich/Templates/ClearBox }} {{:Team:TU_Munich/Templates/YellowBox | text=Promega Kit }}<br />
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<br />
===14.06.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]'''<br />
: 10 µL pSB1A10_TrpTerm Aat2/Nsi1 0.5 ng/uL<br />
: 1 µL R0011_TrpSig_B0014 Aat2/Psb1 11 ng/uL<br />
: 2 µL T4 ligase buffer<br />
: 1 µL T4 Ligase<br />
: 6 µL H2O<br />
::10 min RT<br />
<br><br />
*'''Transformation''' of DH5a with<br />
: Ligation<br />
: T7His#1<br />
: T7Trp#1<br />
<br><br />
*'''over night cultures''' of<br />
: pSB1K3_R0011_TrpSig_B0014<br />
: pSB1K3_R0011_HisSig_B0014<br />
: pSB1A10_TrpTerm<br />
: pSB1A10_HisTerm<br />
<br />
===15.06.2010===<br />
*Over night cultures <br />
*Aliquots of the Promega in vitro expressions kit from ''E. coli'' S30 extract:<br />
: 40 µL with aa mix including all aa.<br />
<br />
===16.06.2010===<br />
'''Fluoresence measurements using in vitro kit'''<br />
* in vitro kit sample <br />
* adding psBA1A10 Trp_Term --> constant over time, no significant changes compared to kit alone --> high efficiency of AraC<br />
* adding L-(+)Arabinose (final concentration 2%) --> after approx. 10 min significant GFP production --> measuring for xxx min --> RFP is slightly increased (to proof if correlated to GFP peak --> crossdetection)<br />
* adding psB1K3 R0011_TrpSig_B0014<br />
'''Cell culture'''<br />
5 ml culture for<br />
* psBA1A10 Trp_Term/HisTerm<br />
* psB1K3 R0011_TrpSig/Hissig_B0014<br />
<br />
===17.06.2010===<br />
''' Cloning '''<br />
<br><br />
Digestion of Trp-Sig with E/P and psB1A10 Trp_Term with E/P<br />
* Gel purification of psB1A10 Trp_Term E/P cut<br />
[[Image:TUM2010_100617_pSB1A10_EPcut_dunkler.jpg|700px]]<br />
* Heat inactivation of Trp-Sig E/P cut<br />
* Ligation for 10 min @ RT and Transformation in DH5-a cells<br />
<br />
===18.06.2010===<br />
'''cloning'''<br />
* Transformation (about 20 colonies) --> picking 5 colonies<br />
* colony PCR<br />
* Gel <br><br />
2 % broad range agarose, 1 h 120 V [[Image:TUM2010_100618_psb1A10-Trp_sig_colonypcr.jpg|600px]]<br />
Sample 2, 4, 5 shows probably Trp-Signal + Pre/Suffix --> send sample 2 for sequencing!<br />
<br />
<br><br><br />
* control digestion of all 10 picked psB1A10-TrpSig in 1% broad range agarose, > 3 h, 120 V<br />
[[Image:TUM2010_100618_psb1A10-Trp_sig_controlverdau.jpg|600px]]<br />
--> digestions worked, but again, no insert can be found, despite gel was at maximum resolution ( 3h 120 V, see LMW)<br />
<br />
''' in vitro measurements '''<br />
f$%&&§ s%§$! Again, nothing worked! Although we saw an increasing "GFP signal" comparable to 16.06.10, taking spectra suggested we DON'T see significant GFP-production! We used new water for preparing the samples, cleaned cuvettes with "new water", used other DNA-samples etc. Somehow, it seems as if we don't express GFP (we compared Christoph's results! We should see a really significant spectrum! <br>Next steps:<br />
* Try in vivo measurements, just using psb1A10_xTerm without Signal (thus just measuring plasmid) to proof if kit or measuring plasmid causes this problem!<br />
<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week12{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} {{:Team:TU_Munich/Templates/BlueBox | text=First steps }} {{:Team:TU_Munich/Templates/ClearBox }} {{:Team:TU_Munich/Templates/YellowBox | text=Promega Kit }}<br />
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<br />
===22.06.2010===<br />
<br />
'''Transformation'''<br />
* psb1A10_HisTerm/TrpTerm into BL21 (DE3) RIL<br />
* psb1A10_Monster-Trp No. 7, 8, 10 (Positiv control) into DH5-a<br />
<br />
'''Liquid culture'''<br />
* psb1A10_TrpSig (Positiv control) No. 2<br />
<br />
===23.06.2010===<br />
<br />
*Miniprep<br />
: pSBN1A10_TrpSig - 40 µL, 12.5 ng/µL -->very low amount of DNA...<br />
: culture is slightly red? -> strange because there cannot be any rfp-insert with constitutive promoter as the construct was built up from pSB1A10_TrpTerm (digested) and TrpSignal (PCR product)<br />
<br><br />
*cultures<br />
**5 ml cultures of DH5a <br />
::with MonsterTrp, #7,8,10 (Carbamp)<br />
:*50 ml cultures of BL21 (DE3) RIL<br />
::pSB1A10_HisTerm<br />
::pSB1A10_TrpTerm<br />
<br><br />
*Arabinose Stock<br />
0.2%<br />
<br />
===24.06.2010===<br />
<br />
* Miniprep<br />
: MonsterTrp #7,8,10<br />
: positive Control<br />
:: concentrations are too low for sequencing --> again we have to set up 5 ml cultures for tomorrow<br />
<br><br />
* measurements<br />
<br />
===25.06.2010===<br />
'''Plasmid purification and sequencing'''<br />
* Monster_Trp 7, 8, 10 and psB1A1ß_TrpSig plasmids are isolated (concentrations up to 110 ng/ul) and sent for sequencing<br />
* psB1A1ß_TrpSig liquid culture was completely pink! still not clear what happend (wrong labeling of digested psB1A10_Trpterm?) --> wait for sequencing details<br />
'''Fluorescence Measurements'''<br />
* Induction by putting Arabinose directly into cuvettes with cells IS NOT WORKING at all! Expression of GFP increases, but marginally. probably, despite stirring, oxygen is lacking?<br />
* Induction on shaker work perfectly --> both Trp and His showed strong GFP-signals, BUT: Probably, too high OD results in not exciting all GFP within the sample (incident beam is already scatterd enormously on the edge of the cuvette --> only small volume is excited correctly). For instance, a sample showing OD of 0.7 shows a signal of 30 a.u., diluted to OD 0.35 signal falls only to 19 a.u.! Thus dilution did not result in a linear decrease of flourescence! a.u. !!! We diluted down to OD 0.1; the result: OD´s smaller than 0.4 show linear change of fluorescence signal --> using OD´s up to 0.4 results in meaningful measurements!!!<br />
<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week13{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} {{:Team:TU_Munich/Templates/BlueBox | text=Testing pSB1A10 }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===28.06.2010===<br />
* Sequencing results<br />
:psB1A10_TrpSig (Positive control) worked: Trp-Sig is inside, directly in front of RFP (without the promotor of the measurement plasmid insert [http://partsregistry.org/Part:BBa_J04450 BBa_J04450], so it seems like everything worked. Furthermore, i performed a promotor prediction with the following tool [http://linux1.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb bacteria promotor prediction tool] to proof if our Trp-Sig in combination with the flanking regions is not forming a promotor,by mischance. According to this, there are two promotors, BUT: <br> one in and after the suffix (so it should be in each of our constructs), but the tools says theres is no known sigma-factor for this promotor! the second one is within the RFP and there is a sigma-factor for this one ( rpoD16). So i don´t see a explanation, why our colonies were pink in contrast to the other "Messplasmids". <br><br> None of the Monsterplasmids contains the signal construct. Probably, the problem is there is no selection methods which allowed us to distinguish uncut plasmids.... --> we should discuss at our next meeting, one possiblity would be connecting our construct to a resistance marker. I summed up all sequences in this document: [[File:25.06.-sequenzierung.doc]] <br><br><br />
<br />
*Induction in cuvette and measuring fluorescence at the same time IS NOT WORKING! (probably cells are not growing and expressing very well, maybe lacking oxygen despite stirring. Bleaching is more unlikely) <br> In vivo, measuring plasmid (at least GFP) works! we optimized the paramters for fluorescence measurment! We tried different OD´s and found out that only measurments below OD 0.4 result in meaningful measurements. <br> as a result, in vitro expression did somehow not work, reasons are unclear, maybe too low DNA-concentrations. <br> positvie control psB1A10_TrpSig was pink again, we have to wait the results from GATC<br />
<br />
===29.06.2010===<br />
<br />
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<br />
===01.07.2010===<br />
*'''in vitro measurement'''<br />
:using Invitrogen kit<br />
:measuring kinetics for 3 h @ 37°C<br />
:40 µL + 5 µL pSB1A10_TrpSig (126 ng/µL) + 0.5 µL 100x L-Arabinose (=0.2%) + 4.5 µL H2O<br />
::observations: GFP signal grows, after 30 min it crashes. RFP grows<br />
::emission spectra for GFP and RFP result in no spectrum<br />
::looks strange, a problem might be evaporation of liquid and hence scattering of light which produces artefacts<br />
<br><br />
*'''over night cultures'''<br />
:pSB1A10_TrpSig (DH5a), 5 ml for miniprep<br />
:pSB1A10 XS (DH5a), 5 ml for miniprep<br />
<br />
===02.07.2010===<br />
*Miniprep<br />
:pSB1A10_XS: 30 µL 10 ng/µL<br />
:pSB1A10_TrpSig: 30 µL 10 ng/µL<br />
<br><br />
*Transformation<br />
:BL21 with pSB1A10_XS (positive control without insert and no without any bio brick site left)<br />
<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week15{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/ClearBox }} {{:Team:TU_Munich/Templates/BlueBox | text=Testing pSB1A10 }} {{:Team:TU_Munich/Templates/ClearBox }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===05.07.2010===<br />
*'''over night cultures'''<br />
:250 ml of DH5a pSB1A10_XS<br />
:20 ml BL21 pSB1A10_XS<br />
<br><br />
*'''in vitro transcription measurement planned'''<br />
:check [[In_vitro_Measurements]]<br />
<br />
===06.07.2010===<br />
*'''In vivo measurement'''<br />
: BL21 pSB1A10_XS - positive control (to check the measurement plasmid...)<br />
: GFP, RFP Fluorescence<br />
:induced with 0.2% arabinose in (1), uninduced (2)<br />
::at OD 0.15: GFP/RFP emissions spectra /100706/spectra/gfp10 and rfp10<br />
::2.5 h kinetic measurement GFP/RFP /100706/kinetics/<br />
::OD 0.7 (1) and 0.64 (2) after 2.5 h --> GFP/RFP emissions spectra /100706/spectra/gfp11,rfp11,gfp21,rfp21<br />
::in addition for 4 h a culture at OD 0.8 induced (with 0.2% Arab), spectra taken afterwards at 1:15 dilution (OD 0.39) gpf_ku and rfp_ku<br />
:observation: measurement plasmid is totale verarsche. RFP is not expressed at all, or this protein is not rfp. whatever.<br />
<br><br />
<br />
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No Lab work this week, everybody is busy studying for their exams...<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week17{{:Team:TU Munich/Templates/ToggleBoxStart2}}The Era of Exams<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
No Lab work this week, everybody is busy studying for their exams...<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week18{{:Team:TU Munich/Templates/ToggleBoxStart2}} The Era of Exams<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
No Lab work this week, everybody is busy studying for their exams...<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week19{{:Team:TU Munich/Templates/ToggleBoxStart2}} The Era of Exams<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
No Lab work this week, everybody is busy studying for their exams...<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week20{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=Construction of new Measurement Plasmid }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===09.08.2010===<br />
*overnight cultures inoculated from Glycerolstock J06702(mCherry generator) in pSB1A2 from Christoph.<br />
===10.08.2010===<br />
*MiniPrep of pSB1A2-mCherry using ZymoKit<br />
*Digestion<br />
**pSB1A2-mCherry E/P<br />
**pSB1A2-mCherry X/P<br />
**pSB1A10-HisTerm S/P<br />
**pSB1A10-TrpTerm S/P<br />
**pSB1A10-HisTerm E/P<br />
*Purified with agarose gel (1%)<br />
**Gel Doc broken => no picture<br />
**Description: mCherry cut was ok, Plasmid was cut at least once (linear DNA), generally contaminated with genomic DNA<br />
*Ligation<br />
**50 ng Plasmid and 34 ng Insert<br />
**ca. 30min @ RT<br />
*Transformation of DH5a cells with ligation samples<br />
(=> no colonies the next day)<br />
<br />
*overnight cultures<br />
**pSB1A2-mCherry from Christoph`s stock<br />
**pSB1A10-HisTerm from earlier plate<br />
**pSB1A10-TrpTerm from earlier plate<br />
(=> pSB1A10-TrpTerm and pSB1A10-HisTerm did not grow until next day)<br />
<br />
<br />
===11.08.2010===<br />
*MiniPrep of pSB1A2-mCherry using ZymoKit<br />
*analytic gel of Mini preps and ligation of the previous day<br />
**preps still hold genomic DNA<br />
**mCherry Plasmid runs at ca. 2400 bp<br />
<br />
*Digestion<br />
**pSB1A2-mCherry E/P<br />
**pSB1A2-mCherry X/P<br />
*Purified with agarose gel (1%)<br />
**Gel Doc broken => no picture<br />
**Description: mCherry cut was ok, stil contaminated with genomic DNA<br />
*Ligation<br />
**Plasmid (from previous day) and mCherry-Insert<br />
**ca. 30 min @ RT<br />
*Transformation of DH5a cells with<br />
**ligation samples (=> no colonies the next day)<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
<br />
*overnight cultures<br />
**pSB1A2-mCherry from Christoph`s stock<br />
<br />
<br />
===12.08.2010===<br />
*MiniPrep of pSB1A2-mCherry using ZymoKit<br />
*analytic gel of Mini preps and ligation of the previous day<br />
**preps still hold genomic DNA<br />
**mCherry Plasmid runs at ca. 2400 bp<br />
**Gel (1%)<br />
[[Image:TUM2010_100812 ligation100811 mChPrep.jpg]] <br />
<br />
<br />
<br />
*Digestion<br />
**pSB1A2-mCherry X/P<br />
**pSB1A10-HisTerm S/P<br />
**pSB1A10-TrpTerm S/P<br />
*Purified with agarose gel (1%)<br />
[[Image:TUM2010_100812 DigestionmCh pSB1A10.jpg|600px]] <br />
<br />
*Ligation<br />
**Plasmid and mCherry-Insert<br />
**ca. 30 min @ RT<br />
*Transformation of DH5a cells with<br />
**ligation samples (=> no colonies the next day)<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
<br />
*overnight cultures<br />
**pSB1A2-mCherry from Christoph`s stock<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
<br />
'''Caution: ran out of gas => not steril?'''<br />
<br />
===13.08.2010===<br />
*MiniPrep using ZymoKit<br />
**pSB1A2-mCherry<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
*analytic gel of Mini preps and ligation of the previous day<br />
**low concentration<br />
**Gel (1%)<br />
[[Image:TUM2010_100813 Prep-mChHisTrp Ligation100812.jpg|600px]]<br />
<br />
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<br />
<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week21{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=Construction of new Measurement Plasmid }}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
<br />
===16.08.2010===<br />
*Concentrating MiniPrep-Samples using ZymoKit<br />
**pSB1A2-mCherry<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
<br />
*Digestion<br />
**pSB1A2-mCherry E/P<br />
**pSB1A2-mCherry X/P<br />
*Purified with agarose gel (1%)<br />
[[Image:TUM2010_100816 Digestion-mCherry.jpg]]<br />
=> no mCherry band!!!<br />
<br />
*Purification using Zymo Concentrator Kit<br />
**pSB1A10-HisTerm S/P<br />
**pSB1A10-TrpTerm S/P<br />
<br />
*Ligation<br />
**Plasmid (from earlier date) and mCherry-Insert ('''from 11.08''')<br />
**ca. 30 min @ RT<br />
**'''used new ligase and new ligase buffer'''<br />
<br />
*Transformation of DH5a cells with<br />
**ligation samples (=> '''colonies found the next day''')<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
**pSB1A10-RFP (BioBrick Standard)<br />
<br />
*overnight cultures<br />
**pSB1A2-mCherry from Christoph`s stock<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
<br />
===17.08.2010===<br />
*MiniPrep using ZymoKit<br />
**pSB1A2-mCherry<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
<br />
*Digestion<br />
**pSB1A2-mCherry X/P<br />
**pSB1A10-HisTerm S/P<br />
**pSB1A10-TrpTerm S/P<br />
: => heating block went up to 50°C<br />
*Purified "digestion" samples with ZymoKit => stored for next day<br />
<br />
<br />
<br />
<br />
*Picked 12 colonies from previous day's ligation<br />
: => Colony PCR => Gel (2%)<br />
[[Image:TUM2010_100817 colonyPCR pSB1A10-mCherry.jpg|600px]]<br />
<br />
<br />
*Purified with agarose gel (1%)<br />
**Gel Doc broken => no picture<br />
**Description: mCherry cut was ok, stil contaminated with genomic DNA<br />
*Ligation<br />
**Plasmid (from previous day) and mCherry-Insert<br />
**ca. 30 min @ RT<br />
*Transformation of DH5a cells with<br />
**ligation samples (=> no colonies the next day)<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
<br />
*overnight cultures<br />
**pSB1A10-RFP (plate from previous day)<br />
**pSB1A2-mCherry<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
<br />
<br />
<br />
'''PROBLEM:<br />
mCherry has a SgrA1-cleavage site! These constructs cannot be used. Starting all over, cloning the linker sequence first...'''<br />
<br />
===18.08.2010===<br />
*MiniPrep using ZymoKit<br />
**pSB1A2-mCherry<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
**pSB1A10-RFP<br />
<br />
*Analytical agarose gel (1%):<br />
[[Image:TUM2010_100818 MiniPreps HisTrpCherryRFP.jpg|600px]]<br />
<br />
*Digestion<br />
**pSB1A10-HisTerm SgrAI/PstI<br />
**pSB1A10-TrpTerm SgrAI/PstI<br />
**pSB1A10-RFP SgrAI/PstI<br />
: =>RFP has a SrgAI cleaving site. Discarded RFP digestion.<br />
*preparativ agarose gel (1%):<br />
[[Image:TUM2010_100818 Digestion HisTrp2.jpg|600px]]<br />
<br />
*Soubilization of SrgAI-PstI Linker<br />
<br />
*Ligation<br />
**Plasmid His-Term(Trp-Term) and Linker<br />
**ca. 30 min @ RT<br />
*Transformation of DH5a cells with<br />
**ligation samples<br />
<br />
*overnight cultures<br />
**pSB1A2-mCherry<br />
**pSB1A10-TrpTerm<br />
<br />
===19.08.2010===<br />
*MiniPrep using ZymoKit<br />
**pSB1A2-mCherry<br />
*analytic agarose gel (1%) from various mCherry Preps<br />
[[Image:TUM2010_100819 MiniPreps mCherry2.jpg]]<br />
<br />
<br />
*Picked 6 colonies from pSB1A10-HisTerm-linker and pSB1A10-TrpTerm-linker each (previous day's ligation)<br />
: => Colony PCR => Gel (1.5%)<br />
[[Image:TUM2010_100819 ColonyPCRlinker2.jpg|600px]]<br />
<br />
<br />
*overnight cultures (600µl)<br />
**pSB1A2-mCherry<br />
**pSB1A2-R0011<br />
**pSB1A10-HisTerm-linker (#7, 11, 12)<br />
**pSB1A10-TrpTerm-linker (#1, 2, 4)<br />
<br />
===20.08.2010===<br />
*MiniPrep using ZymoKit<br />
**pSB1A2-mCherry<br />
**pSB1A2-R0011<br />
**pSB1A10-TrpLinker (picked Colonies)<br />
**pSB1A10-HisLinker (picked Colonies)<br />
<br />
*analytical Digestion<br />
**pSB1A10-HisLinker SgrAI /EcoRI<br />
**pSB1A10-HisLinker NsiI<br />
**pSB1A10-TrpLinker SgrAI /EcoRI<br />
<br />
*analytical agarose gel (1.5%)<br />
[[Image:TUM2010_100820gel1verdau.jpg]]<br />
<br />
*preparativ Digestions<br />
**pSB1A2-mCherry EcoRI /PstI<br />
**pSB1A2-mCherry XbaI /PstI<br />
**pSB1A2-R0011 SpeI /PstI<br />
**pSB1A10-HisLinker SpeI /PstI<br />
**pSB1A10-HisLinker EcoRI /PstI<br />
**pSB1A10-TrpLinker SpeI /PstI<br />
**pSB1A10-TrpLinker EcoRI /PstI<br />
**PCR_BB1006 XbaI /PstI<br />
<br />
*agarose gel (1.5%):<br />
[[Image:TUM2010_100820gel1verdau2.jpg]]<br />
<br />
*agarose gel (1.0%):<br />
[[Image:TUM2010_100820gel3verdau.jpg]]<br />
<br />
*agarose gel (1.0%):<br />
[[Image:TUM2010_100820gel2verdau.jpg|600px]]<br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week22{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=Construction of new Measurement Plasmid }}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
<br />
===23.08.2010===<br />
*Ligation<br />
**pSB1A10-HisLinker SpeI /PstI + mCherry XbaI /PstI<br />
**pSB1A10-HisLinker EcoRI /PstI + mCherry EcoRI /PstI<br />
**pSB1A10-TrpLinker SpeI /PstI + mCherry XbaI /PstI<br />
**pSB1A10-TrpLinker EcoRI /PstI + mCherry EcoRI /PstI<br />
**pSB1A2-R0011 SpeI /PstI + PCR_BB1006 XbaI /PstI<br />
<br />
*Transformation of DH5a cells<br />
<br />
===24.08.2010===<br />
*Picked 2 colonies per plate (previous day's ligation)<br />
**R0011_B1006Sig<br />
**Trp1_mCherry<br />
**Trp2_mCherry<br />
**His7_mCherry<br />
**His11_mCherry<br />
**pSB1A10_mCherry<br />
<br />
*Colony PCR of picked colonies with prefix/suffix primers<br />
<br />
*analytical agarose gel 1 (1.5%)<br />
[[Image:TUM2010_100824ColonyPCRgel1.jpg|600px]]<br />
<br />
*analytical agarose gel 1 (1.5%) <br />
[[Image:TUM2010_100824ColonyPCRgel2.jpg|600px]] <br><br />
Trp=R0011, R0011= Trp :)<br><br />
<br />
Faint bands at the correct length can be guessed. <br />
*overnight cultures (5 ml)<br><br />
** pSB1A10_TrpTerm_mCherry_linker<br />
** pSB1A10_HisTerm_mCherry_linker<br />
** pSB1A10_mCherry_linker<br />
<br />
===25.08.2010===<br />
*Miniprep using Zymo Miniprep-Classic Kit:<br />
** pSB1A10_TrpTerm_mCherry_linker<br />
** pSB1A10_HisTerm_mCherry_linker<br />
** pSB1A10_mCherry_linker<br />
<br />
*analytical digestions<br />
** pSB1A10_TrpTerm_mCherry_linker EcoRI /PstI<br />
** pSB1A10_HisTerm_mCherry_linker EcoRI /PstI<br />
** pSB1A10_mCherry_linker EcoRI /PstI<br />
<br />
*analytical agarose gel 1 (1.0%)<br />
[[Image:TUM2010_100825verdaugel1.jpg|600px]]<br />
<br />
*Picked 2 colonies per plate (day before yesterday's ligation)<br />
**R0011_B1006Sig<br />
**Trp1_mCherry<br />
**Trp2_mCherry<br />
**His7_mCherry<br />
**His11_mCherry<br />
**pSB1A10_mCherry<br />
<br />
*Colony PCR of picked colonies with prefix/suffix primers<br />
** Program: colonypcr<br />
<br />
*analytical agarose gel 1 (1.5%)<br />
[[Image:TUM2010_100825colonypcrGEL1.jpg|600px]]<br />
<br />
*analytical agarose gel 1 (1.5%) <br />
[[Image:TUM2010_100825colonypcrGEL2.jpg|600px]]<br />
<br />
<br />
*overnight cultures (5 ml)<br><br />
** pSB1A10_TrpTerm_mCherry_linker<br />
** pSB1A10_HisTerm_mCherry_linker<br />
** pSB1A10_mCherry_linker<br />
===26.08.2010===<br />
*Miniprep using Zymo Miniprep-Classic Kit:<br />
** pSB1A10_TrpTerm_mCherry_linker<br />
** pSB1A10_HisTerm_mCherry_linker<br />
** pSB1A10_mCherry_linker<br />
**pSB1A2_R0011_B1006<br />
<br />
*PCR<br />
** Trp-Signal (R0011_Sig_B0014)<br />
<br />
** His-Signal (R0011_Sig_B0014)<br />
** Terminator B0014<br />
<br />
*analytical digestions<br />
** pSB1A10_mCherry_linker EcoRI /PstI<br />
*preparative digestion<br />
** pSB1A10_TrpTerm_mCherry_linker SpeI/PstI<br />
** pSB1A10_HisTerm_mCherry_linker SpeI/PstI<br />
**pSB1A2_R0011_B1006 SpeI/PstI<br />
** PCR Trp-Sig XbaI/PstI<br />
** PCR His-Sig XbaI/PstI<br />
** B0014 XbaI/PstI<br />
<br />
*preparative agarose gel 1 (1.0%)<br />
[[Image:TUM2010_100826 prep Verdau.jpg|600px]]<br />
last lane: pSB1A10_His11_mCherry SpeI/PstI<br />
<br />
*analytical agarose gel (1.0 %)<br />
[[Image:TUM2010_100826anaVerdauPCRControl.jpg|600px]]<br />
*Ligations<br />
** pSB1A10_TrpTerm_mCherry_linker + Trp-Signal (R0011_Sig_B0014)<br />
** pSB1A10_HisTerm_mCherry_linker + His-Signal (R0011_Sig_B0014)<br />
**pSB1A2_R0011_B1006 + Terminator B0014<br />
*Transformation of Ligation product in DH5alpha cells<br />
*Transformation of pSB1A10_mCherry_linker in BL21<br />
===27.08.2010===<br />
*Mini-Prep<br />
** pSB1A2_R0011_B1006 <br><br />
for sequencing<br />
*Colony PCR<br />
**2 colonies per plate<br />
<br />
[[Image:TUM2010_100827 coloypcr 2.jpg|600px]]<br />
R0011=R0011_B1006!!!<br />
*Sequencing<br />
** pSB1A2_R0011_B1006 4b with primer Biobrick VR<br />
** pSB1A10mod_mCherry 27b with primer GFP_FP and Biobrick VR<br />
** pSB1A10mod_mCherry 32a with primer GFP_FP and Biobrick VR<br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
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<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week23{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=Construction of new Measurement Plasmid }}{{:Team:TU_Munich/Templates/BlueBox | text=Testing new Measurement Plasmid }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===30.08.2010===<br />
*Colony PCR <br />
**picked two colonies per plate from 26.08' ligation <br />
**Program: colonypcr, modified elongation time: 1.15 instead of 1.00<br />
<br />
*analytical agarose gel (1.5%)<br><br />
<br />
<u>'''Gel 1:'''</u><br> <br />
<br />
[[Image:TUM2010_100830colonyPCR gel1.jpg|600px]] <br />
<br />
=&gt; <span style="color: rgb(255, 0, 0);">samples named "1x" to "8x" for pSB1A2-R0011-BB1006Sig-B0014 colonies</span><br> <br />
<br />
=&gt; <span style="color: rgb(255, 0, 0);">samples named "9x" to "12x" for pSB1A10mod-TrpTerm-mCherry-TrpSig colonies</span><br> <br />
<br />
<u>'''Gel 2:'''</u> <br />
<br />
[[Image:TUM2010_100830colonyPCR gel2.jpg|600px]] <br />
<br />
=&gt; <span style="color: rgb(255, 0, 0);">samples named "13x" to "16x" for pSB1A10mod-TrpTerm-mCherry-TrpSig colonies</span><br> <br />
<br />
=&gt; <span style="color: rgb(255, 0, 0);">samples named "17x" to "24x" for pSB1A10mod-HisTerm-mCherry-HisSig colonies</span> <br />
<br />
'''Interpretation for Gel1 and Gel2''': <br />
<br />
Ligation worked for the samples 1x-6x (pSB1A2-R0011-BB1006Sig-B0014), 9x-16x (pSB1A10mod-TrpTerm-mCherry-TrpSig), 18x-24x (pSB1A10mod-HisTerm-mCherry-HisSig) <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
*over night cultures: <br />
**pSB1A10mod_HisTerm_mCherry_HisSignal <br />
**pSB1A10mod_TrpTerm_mCherry_TrpSignal <br />
**PSB1A2_R0011_BB1006_B0014<br />
<br />
<br />
*Received sequencing results from GATC. All Sequences are okay: <br />
**pSB1A10mod_mCherry (27b) and (32a) <br />
**pSB1A2_R0011_BB1006 (4b)<br />
<br />
===31.08.2010===<br />
*'''Fluorescence measurement (positive control experiment):'''<br />
**Settings: GFP-Excitation: 501 nm; mCherry-Excitation: 587 nm;<br />
**endpoint measurements of:<br />
***Timepoints of measurement: 3 h after induction and 9 h after induction (1.5 h and 7 h for 15x-sample)<br />
***Samples:<br />
****pSB1A10mod-mCherry (27b) in BL21 cells, induced with ca. 0.4% L-Arabinose<br />
****pSB1A10mod-mCherry (27b) in BL21 cells, not induced<br />
**kinetic measurement of induced (0.4% L-Arabinose) BL21 cells carrying pSB1A10mod-mCherry (27b)<br />
<br />
*'''Results:'''<br />
**NO mCherry signal detected at all: The GFP signal shows a nice and strong increase; the RFP channel did not change at all.<br />
**GFP signal looks perfect: strong if induced, neglectable if not!<br />
::=> System seems not capable of serving as a testing system for our switches! <br />
<br />
*'''Glycerol stocks'''<br />
**in DH5a cells:<br />
***pSB1A10mod-TrpTerm-mCherry-TrpSig (9x)<br />
***pSB1A10mod-TrpTerm-mCherry-TrpSig (15x)<br />
***pSB1A10mod-TrpTerm-mCherry-TrpSig (10x) (sequence verified)<br />
***pSB1A10mod-HisTerm-mCherry-HisSig (15x)<br />
***pSB1A10mod-HisTerm-mCherry-HisSig (18x)<br />
***pSB1A10mod-HisTerm-mCherry-HisSig (23x) (sequence verified)<br />
***pSB1A10mod-mCherry (32a) (sequence verified)<br />
***pSB1A10mod-mCherry (27b) (sequence verified)<br />
***pSB1A2mod R0011-BB1006Sig-B0014 (2x) (sequence verified)<br />
***pSB1A2mod R0011-BB1006Sig-B0014 (3x)<br />
***pSB1A2mod R0011-BB1006Sig (4b) (sequence verified)<br />
**in BL21 cells:<br />
***pSB1A10mod-mCherry (27b) (sequence verified)<br />
<br />
:"x" refers to Colony-PCR of 30.08.2010<br />
<br />
*5ml '''Over night cultures'''<br />
**pSB1A10mod-TrpTerm-mCherry-TrpSig (9x, 15x, 10x)<br />
**pSB1A10mod-HisTerm-mCherry-HisSig (18x, 23x, 15x)<br />
**pSB1A2mod R0011-BB1006Sig-B0014 (2x, 3x)<br />
<br />
===01.09.2010===<br />
*'''MiniPrep''' using Zymo classical kit. Samples:<br />
**pSB1A10mod-TrpTerm-mCherry-TrpSig (9x, 15x, 10x)<br />
**pSB1A10mod-HisTerm-mCherry-HisSig (18x, 23x, 15x)<br />
**pSB1A2mod R0011-BB1006Sig-B0014 (2x, 3x)<br />
<br />
*'''Fluorescence measurement (positive control experiment):'''<br />
**endpoint measurements:<br />
***Timepoints of measurement: 3 h after induction and 9 h after induction (1.5 h and 7 h for 15x-smaple)<br />
***Settings: GFP-Excitation: 501 nm; mCherry-Excitation: 587 nm; RFP-Excitation: 584 nm<br />
***Samples:<br />
****pSB1A10mod-mCherry (32a) in DH5a cells, induced with ca. 0.4% L-Arabinose<br />
****pSB1A10mod-mCherry (32a) in DH5a cells, not induced<br />
****pSB1A10mod-mCherry (32a) in BL21 DE3 cells, induced with ca. 0.4% L-Arabinose<br />
****pSB1A10mod-mCherry (32a) in BL21 DE3 cells, not induced<br />
****pSB1A10-RFP, in DH5a cells, induced with ca. 0.4% L-Arabinose<br />
****pSB1A10-RFP, in DH5a cells, not induced<br />
****pSB1A10mod-TrpTerm-mCherry-TrpSig (15x), in DH5a cells, induced with ca. 0.4% L-Arabinose<br />
****pSB1A10mod-TrpTerm-mCherry-TrpSig (15x), in DH5a cells, not induced<br />
**'''Results:'''<br />
***Very strong RFP signal in pSB1A10-RFP, induced and not induced<br />
***For the first time we saw a weak but easily-detectable mCherry signal in positive control samples (pSB1A10mod-mCherry) 3 hours after induction! There was hardly no difference between the uninduced and the induced control samples for mCherry. The GFP signals was strong for induced control experiments and very weak for not induced samples! The pSB1A10mod-TrpTerm-mCherry-TrpSig sample also showd a small mCHerry signal.<br />
***After 9 hours the mCherry signals were generally reduced, whereas the GFP signals were still high in all induced samples and low in all uninduced samples.<br />
:::=> Although we saw mCherry for the first time (!), the signal is to weak not reproducable! As a consequence the system can not be used to serve as a measure for our switches! Furthermore the settings of the fluorometer are ok, since we saw strong RFP signal.<br />
===02.09.2010===<br />
'''Starting the cloning of pBAD (BioBrick I13453) downstream of GFP'''<br />
<br />
*'''Amplifing''' the Arabinose-inducable promotor pBAD<br />
**Resuspending the BioBrick I13453 with 10 µl in well 1F in the 2010 Distribution<br />
**PCR using 1 µl template (programm "igempcr")<br />
**Purified using DNA Concentrator (ZymoKit)<br />
<br />
*Digestion (EcoRI and SpeI) of PCR product and heat inactivation (20 min @ 80°C)<br />
<br />
*Digestion of the target vectors using EcoRI and XbaI<br />
**Samples:<br />
***pSB1A10mod-TrpTerm-mCherry-TrpSig (9x, 10x)<br />
***pSB1A10mod-HisTerm-mCherry-HisSig (23x, 24x)<br />
***pSB1A10mod-mCherry (32a, 27b)<br />
***pSB1A10mod-TrpTerm-mCherry (10b, 13b)<br />
***pSB1A10mod-HisTerm-mCherry (18b, 24b)<br />
**Purified using 1% agarose gel:<br />
<br />
[[Image:TUM2010_100902 Digestion 1.jpg|700px]]<br />
<br />
:=> '''Interpretation:''' Gel is overloaded! However digestion seemed to work since the bands show correct masses.<br />
<br />
*Extraction of bands at ca. 6000 bp<br />
<br />
*10 µl '''ligation''' of 50 ng of each digested vector with 8ng insert<br />
<br />
*'''Transformation''' of DH5a cells using 8 µl ligation sample<br />
===03.09.2010===<br />
*Colony PCR <br />
**picked two colonies per plate from 02.09' ligation <br />
**Note: no PCR because Thermocycler was occupied<br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
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<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week24{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} {{:Team:TU_Munich/Templates/BlueBox | text=Testing HisTrp }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===06.09.2010===<br />
*Colony PCR <br />
**using colonies picked on 03.09.<br />
<br />
*analytical agarose gel (1.5%)<br />
[[Image:TUM2010_100906ColonyPCR 1.jpg|600px]]<br />
<br />
*K1= pSB1A10mod_HisTerm_mCherry_HisSig<br />
*K2 = pSB1A10mod_TrpTerm_mCherry_TrpSig<br />
<br />
*5ml over night cultures:<br />
**positive control pSB1A10mod_Pbad_mCherry<br />
**negative His control pSB1A10mod_Pbad_HisTerm_mCherry<br />
**His-Switch pSB1A10mod_Pbad_HisTerm_mCherry_HisSig<br />
**Trp-Switch pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSig<br />
<br />
===07.09.2010===<br />
*MiniPrep using Zymo Classical kit<br />
**pSB1A10mod_Pbad_mCherry<br />
**pSB1A10mod_Pbad_HisTerm_mCherry<br />
**pSB1A10mod_Pbad_HisTerm_mCherry_HisSig<br />
**pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSig<br />
<br />
<br />
*Fluorescence measurements<br />
**Induction with 0.2% L-arabinose<br />
**Measurement of OD600<br />
**Fluorescence measurement at 30 min, 150 min (only Pos.Control) and 4.5 h<br />
**Samples:<br />
::pSB1A10mod_Pbad_mCherry<br />
::pSB1A10mod_Pbad_HisTerm_mCherry<br />
::pSB1A10mod_Pbad_HisTerm_mCherry_HisSig<br />
::pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSig<br />
<br />
*Transformation of BL21 (DE3)<br />
**pSB1A10mod_Pbad_mCherry<br />
**pSB1A10mod_Pbad_HisTerm_mCherry<br />
**pSB1A10mod_Pbad_HisTerm_mCherry_HisSig<br />
**pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSig<br />
<br />
===08.09.2010===<br />
*Glycerolstocks<br />
:30%Glycerol in LB_Carb<br />
**pSB1A10mod_Pbad_mCherry<br />
**pSB1A10mod_Pbad_HisTerm_mCherry<br />
**pSB1A10mod_Pbad_HisTerm_mCherry_HisSig<br />
**pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSig<br />
<br />
*Fluorescence measurements<br />
**Induction with 0.2% L-arabinose<br />
**Measurement of OD600<br />
**Fluorescence measurement at 24 h and different OD600<br />
**Samples:<br />
::pSB1A10mod_Pbad_mCherry<br />
-->OD600 0.05 reasonable for our cell measurements. Positive control works fine after 24 h induction.<br />
<br />
[[Image:TUM2010_Graph2.jpg|600px]]<br />
[[Image:TUM2010_Graph3.jpg|600px]]<br />
*5ml cultures of BL21 cells<br />
**pSB1A10mod_Pbad_mCherry<br />
**pSB1A10mod_Pbad_HisTerm_mCherry<br />
**pSB1A10mod_Pbad_HisTerm_mCherry_HisSig<br />
**pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSig<br />
<br />
*ColonyPCR<br />
**picked 4 Colonies per Neg.TrpControl from 02.09' ligation plates<br />
**PCR using Program 'colonyPCR'. Elongation time modified to 1:20min<br />
<br />
===09.09.2010===<br />
*Fluoresence measurement<br />
**using BL21 cells<br />
**samples:<br />
***Positive control (pSB1A10mod_pBAD_mCherry)<br />
***Negative control (pSB1A10mod_pBAD_HisTerm_mCherry)<br />
***pSB1A10mod_pBAD_HisTerm_mCherry_HisSignal<br />
***pSB1A10mod_pBAD_TrpTerm_mCherry_TrpSignal<br />
**Induction with 0.4% L-arabinose and 1mM IPTG<br />
**Timepoints: 1 h, 2 h, 4 h, 10 h, 16 h (induced the day before)<br />
::(OD checked seperately each time; average ODs=0.02-0.06)<br />
<br />
*30% glycerol stocks of BL21 cells carrying:<br />
**Positive control (pSB1A10mod_pBAD_mCherry)<br />
**Negative control (pSB1A10mod_pBAD_HisTerm_mCherry)<br />
**pSB1A10mod_pBAD_HisTerm_mCherry_HisSignal<br />
**pSB1A10mod_pBAD_TrpTerm_mCherry_TrpSignal<br />
<br />
===10.09.2010===<br />
*Fluorescence measurements<br />
**Induction with 0.4% L-arabinose and 1 mM IPTG<br />
**Measurement of OD600<br />
**Fluorescence measurement at 15 h and 26 h <br />
*Samples:<br />
::pSB1A10mod_Pbad_mCherry - Positive Control<br />
::pSB1A10mod_Pbad_HisTerm_mCherry - HisNeg. Control<br />
::pSB1A10mod_Pbad_HisTerm_mCherry_HisSignal<br />
::pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSignal<br />
[[Image:TUM2010_Graph100910 2.jpg|600px]]<br />
[[Image:TUM2010_Graph100910 3.jpg|600px]]<br />
[[Image:TUM2010_Graph100910 1.jpg|600px]]<br />
[[Image:TUM2010_Graph100910 4.jpg|600px]]<br />
<br />
*Conclusion:<br />
His-Switch DOES NOT seem to work!!! Measurements of Trp-Switch look weird, since Ara-induced culture showed strong mCherry signal than Ara+IPTG-induced culture. Maybe growing condition were not sufficient. We will use 50 ml flask next time!<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week25{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=Cloning }} {{:Team:TU_Munich/Templates/BlueBox | text=HisTerm/TrpTerm }} {{:Team:TU_Munich/Templates/GreenBox | text=Ordering Aptamer }} {{:Team:TU_Munich/Templates/YellowBox | text=New Measurement Plasmid }}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===13.09.2010===<br />
*<i>In vitro</i> Kit measurement:<br />
**Test of <i>In vitro</i> kit with positive control<br />
**1 h on 37°C<br />
::--> no significant mCherry signal detectable. <i>In vitro</i> Kit measurements aborted. <br />
<br />
*20ml cultures for Fluorescence measurements<br />
**Samples:<br />
::pSB1A10mod_Pbad_mCherry - Positive Control<br />
::pSB1A10mod_Pbad_HisTerm_mCherry - HisNeg. Control<br />
::pSB1A10mod_Pbad_TrpTerm_mCherry - TrpNeg. Control<br />
::pSB1A10mod_Pbad_HisTerm_mCherry_HisSignal<br />
::pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSignal<br />
<br />
:*Growth on 37°C until OD600 = 0.6, then induction and growth on 25°C over night<br />
:*each sample uininduced, induced with 0.4% L-arabinose and 0.4% L-arabinose + 1 mM IPTG,respectively<br />
<br />
* 30% Glycerol stock in LB<br />
**pSB1A10mod_Pbad_TrpTerm_mCherry - TrpNeg. Control<br />
<br />
===14.09.2010===<br />
*Fluorescence measurements<br />
**Induction with 0.4% L-arabinose and 1mM IPTG<br />
**Measurement of OD600<br />
**Fluorescence measurement at 16 h<br />
*Samples:<br />
::pSB1A10mod_Pbad_mCherry - Positive Control<br />
::pSB1A10mod_Pbad_HisTerm_mCherry - HisNeg. Control<br />
::pSB1A10mod_Pbad_TrpTerm_mCherry - TrpNeg. Control<br />
::pSB1A10mod_Pbad_HisTerm_mCherry_HisSignal<br />
::pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSignal<br />
<br />
[[Image:TUM2010_PosControl140910.jpg|500px|inline]][[Image:TUM2010_PosControlklein.JPG|200px|inline]]<br />
[[Image:TUM2010_TrpNegativeControl140910.jpg|500px]][[Image:TUM2010_TrpNegativeControlklein.JPG|200px]]<br />
[[Image:TUM2010_HisNegativeControl140910.jpg|500px]][[Image:TUM2010_HisNegativeControlklein.JPG|200px]]<br />
[[Image:TUM2010_TrpSwitch140910.jpg|500px]][[Image:TUM2010_TrpSwitchklein.JPG|200px]]<br />
[[Image:TUM2010_HisSwitch140910.jpg|500px]][[Image:TUM2010_HisSwitchklein.JPG|200px]]<br />
===15.09.2010===<br />
*Waiting for Mr.Gene...<br />
<br />
===16.09.2010===<br />
*Searching for Mr.Gene package...<br />
::--> our new Testsystem pMalachitApt_BB1006<br />
*PCR and PCR purification with Zymo DNA Clean&Concentrator<br />
**pMalachitApt_BB1006 with terminator (Primer: Apt_For and AptFull_wT_Rev)<br />
**pMalachitApt_BB1006 without terminator (Primer: Apt_For and AptPart_woT_Rev)<br />
**Switches<br />
::Trp-Switch<br />
::His-Switch<br />
:*Signals<br />
::His-Sig with Term (template: pSB1A2_R0011_HisSig_B0014)<br />
::Trp-Sig with Term (template: pSB1A2_R0011_TrpSig_B0014)<br />
::BB1006Sig with Term (template: pSB1A2_R0011_BB1006Sig_B0014)<br />
::BB1006Sig without Term (template: pSB1A2_R0011_BB1006Sig)<br />
::HisSig (ssDNA) (template: original biomers order)<br />
::TrpSig (ssDNA) (template: original biomers order)<br />
<br />
===17.09.2010===<br />
*Digestion<br />
**pMalachitApt_BB1006 with EcoRI/PstI<br />
**pMalachitApt_BB1006 with XbaI/SpeI<br />
**Switches<br />
::Trp-Switch with EcoRI/PstI<br />
::His-Switch with EcoRI/PstI<br />
:*Signals<br />
::His-Sig with XbaI/PstI<br />
::Trp-Sig with XbaI/PstI<br />
<br />
*Purification using Qiagen Kit<br />
::--> Cut off size of Qiagen kit too high. Lost all Switches and Signals<br />
<br />
*Purification pMalachitApt using preparative agarose gel<br />
<br />
*new PCR:<br />
**Switches<br />
::Trp-Switch<br />
::His-Switch<br />
:*Signals<br />
::HisSig (ssDNA) (template: original biomers order)<br />
::TrpSig (ssDNA) (template: original biomers order)<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week26{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=Cloning of Malachit green constructs }} {{:Team:TU_Munich/Templates/ClearBox | text=Measurements }} {{:Team:TU_Munich/Templates/GreenBox | text=HisTerm/TrpTerm }} <br />
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===20.09.2010===<br />
*PCR Purification using Qiagen MinElute Kit<br />
**Trp-Signal<br />
**His-Signal<br />
**Trp-Switch<br />
**His-Switch<br />
<br />
*Digestions 37°C, 2h<br />
**Trp-Signal XbaI/PstI<br />
**His-Signal XbaI/PstI<br />
**Trp-Switch EcoRI/PstI<br />
**His-Switch EcoRI/PstI<br />
::--> Heat inactivation 20 min, 80°C<br />
<br />
*Ligation<br />
**pSB1A2_R0011 SpeI/PstI + Trp-Signal XbaI/PstI<br />
**pSB1A2_R0011 SpeI/PstI + His-Signal XbaI/PstI<br />
**pMalachit EcoRI/PstI + Trp-Switch EcoRI/PstI<br />
**pMalachit EcoRI/PstI + His-Switch EcoRI/PstI<br />
**pMalachit XbaI/SpeI<br />
<br />
*Transformation of DH5a cells with ligation<br />
<br />
===21.09.2010===<br />
*Picked 2 colonies per plate of yesterday's transformation => we call them z-Series<br />
*ColonyPCR of z-Series<br />
*analytical agarose gel (2.5%)<br />
:=> ColonyPCR did not work. Probably not enough template.<br />
<br />
*Picked futher colonies<br />
*2nd ColonyPCR of z-Series<br />
<br />
===22.09.2010===<br />
*analytical agarose gel (2.5%) of 2nd colonyPCR of z-Series<br />
<br />
:pSB1A2-R0011-TrpSignal looks good. We removed all pSB1A2-R0011-HisSignal samples since there is a contamination on the gel and colonies turned redish. No conclusion regarding the other ligations of z_series can be drawn. LB control produces similar bands as to what we expected => Choose new Primers for PCR.<br />
<br />
*3rd colonyPCR of z-Series (samples 1-16z)<br />
**used Primers Apt_For and Apt_Part_woT instead of BioBrick Primers (G1005 and G1004). Bands should be about 100bp longer.<br />
<br />
*analytical agarose gel (2%)<br />
<br />
===23.09.2010===<br />
*PCR to obtain DNA for malachite green <i>in vitro</i> measurements<br />
**samples from z-Series (see [[21.09.2010]])<br />
::2z (XbaI/SpeI religated positive control)<br />
::16z (pMalachitApt_HisSig positive control)<br />
:*each samples was amplificated with two sets of primers (including and excluding the terminator BB0014):<br />
::Apt_For and AptFull_wT_Rev<br />
::Apt_For and AptPart_woT_Rev<br />
<br />
*Purified with Qiagen MinElute Kit<br />
<br />
*analytical digestion of z-series ligation:<br />
**EcoRI/PstI:<br />
::2z (XbaI/SpeI religated positive control)<br />
::5z (pMalachitApt_TrpTerm)<br />
::12z (pMalachitApt_HisTerm)<br />
::16z (pMalachitApt_HisSig positive control)<br />
::28z (pSB1A2_R0011_TrpSig)<br />
:*NsiI only (HisTerm contains one cutting site for NsiI, so does pMalachiteApt => we expect 2 fragments):<br />
::12z (pMalachitApt_HisTerm)<br />
<br />
*analytical agarose gel:<br />
<br />
<br />
*10 µl ligations using digested samples from [[21.09.2010]]<br />
**pSB1A2_R0011 (SpeI/PstI cut) + HisSig (XbaI/PstI cut)<br />
**pMalachiteApt (EcoRI/PstI cut) + TrpSwitch (EcoRI/PstI cut)<br />
**pMalachiteApt (EcoRI/PstI cut) + HisSwitch (EcoRI/PstI cut)<br />
<br />
*Transformed DH5a cell with 8µl of the above ligation samples<br />
===24.09.2010===<br />
*Fluorescence measurement: Malachite Green Assay<br />
**Samples<br />
::2z positive control (X/S religated: tac_MalachitApt) (PCR-amplified without BB0014 Terminator)<br />
::2z positive control (X/S religated: tac_MalachitApt_BB0014) (PCR-amplified with BB0014 Terminator)<br />
::negative control (tac_BB1006Switch_MalachitApt_BB0014) (PCR-amplified with BB0014 Terminator)<br />
::tac_BB1006Switch_MalachitApt + tac_BB1006Signal (both PCR-amplified without BB0014 Terminator)<br />
:*Sample Mix:<br />
::2 µl sigma70 satured RNA <i>E.Coli</i> Polymerase (epicenter Biozyme) (2U)<br />
::ca. 1µg DNA template<br />
::5 µM Malachite Green<br />
::10 µM DTT<br />
::4 µM NTPs<br />
::40 mM Tris-HCl pH = 7.1 @ 37°C; 7.5 @ 22°C<br />
::10 mM MgCl2<br />
::150 mM KCl<br />
::added H20 to total volume of 100 µl<br />
:*Kinetics measurement @ 37°C<br />
::*Excitation 630 nm<br />
::*Emission 655 nm<br />
[[Image:TUM2010_100924 MalachitKineticsPosControl BB1006.jpg|600px]]<br />
::*Results: <br />
:::no apatmer formation observed => positive controls did not work at all => assay did not work!!<br />
<br />
:*Scanning measurements<br />
::*Exitation: 630 nm<br />
::*Results: <br />
:::no detactable peaks between 640nm and 800nm in any sample at any measured time (Settings were checked by detecting scattering peaks).<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week27{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/ClearBox | text=Cloning }} {{:Team:TU_Munich/Templates/ClearBox | text=Measurements }} {{:Team:TU_Munich/Templates/GreenBox | text=T7 Switch }} <br />
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<br />
===27.09.2010===<br />
*Fluoresence measurements<br />
**Cary Spectrometer<br />
**samples:<br />
***with T7 RNA Polymerase (NEB)<br />
:::T7 positive control (T7Promotor_MalachitAptamer)<br />
::*with Epicenter E coli RNA Polymerase<br />
:::2z positive control (X/S religated: tac_MalachitApt) (PCR-amplified without BB0014 Terminator)<br />
:::2z positive control (X/S religated: tac_MalachitApt_BB0014) (PCR-amplified with BB0014 Terminator) (NO KINETICS RECORDED)<br />
::*with in vitro kit (cell lysate)<br />
:::2z positive control (X/S religated: tac_MalachitApt) (PCR-amplified without BB0014 Terminator)<br />
:::2z positive control (X/S religated: tac_MalachitApt_BB0014) (PCR-amplified with BB0014 Terminator)<br />
:*Scanning measurements: excitation at 630nm<br />
:*Kinetics measurements: excitation at 630nm; Emission at 655nm<br />
<br />
[[Image:TUM2010_100927Beginn.jpg]]<br />
<br />
<br />
[[Image:TUM2010_100927kinetik2.jpg]]<br />
<br />
<br />
'''Results:'''<br />
T7 Polymerase works perfectly. E coli Polymerase also produced RNA but much less (however enzyme might show reduced activity due to storage problems). In vitro (Cell lysate) kits do not work at all.<br />
<br />
===01.10.2010===<br />
'''T7-Measurments'''<br />
* PCR of switch_phi_T7-construct to obtain dsDNA (conditions: igemPCR and 30s elongation time). Samples were purified using Qiagen MinElute.<br />
<br />
* 1th measurment: <br> Maxi´s NEB buffer conditions: 40 mM Tris pH 7.4 @ RT, 40mM Mg2Cl, 5 µM Malachit-green, 4 mM NTPs, 2,5 U RNA-Polymerase <br>All DNA templates were all added to a final concentration of 200nM.<br />
**sample 1: Positive control<br />
**sample 2: negative control (= switch without any signal) ('''new DTT''' used for this sample)<br />
**sample 3: switch + SigA1a<br />
**sample 4: switch + SigA1c<br />
*Results<br />
**kinetic results <br> [[Image:TUM2010_kinetik-T7.JPG|600px]]<br />
**Spectra <br> [[Image:TUM2010_spektren.JPG|Spektren|600px]]<br />
<br />
=> Switches do look quite good. However DTT was not the same in all samples.<br />
<br />
<br />
* 2th measurment:<br />
**"Paper´s" buffer conditions: 40 mM Tris pH 7.9 @ RT, 6mM Mg2Cl, 5 µM Malachit-green, 100 mM KCl, 0.8 mM NTPs, 2,5 U RNA-Polymerase<br />
***sample 1: Positive control<br />
***sample 2: negative control (= switch without signal)<br />
**Maxi´s NEB buffer conditions: 40 mM Tris, 40 mM Mg2Cl, 10 µM Malachit-green, ph 7.4 @ RT<br />
***sample 3: Positive control ('''new DTT''' used for this sample)<br />
***sample 4: Negative control (= switch without signal)<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week28{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/ClearBox | text=Cloning }} {{:Team:TU_Munich/Templates/ClearBox | text=Measurements }} {{:Team:TU_Munich/Templates/GreenBox | text=T7 Switch }}<br />
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===04.10.2010===<br />
*1st fluorescence measurement<br />
:*Samples:<br />
::*200 nM T7-Switch (100 nM each strand) + SignalA_1a<br />
::*200 nM T7-Switch (100 nM each strand) + SignalA_2a<br />
::*200 nM T7-Switch (100 nM each strand) + SignalA_2b<br />
::*200 nM T7-Switch (100 nM each strand) + SignalA_2c<br />
:*Buffer:<br />
::40 mM Tris pH 7.1<br />
::40 mM MgCl2<br />
::10 mM DTT<br />
::1.6 mM NTPs<br />
::5 µM Malachite green<br />
::2.5 U T7 RNA Polymerase<br />
:*Results:<br />
:[[Image:TUM2010_10104_Kinetik.jpg|600px]]<br />
:Fluorescence increased in all samples in a very similar way, suggesting all signal to work equally well.<br />
<br />
*2nd fluorescence measurement<br />
:*Samples:<br />
::*200 nM T7-Switch only (Buffer A)<br />
::*200 nM T7-Switch only (Buffer B)<br />
::*positiv control (T7Promoter_MalchitAptamer) (Buffer A)<br />
::*positiv control (T7Promoter_MalchitAptamer) (Buffer B)<br />
:*Buffer A:<br />
::40 mM Tris pH 7.1 @ RT<br />
::40 mM MgCl2<br />
::10 mM DTT<br />
::1.6 mM NTPs<br />
::'''10 µM''' Malachite green<br />
::2.5 U T7 RNA Polymerase<br />
:*Buffer B:<br />
::40 mM Tris pH 7.9 @ RT<br />
::6 mM MgCl2<br />
::100 mM KCl<br />
::10 mM DTT<br />
::1.6 mM NTPs<br />
::'''10 µM''' Malachite green<br />
::2.5 U T7 RNA Polymerase<br />
:*Results:<br />
:[[Image:TUM2010_10104_Kinetik2.jpg|600px]]<br />
::*Very strange results!! Both negative controls increased strongly compared to the positive controls that only slightly increased (buffer A seems to be better for transcription than buffer B).<br />
:::=> Maybe something went very wrong. However, last Friday we observed a similar, strange behavior. Maybe the positive control is no good choice. It would make sence to use T7-Switch+SignalA_1a as a reference.<br />
::*Signals were generally stronger. 10 µM of malachite green seems to be quite good.<br />
<br />
===05.10.2010===<br />
* T7 ''in vitro measurements''<br />
<br />
** First measurement of the day: Signal, 1d, 2c, 3b<br />
*** Master Mix:<br />
*** 204 µl 2x Buffer<br />
*** 40.8 µl DTT<br />
*** 8.16 µl NTPs<br />
*** 21.05 µl Switch (1.94 µM)<br />
*** signals: # volume µl<br />
*** negative control - 1.213<br />
*** 1d - 1.290<br />
*** 2c - 1.450<br />
*** 3b - 1.376<br />
[[Image:TUM2010_10105_Kinetik1.jpg|600px]]<br />
<br />
** PCR with positive control and T7 switch<br />
*** Mastermix:<br />
PCR T7 switch/positive signal<br />
** 32 x Mastermix: 1600 µl total volume<br />
*** 32 µl dNTPs<br />
*** 32 µl T7 forward primer<br />
*** 32 µl T7 reverser primer<br />
*** 160 µl 10x buffer<br />
*** 96 µl MgCl2<br />
*** 6.4 µl Taq Polymerase<br />
*** 10 µl Template (some older PCR)<br />
*** 1209.6 µl H2O<br />
<br />
** Second measurement of the day: Signal negative control, nonsense signal, 1a (with 400 µM signal), 1a (with 2 µM signal)<br />
*** *** Master Mix:<br />
*** 204 µl 2x Buffer<br />
*** 40.8 µl DTT<br />
*** 8.16 µl NTPs<br />
*** 21.05 µl Switch (1.94 µM)<br />
*** signals: # volume µl<br />
*** negative control - 30.34<br />
*** 1d - 1.213<br />
*** 2c - 2.672<br />
*** 3b - 13.36<br />
<br />
[[Image:TUM2010_10105_Kinetik2.jpg|600px]]<br />
<br />
** Evaluation of different malachite green concentrations<br />
*** 5 µM, 10 µM, 15 µM, 25 µM chosen<br />
*** high temperature dependency<br />
*** Equilibration of samples important<br />
<br />
===06.10.2010===<br />
* PCR<br />
** yesterday's PCR Purification using Zymo Clear and Concentrated<br />
*** yields<br />
*** Switch: 164 ng/µl<br />
*** positive control: 28 ng/µl<br />
*** problems with the temperature? Yield too low!<br />
<br />
** PCR of positive control<br />
*** annealing temperature set to 48°C<br />
*** 20 x Mastermix:<br />
*** 20 µl dNTPs<br />
*** 20 µl T7 forward primer<br />
*** 20 µl T7 reverser primer<br />
*** 100 µl 10x buffer<br />
*** 60 µl MgCl2<br />
*** 4 µl Taq Polymerase<br />
*** 1 µl Template (some older PCR)<br />
*** 775 µl H2O<br />
<br />
===07.10.2010===<br />
* T7 Trancription<br />
** new buffer:<br />
*** Stocks, volume for 20 ml, endconcentration in 1x<br />
*** 1M Tris/HCl, 1.6 ml, 40 mM<br />
*** 500 mM MgCl2, 3.2 ml, 40 mM<br />
*** 250 mM malachite green, 1.6 ml, 20 µM<br />
*** H20<br />
** Measurement<br />
*** 4.1 x, switch, switch + nonsense, switch + 1a, switch + 1c<br />
*** 21.16 µl switch, 1.93 µM<br />
*** 10.25 µl RPO<br />
*** 205 µl buffer<br />
*** 41 µl DTT 100 µM<br />
*** 20.5 µl rNTPs<br />
*** H2O<br />
<br />
[[Image:TUM2010_10107_Kinetik1.jpg|600px]]<br />
<br />
**second measurement<br />
** Measurement<br />
*** 4.1 x, switch, switch + nonsense, positive control + 1b, positive control + 1b<br />
*** 21.16 µl switch, 1.93 µM<br />
*** 10.25 µl RPO<br />
*** 205 µl buffer<br />
*** 41 µl DTT 100 µM<br />
*** 20.5 µl rNTPs<br />
*** H2O<br />
<br />
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[[Image:TUM2010_10107_Kinetik2.jpg|600px]]<br />
<br />
===08.10.2010===<br />
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[[Image:TUM2010_101008_Kinetik2.jpg|600px]]<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week29{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/ClearBox | text=Cloning }} {{:Team:TU_Munich/Templates/ClearBox | text=Measurements }} {{:Team:TU_Munich/Templates/GreenBox | text=T7 Switch }}<br />
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<br />
===11.10.2010===<br />
* PCR T7 switch<br />
** 32 x Mastermix: 1600 µl total volume<br />
*** 32 µl dNTPs<br />
*** 32 µl T7 forward primer<br />
*** 32 µl T7 reverser primer<br />
*** 160 µl 10x buffer<br />
*** 96 µl MgCl2<br />
*** 6.4 µl Taq Polymerase<br />
*** 10 µl Template (some older PCR)<br />
*** 1209.6 µl H2O<br />
<br />
** Standart iGEM PCR program:<br />
*** 95°C, 2' <br />
*** 1) 95°C, 0,5'<br />
*** 2) 58°C, 0.5'<br />
*** 3) 71°C, 1'<br />
*** 71°C, 7'<br />
<br />
** repeat 1)-3) 35 times<br />
** yield after purification: 80 µl, 1.14 µM<br />
<br />
*Malchitegreen measurement<br />
** for 4.1 x 100 µl<br />
*** 35.88 µl PCR product<br />
*** 20.05 dNTPs<br />
*** 41 µl DTT<br />
*** 10.25 µl T7 RPO<br />
*** 8.75 µl switch<br />
*** 2 µl signal per 100 µl<br />
<br />
** measured signals: (1) nonsense, (2) 1a, (3) 1b, (4) 2a<br />
** Voltage set to 1000 (maximum)<br />
** 38°C, every 15 s measurement<br />
**[[Image:TUM2010_101011_Kinetik.jpg|600px]]<br />
** start at 5 au, end at 25 au<br />
** rise visible over time in all samples, nonsense signal with the fastest rise, 1a similiar, end signal similiar in all measurements<br />
<br />
===12.10.2010===<br />
* PCR T7 switch<br />
** 32 x Mastermix: 1600 µl total volume<br />
*** 32 µl dNTPs<br />
*** 32 µl T7 forward primer<br />
*** 32 µl T7 reverser primer<br />
*** 160 µl 10x buffer<br />
*** 96 µl MgCl2<br />
*** 6.4 µl Taq Polymerase<br />
*** 10 µl Template (some older PCR)<br />
*** 1209.6 µl H2O<br />
<br />
** '''changed''' iGEM PCR program:<br />
*** 95°C, 2' <br />
*** 1) 95°C, 0,5'<br />
*** 2) 50°C, 0.5'<br />
*** 3) 71°C, 1'<br />
*** 71°C, 10'<br />
*** repeat 1) - 3) 35 times<br />
<br />
** --> melting temperature primers: 55/54°C!!!<br />
** yield after purification: 300 ng/µl in 60 µl, 3.66 µM<br />
<br />
* Malachitegreen measurement with preincubation of transcription stuff with signals<br />
<br />
** for 4.1 x 100 µl<br />
*** 35.88 µl PCR product<br />
*** 20.05 dNTPs<br />
*** 41 µl DTT<br />
*** 10.25 µl T7 RPO<br />
*** 2 µl signal per 100 µl<br />
<br />
** preincubation of signal with RPO for one hour, 37°C <br />
** addition of signal after one hour (2.735 µl)<br />
** once done in eppis (low bind), once in cuvettes<br />
** no rise in both<br />
** incubation and measurement of cuvette-incubated mix over night: no rise visible<br />
<br />
* 15 % denaturing acrylamide gels<br />
** for 50 ml<br />
*** 15 % acrylamide: 18.75 ml 40 % acrylamide<br />
*** 6M urea: 18 g<br />
*** 1x TBE: 5 ml 10x TBE<br />
*** 500 µl APS<br />
*** 50 µl TEMED<br />
*** H20 till 50 ml<br />
<br />
** only 30 ml needed for two gels<br />
** big combs for a lot of sample :)<br />
** glass plates cleaned with RNAseZip before pouring the gel<br />
===13.10.2010===<br />
* in vitro T7 transcription, check by polyacrylamide gel electrophoresis <br />
** 10 ml 5 x paper buffer<br />
*** 200 mM Tris/HCl, pH=7.85: 2 ml 1M Tris/HCl, pH=7.85<br />
*** 30 mM MgCl2: 600 µl 0.5 M MgCl2<br />
*** 500 mM KCl: 5 ml 1 M KCl<br />
*** 2.4 ml H2O<br />
<br />
** for in vitro transcription<br />
*** 0.5 µl T7 RPO: 25 U<br />
*** 0.5 µl rNTPs : 20 mM<br />
*** 1.36 µl switch: 250 µM<br />
*** 1 µl signal: 250 µM<br />
*** 2 µl DTT: 10 µM<br />
*** 0.2 µl RNase inhibitor<br />
*** 4 µl 5x buffer<br />
*** 10.44 µl H2O<br />
<br />
**5.1 times<br />
** no signal, nonsense, 1a, 1c, 2a<br />
** in low bind tubes<br />
** 2 hour, 37°C<br />
** actually 2 hours and half a hour pocket cleaning time<br />
<br />
** for PAGE:<br />
*** don't try to run yourgel in the pouring device - if you do so: feel very stupid and embarrassed (I guess I've ran over 100 gels in my life yet... still too stupid...) <br />
*** don't feel tempted to use the Dietz' group's 0.5 x TBE buffer - if you do so: feel very stupid and embarassed, discard buffer carefully and use 1 x TBE<br />
*** don't use a comb which is thinner than your spacers - if you do so: scrap gel pieces out of the pockets for half an hour<br />
*** cook your sample for 5 minutes, 95°C - if you do not so, feel stupid, embarassed and hope that it won't matter so much<br />
*** I did not cool the samples<br />
*** gel runs at 100 V (~ 10V/cm)<br />
*** 20 µl sample + 20 µl ambion loading buffer (1-2 x loading buffer - who does that?) --> 40 µl fits nicely<br />
*** 2 µl low molecular weight marker + 20 µl loading buffer + 18 µ H2O<br />
*** in 1 x TBE<br />
<br />
** M, control (same amounts switch and signals as in the samples), no signal, random, 1a, 2a, 1c, random overnight, 1a overnight, 1c overnight<br />
** overnight samples: measurement with malachitegreen sample over night: no rise visible<br />
** Xylene Cyanol: Comigrating with 60, Bromphenol BLue: comigrating with 15 (http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=845) - Maxi: bei Hälfte ungefähr<br />
** run for 1:45 hours, Bromphenoleblue at about half of the gel<br />
<br />
[[Image:TUM2010_101013 PAGE1.png|600px]]<br />
<br><br />
:--> c=control, ns=no signal, r=random<br />
: --> weird smear everywhere: degraded RNA?<br />
: --> no switch visible on the gel: 133 bp!<br />
: --> signal length: 29-43 bp visible<br />
: --> 25 bp: signal sense, between 50-25 bp: signals: 1a and 1c with approximately the same length, 2a is longer<br />
: --> Wie Sie sehen, sehen Sie nichts.<br />
<br />
*PCR of switch: Biomers original used as template<br />
* PCR T7 switch<br />
** 32 x Mastermix: 1600 µl total volume<br />
*** 32 µl dNTPs<br />
*** 32 µl T7 forward primer<br />
*** 32 µl T7 reverser primer<br />
*** 160 µl 10x buffer<br />
*** 96 µl MgCl2<br />
*** 6.4 µl Taq Polymerase<br />
*** 5 µl Template (some older PCR)<br />
*** 1214.6 µl H2O<br />
<br />
** '''changed''' iGEM PCR program:<br />
*** 95°C, 2' <br />
*** 1) 95°C, 0,5'<br />
*** 2) 50°C, 0.5'<br />
*** 3) 71°C, 1'<br />
*** 71°C, 10'<br />
*** repeat 1) - 3) 35 times<br />
===14.10.2010===<br />
* yesterday's measurement<br />
**[[Image:TUM2010_101014_Kinetik.jpg|600px]]<br />
** rise visible, comparable to tuesday measurement<br />
** samples frozen, put on 15 % PAGE<br />
<br />
* purification of yesterday's PCR<br />
** yield: <br />
<br />
* in vitro T7 transcription, check by polyacrylamide gel electrophoresis <br />
** 10 ml 5 x paper buffer<br />
*** 200 mM Tris/HCl, pH=7.85: 2 ml 1M Tris/HCl, pH=7.85<br />
*** 30 mM MgCl2: 600 µl 0.5 M MgCl2<br />
*** 500 mM KCl: 5 ml 1 M KCl<br />
*** 2.4 ml H2O<br />
<br />
** for in vitro transcription<br />
*** 0.5 µl T7 RPO: 25 U<br />
*** 0.5 µl rNTPs : 20 mM<br />
*** 1.36 µl switch: 250 µM<br />
*** 1 µl signal: 250 µM<br />
*** 2 µl DTT: 10 µM<br />
*** 0.2 µl RNase inhibitor<br />
*** 4 µl 5x buffer<br />
*** 10.44 µl H2O<br />
<br />
**5.1 times<br />
** no signal, nonsense, 1a, 1c, 2a<br />
** in low bind tubes<br />
** 2 hour, 37°C<br />
** actually 2 hours and half a hour pocket cleaning time<br />
<br />
* 2 % agarose gel to check previous PCR products<br />
[[Image:TUM2010_101014 .png]]<br />
:--> yesterdays results bad because no switch :) <br />
<br />
*15 % 6 M urea PAGE<br />
<br />
[[Image:TUM2010 PAGE 101014.png|600px]]<br />
<br />
:--> M = low molecular weight marker (NEB)<br />
:--> c=control=all DNAs mixed together in used concentrations: switch and all signals, ns=no signal, r=random=nonsense<br />
:--> r, 1a, 2a on the left: overnight incubation with malchitegreen<br />
:--> r, 1a, 1c, 2a on the right: two hour in vitro transcription without malachitegreen<br />
<br><br />
: --> switch: 133 bp! <br />
: --> signal length: 29-43 bp<br />
: --> T7 promoter sense: ca. 20 bp (I can't look it up right now) <br />
<br><br />
:--> extra bands visible after overnight transcription (rise in malchitegreen fluorescence visible)<br />
:--> switch runs at a strange height: WHY? looks normal on 2 % agarose gel<br />
:--> upper band: RPO bound to DNA? Compare EMSA<br />
:--> DNA ladder, RNA bands: How to compare?<br />
<br><br />
: Okay, let's try to interpret this:<br />
: Denaturing conditions, everything precooked: No guarantee for double stranded DNA/RNA<br />
: control: switch and signals from tubes on gel - otherwise treated the same<br />
: --> internal standart: switch at about 80 bp (low molecular weight standart) equals 133 bp<br />
: --> band seen in all samples at the height of random/1c --> termination product of switch??? (expected size: about 90 bp? does not fit at all?!)<br />
: --> lower band visible, what is it?<br />
: --> What is left: NO Differences Between Random control (=nonsense) and Designed Switches...<br />
<br />
<br />
* New malachitegreen assay (overnight in vitro transcription) with new PCR product<br />
<br />
** for 4.1 x 100 µl<br />
*** 35.88 µl PCR product<br />
*** 20.05 dNTPs<br />
*** 41 µl DTT<br />
*** 10.25 µl T7 RPO<br />
*** 2 µl signal per 100 µl<br />
<br />
** preincubation of signal with RPO at 37°C<br />
** no rise during preincubation (makes sense)<br />
** addition of signal after about one and a half hour<br />
<br />
===15.10.2010===<br />
* in vitro transcription - malachite green<br />
<br />
** slight rise visible after overnight incubation after preincubation of signal<br />
** only 1/4 of the intensity measured yesterday<br />
** spectra fit but very weak signal, scattered spectra<br />
<br />
*Next step: addition of DNaseI (RNase free) to overnight transcription products<br />
<br />
*Felt ill, went home soon<br />
<br />
===17.10.2010===<br />
*Malachite-green measuring assay<br />
<br />
** 1x:<br />
*** 2 µl signal, 5 µM<br />
*** 4.66 µl switch, 176 µM<br />
*** 2.5 µl RPO<br />
*** 50 µl Paperbuffer<br />
*** 10 µl DTT 100 µM<br />
*** 5 µl rNTPs<br />
*** 25.84 µl H2O<br />
<br><br><br />
<br />
** 4.1 x<br />
*** 19.11 µl switch, 176 µM<br />
*** 10.25 µl RPO<br />
*** 205 µl Paperbuffer<br />
*** 41 µl DTT 100 µM<br />
*** 20.5 µl rNTPs<br />
*** 105.94 µl H2O<br />
<br><br><br />
** Signals: random, 1a, 2a, 1c<br />
<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week30{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=Cloning of Parts into pSB1C3}} {{:Team:TU_Munich/Templates/ClearBox | text=Measurements }} {{:Team:TU_Munich/Templates/GreenBox | text=T7 & E. coli }} <br />
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===18.10.2010===<br />
*Yesterday's malachitegreen-binding assay<br />
**Kinetics<br />
[[Image:TUM2010_101018_Kinetics.png|600px]]<br />
<br />
--> Favourite one ever so far!<br />
<br />
** Emission and Excitation: Exc=530 nm, Em=552 nm<br />
<br />
[[Image:TUM2010_101018_Scan.png|600px]]<br />
<br />
*Biomers order arrived<br />
** DNA resolved according to manual, 100 µM endconcentration<br />
<br />
* PCR T7 switch/positive signal<br />
** 32 x Mastermix: 1600 µl total volume<br />
*** 32 µl dNTPs<br />
*** 32 µl T7 forward primer<br />
*** 32 µl T7 reverser primer<br />
*** 160 µl 10x buffer<br />
*** 96 µl MgCl2<br />
*** 6.4 µl Taq Polymerase<br />
*** 10 µl Template (some older PCR)<br />
*** 1209.6 µl H2O<br />
<br />
** '''changed''' iGEM PCR program:<br />
*** 95°C, 2' <br />
*** 1) 95°C, 0,5'<br />
*** 2) 50°C, 0.5'<br />
*** 3) 71°C, 1'<br />
*** 71°C, 10'<br />
*** repeat 1) - 3) 35 times<br />
<br />
** yields positive control:<br />
*** Mr=67234,6 g/mol <br />
***230 ng/µl --> 3.42 µM<br />
*** 198 ng/µl --> 2.94 µM<br />
*** 209 ng/µl --> used for cloning<br />
*** 178 ng/µl --> 2.65 µM<br />
<br />
** yields switch:<br />
*** Mr=82059 g/mol<br />
*** 170 ng/µl --> 2.07 µM<br />
*** 204 ng/µl --> 2.49 µM<br />
*** 179 ng/µl --> 2.18 µM<br />
*** 160 ng/µl --> 1.95 µM<br />
<br />
* 6 M urea 15 % acrylamid PAGE<br />
** DNase I digestion<br />
*** 1 µl 10 x DNase buffer<br />
*** 1 µl DNaseI <br />
*** 20 µl reaction product from malachitegreen assay, 17.10.10<br />
*** 37°C, 90 minutes<br />
<br />
** Gel:<br />
*** 20 µl loading buffer and 20 µl sample<br />
*** <br />
<br />
* Malachitegreen assay with double stranded signals<br />
<br />
** Concentration verification of sense and antisense in ng/µl<br />
*** diluted 1:8 (5+35 µl)<br />
*** signal - sense ng/µl - antisense ng/µl<br />
*** 1a - 1391 - 1584<br />
*** 2b - 1812 - 1823<br />
*** 1c - 2208 - 1575<br />
*** random2=nonsense2 - 2480 - 1903<br />
<br><br />
** Concentration verification of sense and antisense in µM<br />
*** signal - sense µM - antisense µM<br />
*** 1a - 15.47 - 17.99<br />
*** 2b - 15.43 - 15.70<br />
*** 1c - 16.95 - 12.31<br />
*** r2 - 21.27 - 16.28<br />
<br />
<br><br />
** for 10 µM of both sense and antisense and to put it together to equal 5 µM in total...<br />
*** signal - sense µl - antisense µl - water µl<br />
*** 1a - 32.32 - 27.79 - 38.89<br />
*** 2b - 32.40 - 31.85 - 35.75<br />
*** 1c - 29.50 - 40.62 - 29.88<br />
*** r2 - 23.51 - 30.71 - 45.78<br />
<br />
** Measurement<br />
** 1x switch<br />
<br />
*** 2 µl signal, 5 µM, double stranded!<br />
*** 4.17 µl switch, 2.49 µM<br />
*** 2.5 µl RPO<br />
*** 50 µl Paperbuffer<br />
*** 10 µl DTT 100 µM<br />
*** 5 µl rNTPs<br />
*** 26.3 µl H2O<br />
<br />
<br />
** 3.1 x<br />
*** 12.93 µl switch, 176 µM<br />
*** 7.75 µl RPO<br />
*** 155 µl Paperbuffer<br />
*** 31 µl DTT 100 µM<br />
*** 15.5 µl rNTPs<br />
*** 81.53 µl H2O<br />
*** signals: r2, 1a, 1c<br />
** 1x positive control<br />
*** 2 µl signal, 5 µM, double stranded!<br />
*** 2.94 µl switch, 2.94 µM<br />
*** 2.5 µl RPO<br />
*** 50 µl Paperbuffer<br />
*** 10 µl DTT 100 µM<br />
*** 5 µl rNTPs<br />
*** 27.57 µl H2O<br />
*** signal: r2<br />
<br />
* Cloning Malachitegreen-binding aptamer into pB1C3<br />
** E/P digestion<br />
*** 2 µl 10x NEB 4<br />
*** 2 µl 10x BSA<br />
*** 0.5 µl EcoRI<br />
*** 0.5 µl PstI<br />
*** 15 µl linearized pB1C3 (50 ng/µl)/malachitegreen binding aptamer (203 ng/µl)<br />
*** 37°C, 1 h<br />
*** purification afterwards using DNA clean and concentrated (or something)<br />
<br />
** Concentrations<br />
*** backbone: 11 ng/µl<br />
*** insert: <br />
<br />
** Ligation<br />
*** 5 µl digested plasmid<br />
*** 2 µl malachitegreen binding aptamer, 1:10 diluted<br />
*** 2 µl T4 ligase buffer<br />
*** 1 µl T4 ligase<br />
*** 10 µl water<br />
<br />
** no transformation not possible: no chloramphenicol plates in physic's department...<br />
<br />
===19.10.2010===<br />
* Yesterday's malachitegreen assay<br />
** Kinetics:<br />
[[Image:TUM2010_101019_Kinetik.png|600px]]<br />
** Emission spectra: Exc=530 nm<br />
[[Image:TUM2010_101019_Em.png|600px]]<br />
<br />
** Excitation spectra: Em=552 nm<br />
[[Image:TUM2010_101019_Exc.png|600px]]<br />
<br />
* 7 M urea, 15 % PAGE<br />
<br />
** DnaseI testdigestion<br />
*** plasmid digestion to test DnaseI activity: <br />
*** 10 µl paper buffer<br />
*** 0.2 µl DTT<br />
*** 2 µl 10x DnaseI buffer<br />
*** 1 µl Dnase<br />
*** 1 µl plasmid (some random thing from Wuschel)<br />
*** 5.8 µl water<br />
*** 37°C, 2h<br />
<br />
** samples for DNaseI digestion<br />
*** 2 µl 10x DnaseI Buffer<br />
*** 1 µl DnaseI<br />
*** 17 µl reaction product from malachitegreen assay, 18.10.10<br />
*** 37°C, 2 h<br />
<br />
** 7M urea, 15 % PAGE<br />
*** 30 ml<br />
*** 11.25 ml acrylamide<br />
*** 12.6 g urea<br />
*** 3 ml 10x TBE<br />
*** 300 µl 10 % APS<br />
*** 30 µl TEMED<br />
*** H20 to 30 ml<br />
*** heat a bit and sonificate<br />
** gel could not be run: pockets not solid...<br />
*** most likely reason: too hot when radical starter were added: instant polymerization were they hit the mixture...<br />
<br />
* malachitegreen assay with more malachitegreen this time!<br />
** --> 3x more than usual<br />
** 30 mM 2x malachitegreen paper buffer<br />
*** 1 ml 5x paper buffer without malachitegreen<br />
*** 0.6 ml 250 mM malachitegreen<br />
*** 0.9 ml Water<br />
<br />
** 1x switch<br />
*** 2 µl signal, 5 µM, double stranded!<br />
*** 4.17 µl switch, 2.49 µM<br />
*** 2.5 µl RPO<br />
*** 50 µl Paperbuffer<br />
*** 10 µl DTT 100 µM<br />
*** 5 µl rNTPs<br />
*** 26.3 µl H2O<br />
*** signals: 1a, r2<br />
** 1x positive control<br />
*** 2 µl signal, 5 µM, double stranded!<br />
*** 2.94 µl switch, 2.94 µM<br />
*** 2.5 µl RPO<br />
*** 50 µl Paperbuffer<br />
*** 10 µl DTT 100 µM<br />
*** 5 µl rNTPs<br />
*** 27.57 µl H2O<br />
*** signal: r2, none<br />
<br />
* Cloning Malachitegreen-binding aptamer into pB1C3<br />
<br />
** Ligation<br />
*** 5 µl digested plasmid<br />
*** 2 µl malachitegreen binding aptamer, 1:10 diluted<br />
*** 2 µl T4 ligase buffer<br />
*** 1 µl T4 ligase<br />
*** 10 µl water<br />
<br />
** Transformation<br />
*** borrowed plates from the Prof. Groll's department<br />
*** and from Prof. Becker's<br />
*** the one from Prof. Becker's once contained tetracyclin...<br />
*** in DH5alpha cells<br />
*** 200 µl plated<br />
*** overnight, 37°C<br />
<br />
===20.10.2010===<br />
* Yesterday's malachite green binding assay<br />
** Kinetics: <br />
[[Image:TUM2010_101020 kinetics.png|600px]]<br />
<br />
** Emission spectra: Exc=530 nm<br />
[[Image:TUM2010_101020 em.png|600px]]<br />
<br />
** Excitation spectra: Em=552 nm<br />
[[Image:TUM2010_101020_exc.png|600px]]<br />
<br />
* Cloning Malachitegreen-binding aptamer into pB1C3<br />
** many colonies grown<br />
** Well done, Flo!<br />
** Colony PCRl<br />
** 10 x Mastermix: 500 µl total volume<br />
*** 10 µl dNTPs<br />
*** 10 µl G1004<br />
*** 10 µl G1005<br />
*** 50 µl 10x buffer<br />
*** 30 µl MgCl2<br />
*** 2 µl Taq Polymerase<br />
*** 2 µl Template per reaction<br />
*** H2O<br />
<br><br />
** PCR program<br />
*** iGEM program!<br />
*** 58°C annealing<br />
<br />
<br />
** 2.5 % agarose gel<br />
[[Image:TUM2010_101020_ColonyPCR.png|600px]]<br />
*** lmw: low molecular weight (ladder)<br />
*** 100: 100 kb (ladder)<br />
*** 7 clones chosen to be checked by Colony PCR<br />
*** LB=negative control=LB-Chloramphenicol<br />
*** positive control: PCR with Biomer product<br />
** also on the gel:<br />
*** S=switch=PCR of switch used for measurement<br />
*** P=positive control=PCR product used for measurement and cloning<br />
<br><br />
** Interpretation<br />
*** stupid negative control looks just the same like everything else<br />
*** two bands at approximately the right height, but exactly above and below positive control band<br />
*** shit.<br />
<br />
** Further proceedings<br />
*** 5 ml cultures of clones 3, 4, 5, 7<br />
*** 5 ml cultures of 6 new colonies<br />
*** minipreps and analytical digestions tomorrow<br />
<br />
<br />
<br />
* 7 M urea 15% PAGE<br />
<br />
** samples from 19.10.10<br />
[[Image:TUM2010_101020 page.png|600px]]<br />
<br />
** controls: <br />
*** switch: PCR of switch, used in measurements<br />
*** P: PCR of positive control, used in measurements<br />
*** Plas: random test plasmid used for evaluation of DnaseI activity<br />
** DNase digested samples: 20 µl<br />
*** Plas: DnaseI digested random plasmid, for conditions check previous day<br />
*** P+r2: Positive control together with double stranded nonsense 2/random 2 signal after overnight transcription in paper buffer (check malachitegreen assay, previous day), DnaseI digested<br />
*** S+r2: Switch together with double stranded nonsense 2/random 2 signal after overnight transcription in paper buffer, DnaseI digested<br />
*** S+1a: Switch together with double stranded 1a signal after overnight transcription in paper buffer, DnaseI digested<br />
*** S+1c: Switch together with double stranded 1c signal after overnight transcription in paper buffer, DnaseI digested<br />
** other: 15 µl<br />
*** P+r2: Positive control together with double stranded nonsense 2/random 2 signal after overnight transcription in paper buffer (check malachitegreen assay, previous day)<br />
*** S+r2: Switch together with double stranded nonsense 2/random 2 signal after overnight transcription in paper buffer<br />
*** S+1a: Switch together with double stranded 1a signal after overnight transcription in paper buffer<br />
** no marker this time! Only 12 lanes :)<br />
<br><br />
** Interpretation:<br />
*** no real differences between DnaseI digested and not<br />
*** It is rather stupid to use a plasmid to check for DnaseI activity if you want to run the result on a 15 % gel. A plasmid with some kb does not seperate... But: DNA visible, without Dnase I, no DNA visible with DnaseI (stuck in the upper lane, 1)<br />
*** Next try with PCR product maybe<br />
*** weird lane in all PCR reactions at 2 in both cases and in all other gels and a bit also in agarose gels visible<br />
*** positive control runs totally elsewhere than switch even though they do not really vary in length (3)<br />
<br />
<br />
* Malachitegreen binding assay<br />
** Measurement<br />
** 1x switch<br />
*** 2 µl signal, 5 µM, double stranded!<br />
*** 4.17 µl switch, 2.49 µM<br />
*** 2.5 µl RPO<br />
*** 50 µl Paperbuffer<br />
*** 10 µl DTT 100 µM<br />
*** 5 µl rNTPs<br />
*** 26.3 µl H2O<br />
<br />
** measured: signals all doublestranded, everything in paper buffer, 10 µM malachitegreen<br />
*** positive control<br />
*** positive control with nonsense2/random2<br />
*** switch with nonsense2/random2<br />
*** switch with 1a<br />
<br />
===21.10.2010===<br />
* Yesterday's measurement<br />
[[Image:TUM2010_101021_Kinetik.png|600px]]<br />
<br />
** Fluorescence spetrum, Exc=530 <br />
[[Image:TUM2010_101021_Em.png|600px]]<br />
<br />
** Fluorescence excitation spectrum, Em= 552<br />
[[Image:TUM2010_101021_Exc.png|600px]]<br />
<br />
<br />
* Amplifying in vivo positive control to send for PartsRegistry<br />
** Miniprep of 5 ml overnight control<br />
*** P1 185 ng/µl <br />
*** P2 416 ng/µl<br />
*** check below for gel picture and further proceeding<br />
<br />
*Cloning Malachitegreen-binding aptamer into pSB1C3 <br />
** Mini-prep of 5 ml overnight cultures<br />
*** # - concentration in ng/µl<br />
*** 3 - 272 <br />
*** 4 - 59<br />
*** 5 - 100<br />
*** 7 - 95.56<br />
*** a - 86<br />
*** b - 165<br />
*** c - 114<br />
*** d - 131<br />
*** e - 232<br />
*** f - 144<br />
*** P1 - 185<br />
*** P2 - 416<br />
<br />
** Control digestion<br />
*** 4 µl plasmid<br />
*** 2 µl NEB 3<br />
*** 0.5 µl PstI<br />
*** 0.5 µl EcoRI-HF<br />
*** 13 µl H2O<br />
*** 1 h, 37°C<br />
<br />
** 2 % agarose gel<br />
[[Image:TUM2010_101021_control_digestion.png|600px]]<br />
*** lmw: low molecular weight ladder (NEB)<br />
*** 100 bp: 100 bp ladder (NEB)<br />
*** 3,4,5,7: yesterday's clones, checked and falsified by colony PCR<br />
*** a-g: yesterday picked clones<br />
*** P1, P2: minpreps of in vivo measurement positive control, to be sent to partsregistry<br />
<br />
** Interpreation<br />
*** well, something went seriously wrong while cloning<br />
*** asked at physic's department: DH5alpha cells in use were pretty old: cell from old -80°C are baaaad!<br />
*** showed us new stock<br />
*** further possibilities: EcoRI nearly empty --> not cutting properly anymore<br />
*** T4 ligation buffer: went through 4 tubes until I found one which was not totally full of precipitates<br />
*** Chloramphenicol-plates: got plates from Groll group, plates from 2008<br />
<br />
** Further cloning procedure<br />
*** digestion of linearized pSB1C3 (from parts registry) and positive control with EcoRI-HF (full enzyme)<br />
*** digestion of T7 positive control<br />
*** 15 µl linearized pSB1C3 (25 ng/µl, damn it, I thought there are 50 ng/µl while preparing the ligation...)/5 µl of positive control, PCR product (c=173 ng/µl) + 10 µl H2O<br />
*** 2 µl 10x NEB 3<br />
*** 2 µl 10x BSA<br />
*** 0.5 µl EcoRI<br />
*** 0.5 µl PstI<br />
*** 37°C, 1 h<br />
<br />
** heat inactivation<br />
*** 80°C, 20 minutes<br />
<br />
** ligation<br />
*** 1 µl T4 ligase<br />
*** 2 µl T4 ligase buffer: tested three buffers, one was okay, made aliquots: what happened to all the buffers???<br />
*** 1.33 µl backbone (I thought 50 ng, actually 25 ng)<br />
*** 0.23 µl insert, 1:10 diluted<br />
*** 17.37 µl H2O<br />
*** incubated till transformation<br />
<br />
** Extracting pSB1C3 from iGEM 2010 distribution<br />
*** 10 µl H20 in A3<br />
*** 3 µl used for transformation<br />
<br />
** transformation<br />
*** in DH5alpha cells from new -80°C fridge...<br />
*** also in XL-1 blue: Tetracycline resistance<br />
*** 15 minutes in ice with ligation product<br />
*** 1 minute heat shock, 42°C<br />
*** 2 minutes on ice<br />
*** 500 µl LB0 added<br />
*** 1.5 hours at 37°C, shaking<br />
*** control: digested, linearized pSB1C3 into XL-1 blue<br />
*** reason: DpnI digestion recommended, we don't have any DpnI <br />
*** on chloramphenicol agar plates from Prof. Buchner's lab --> new and shiny<br />
*** XL-1 blue plated on Tetracycline/Chloramphenicol plates from Becker's lab: not used in Becker's lab because Tetracycline broken, does not matter for us...<br />
<br />
* E. coli RPO malachitegreen assay<br />
** E. coli RPO finally arrived<br />
*** called Biozym: RPO was supposed to arrive yesterday, delivery cimpany signed for yesterday<br />
*** called Materialausgabe two times everyday for the last week --> In the end they already knew, when they heard E14<br />
*** nevertheless when the enzyme arrived, somebody forgot to put it into the large book of incoming stuff<br />
*** only one person knew, that the enzyme already arrived <br />
*** stored at -20 °C meantime<br />
*** dry ice already vaporated...<br />
*** people were first mad at me, because I insisted that the package arrived although it was not written in the main book<br />
*** then mad at each other because somebody did not put it into the main book<br />
*** then I was slightly mad because we called two times a day for seven days, argued about -80°C storage and still...<br />
*** bought dry ice, transport into ZNN, stored at -20°C covered in dry ice (-80°C fridge is already standing in the lab but not installed yet) until measurements<br />
*** now stored at old -80°C in physic's department in iGEM box<br />
<br />
** preperation of measuring constructs - without terminator<br />
*** PCR of 12z (His-Terminator in plasmid)<br />
*** 5z (TrpTerm in plasmid)<br />
*** 28z (pSB1A2-R0011-TrpSig)<br />
*** pSB1K3-R0011-HisSig-B0014<br />
*** 4x 8x PCR mix<br />
*** 8 µl dNTPs<br />
*** 8 µl Apt forward<br />
*** 8 µl Apt reverse - without terminator!<br />
*** 40 µl 10x buffer<br />
*** 24 µl MgCl2<br />
*** 1.6 µl Taq Polymerase<br />
*** 302.4 µl Template per reaction<br />
*** H2O<br />
<br><br />
*** recognized that wrong primers were used for the signals, thrown away after PCR<br />
*** protocol iGEM PCR, 52°C annealing temp.<br />
*** purified using Clean and Concentrator<br />
*** yields: 220 ng/µl TrpTerm, 178 ng/µl (or something like that) HisTerm<br />
<br />
** Buffer preparation for E. coli RPO<br />
*** Instructions from Epicentre Biotechnologies Datasheet (included in package)<br />
*** 5x transcription buffer<br />
*** 2 ml Tris, pH=7.49<br />
*** 1 ml 500 mM MgCl2<br />
*** 5 µl Triton X-100<br />
*** 3.75 ml 2 M KCl<br />
*** water to 10 ml<br />
*** stored in fridge<br />
<br><br />
*** 2x transcription buffer with malachitegreen<br />
*** 4 ml 5x buffer<br />
*** 800 µl 250 mM malachitegreen stock<br />
*** water to 10 ml<br />
*** wrapped in aluminium foil, stored in fridge<br />
<br />
** measurement with E. coli RNA Polymerase<br />
*** measured: 12z (TrpTerm - negative control), 5z (HisTerm, negative control), (positive control)<br />
*** 1x measurement<br />
*** 2.5 µl RPO (2.5 U)<br />
*** 10 µl DTT (100 mM stock, 10 mM end)<br />
*** 50 µl 2x buffer <br />
*** 2.5 µl rNTPs (80 mM stock, 2 mM end)<br />
*** 1 µg DNA template<br />
*** water to 100 µl<br />
<br><br />
*** 3.1 x<br />
*** 7.75 µl RPO<br />
*** 31 µl DTT<br />
*** 7.75 µl rNTPs<br />
*** 155 µl 2x buffer<br />
*** 77.5µl H20<br />
*** 4.5 µl 5z/5.5 µl H2O<br />
*** 5.35 µl 12z/4.65 µl H2O<br />
*** 5.88 µl ??? /4.12 µl H20<br />
<br><br />
*** 38°C, Exc=630 nm, Em=650/655 nm<br />
<br />
** Cloning of R0011 and B1006 in pSB1K3<br />
*** digestion of R0011 with EcoRi and SpeI (check above, same procedure)<br />
*** ligation<br />
*** 0.3 µl R0011<br />
*** 0.2 µl B1006 (signal)<br />
*** 5 µl pSB1K3<br />
*** 2 µl T4 ligation buffer<br />
*** 1 µl T4 ligase<br />
*** 11.5 µl H2O<br />
*** Transformation into DH5alpha cells<br />
*** put on Kana-Plates<br />
<br><br />
** Cloning of signal B1006 in pSB1A2_R0011<br />
*** 1.82 µl pSB1A2_R0011<br />
*** 0.2 µl Signal B1006<br />
*** 2 µl T4 DNA ligation buffer<br />
*** 1 µl T4 DNA ligase<br />
*** 15 µl H2O<br />
*** Transformation into DH5alpha cells<br />
** put on Amp-plates<br />
*** check above<br />
<br />
===22.10.2010===<br />
* Yesterday's malachitegreen assay using E. coli RPO<br />
<br />
[[Image:TUM2010_101022_Kinetik.png|600px]]<br />
<br />
** Fluorescence spetrum, Exc=630 <br />
[[Image:TUM2010_101022_Em_skal.png|600px]]<br />
<br />
** Fluorescence excitation spectrum, Em= 652<br />
[[Image:TUM2010_101022_Exc.png|600px]]<br />
<br />
* 7 M 15 % PAGE<br />
** DnaseI digestion of E. coli RPO transcription<br />
*** control: T7 positive control<br />
*** 17 µl Transcription product<br />
*** 2 µl 10 x DnaseI buffer<br />
*** 1 µl Dnase<br />
<br />
[[Image:TUM2010_101022_page.png|600px]]<br />
<br />
*** lmw: low molecular weight ladder, NEB<br />
*** H: His-switch<br />
*** W: Trp-Switch<br />
*** 16z: Positive control (with TrpSignal); using Primer Apt_For+AptPart_woT_Rev<br />
<br />
*** Ladies and Gentleman, we definetly see RNA this time<br />
*** positive control completely transcriped, terminators terminate (finally some terminator terminate...)<br />
*** DnaseI digestion works. DnaseI control completely clean.<br />
*** I think termination products are visible<br />
*** I also think, that RPO/DnaseI/protein in general bound RNA is stuck in pockets<br />
*** 1h. 37°C, Dnase digestion conditions: RNA suffers a bit, paradise for RNases?<br />
<br />
<br />
* Cloning Malachitegreen-binding aptamer into pSB1C3 <br />
** no clones on plates<br />
*** clones expected to show up on A3 transformed cells --> part from partsregistry!<br />
*** weird clones on Tetracycline/Chloramphenicol plates from Becker's department --> maybe both antibiotics not working anymore<br />
*** talked to Moni from E 22 <br />
*** still old cells in new -80°C --> cells from 2008, maybe dead, certainly not competent...<br />
<br />
** repeat transformation (see yesterday's labbook)<br />
<br />
<br />
===23.10.2010===<br />
*''In vitro'' transcription using ''E. coli'' RPO <br />
** PCR of HisSig, TrpSig, HisTerm, TrpTerm, 16z (positive control)<br />
*** 100 µl per template, 800 µl total<br />
*** 16 µl dNTPs<br />
*** 16 µl forward primer<br />
*** 16 µl reverse primer<br />
*** 80 µl Taq-buffer Mg-free<br />
*** 3.2 µl Taq Polymerase<br />
*** 48 µl MgCl2<br />
*** 5 µl template per 100 µl<br />
*** 615.8 µl H2O<br />
** PCR Purification using DNA Quick and Clean<br />
*** yield: Template - # - concentration [ng/µl]<br />
*** 16z - 1 - 146<br />
*** 16z - 2 - 138<br />
*** 16z - 3 - 138<br />
*** 16z - 4 - 128<br />
*** HisTerm - 1 - 96<br />
*** HisTerm - 2 - 160<br />
*** TrpTerm - 1 - 204<br />
*** TrpTerm - 2 - 190<br />
*** HisSig - 1 - 160<br />
*** HisSig - 2 - 165<br />
*** TrpSig - 1 - 83.5<br />
*** TrpSig - 2 - 166<br />
<br />
**Measurement<br />
*** 16z positive control, TrpTerm, both with and without signal<br />
*** 4.1 x Master Mix<br />
*** 105 µl Buffer<br />
*** 41 µl DTT<br />
*** 10.25 µl RPO<br />
*** 10.25 µl rNTPs<br />
*** 77.9 µl H2O<br />
*** take 84 µl Master Mix<br />
*** add to: <br />
*** 10.85 µl 16 z + 6.1 µl H20<br />
*** 10.85 µl 16 z + 6.1 µl TrpSig<br />
*** 10.92 µl TrpTerm + 6.1 µl H20<br />
*** 10.92 µl TrpTerm + 6.1 µl TrpSig<br />
[[Image:TUM2010_101023_kinetik.png|600px]]<br />
Only the positive control without Signal increases!<br />
<br />
<br />
* Cloning of MPA into pSB1C3<br />
** Checked plates, no clones<br />
** being sad<br />
** said stupid plates <br />
** Repetition of ligation<br />
*** for 50 ng plasmid backbone<br />
*** 2.6 µl pBS1C3 E/P digested<br />
*** 0.23 µl MPA, E/P digested<br />
*** 2 µl T4 ligase buffer - I checked four buffers, all were precipitated, took a completely new one and aliquoted it!<br />
*** 1 µl T4 Ligase<br />
*** for 100 ng plasmid backbone<br />
*** 5.2 µl pBS1C3 E/P digested<br />
*** 0.46 µl MPA, E/P digested<br />
*** 2 µl T4 ligase buffer - I checked four buffers, all were precipitated, took a completely new one and aliquoted it!<br />
*** 1 µl T4 Ligase<br />
*** transformation into pSB1K3 (just in case...)<br />
*** 4.5 µl pSB1K3 E/P digested<br />
*** 0.23 µl MPA, E/P digested<br />
*** 2 µl T4 ligase buffer - I checked four buffers, all were precipitated, took a completely new one and aliquoted it!<br />
*** 1 µl T4 Ligase<br />
** found some pSB1C3_RFP clones in the evening, 5 ml cultures<br />
** stroke over the plate with a pipette tip, 5 ml culture<br />
<br />
===24.10.2010===<br />
*Cloning of MPA into pSB1C3<br />
** Miniprep of pSB1C3_RFP and something random from the plate of no clones<br />
*** mixed up samples: yields were good, but I guess they don't matter<br />
** very tiny clones found in the morning<br />
** slightly bigger ones by noon<br />
** pickable clones by evening<br />
** picked 10 clones, 5 ml cultures<br />
<br />
* BBa_K494001-BBa_K494006<br />
** 2x 5 ml LB_Amp from glycerin stocks<br />
<br />
* Measurements with T7 RPO and ''E. coli'' constructs<br />
** 16z positive control, TrpTerm, both with and without signal<br />
*** 4.1 x Master Mix<br />
*** 205 µl Buffer<br />
*** 41 µl DTT<br />
*** 10.25 µl RPO<br />
*** 10.25 µl rNTPs<br />
*** 77.9 µl H2O<br />
<br />
*** take 76.2 µl Master Mix<br />
*** add to: <br />
*** 10.07 µl 16 z + 13.7 µl H20<br />
*** 10.07 µl 16 z + 9.61 µl TrpSig + 4 µl H2O<br />
*** 23.67 µl HisTerm (1)<br />
*** 14.2 µl HisTerm (2) + 9.61 µl TrpSig<br />
<br />
[[Image:TUM2010_101024_kinetik.JPG|600px]]<br />
It looks like an increase in 3 of 4 traces, but spectr show no sign of malachite green binding:<br />
[[Image:TUM2010_101024_scan.JPG|600px]]<br />
<br />
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<br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week31{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=Cloning of Parts into pSB1C3}} {{:Team:TU_Munich/Templates/ClearBox | text=Measurements }} {{:Team:TU_Munich/Templates/GreenBox | text=T7 & E. coli }} <br />
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The Week of Wiki freeze!<br />
<br />
===25.10.2010===<br />
* Submitting Parts<br />
** Miniprep of yesterday's 5 ml cultures using Zymo Classic<br />
** MPA into pSB1C3<br />
*** # - concentration (ng/µl)<br />
*** 1 - 45.5<br />
*** 2 - 96.5<br />
*** 3 - 19.5<br />
*** 4 - 31<br />
*** 5 - 122<br />
*** 6 - 164<br />
*** 7 - 30<br />
*** 8 - 39.5<br />
*** 9 - 20.5<br />
*** 10 - 113<br />
<br />
** BBa_K494001-BBa_K494006<br />
*** BioBrick - # - concentration in ng/µl<br />
*** BBa_K494001 - 1 - 179<br />
*** BBa_K494001 - 2 - 182<br />
*** BBa_K494002 - 1 - 214<br />
*** BBa_K494002 - 2 - 288<br />
*** BBa_K494003 - 1 - 160<br />
*** BBa_K494003 - 2 - 202<br />
*** BBa_K494004 - 1 - 308<br />
*** BBa_K494004 - 2 - 322<br />
*** BBa_K494005 - 1 - 290<br />
*** BBa_K494005 - 2 - 126<br />
*** BBa_K494006 - 1 - 290<br />
*** BBa_K494006 - 2 - 232<br />
<br />
** control digestion of everything<br />
*** EcoRI/PstI<br />
*** MPA clones 1, 3, 4, 7, 8, 9: 8 µl<br />
*** all other: 4 µl<br />
*** 7 x for 4 µl<br />
*** 14 µl 10 x BSA<br />
*** 14 µl 10 NEB3<br />
*** 2.1 µl EcoRI HF<br />
*** 2.1 µl PstI<br />
*** water as needed<br />
<br />
*** 19 x for 4 µl<br />
*** 38 µl 10x BSA<br />
*** 38 µl 10x NEB3<br />
*** 5.7 µl EcoRI HF<br />
*** 5.7 µl PstI<br />
*** water as needed<br />
<br />
*** 37°C, 1 hour, fitted just perfectly into the heat block<br />
<br />
*** recognized later that MPA 2, 5, 6, 10 were digested without DNA<br />
*** NotI Digestion with --> PstI empty... Time to finish :)<br />
*** 10 µl NEB<br />
*** 10 µl 10x BSA<br />
*** NotI<br />
<br />
** pSB1C3_MPA (BBa_K494000) run on 2 % agarose gel<br />
[[Image:TUM2010 101025 MPA.PNG|center|500 px]]<br />
<br />
**--> clone 1 picked for submission: Looked better in reality!<br />
<br />
** BBa_K494001-BBa_K494006 run on 1.5 % agarose gel<br />
[[Image:TUM2010 101025 backbones.PNG|center|500 px]]<br />
<br />
** pSB1C3_MPA (BBa_K494000) missed preps run on 2 % agarose gel<br />
<br />
===26.10.2010===<br />
* Waited for the FedEx man from 8:00-15:00. Called at 15:15. Said, oh, they forgot to pick it up! Sent somebody immediately who arrived at 15:45. Happyness --> Parts submitted, not our fault anymore :) Track: 871353522440<br />
<br />
===27.10.2010===<br />
* Ran around to shoot weird ''E. coli'' pics<br />
<br />
* Not in the lab anymore... FedEx delivered our parts! 9.14 am EDT, we recognized it at 16:22 MEST! More Happyness, now back to serious working maybe...<br />
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<br />
<br><br />
<br />
=Protocols=<br />
<br />
==Molecular Biology==<br />
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*PCR <br />
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<br />
<center>'''Taq Polymerase Hot Start '''</center><br />
<br />
'''PCR Pippeting plan: '''<br> <br />
<br />
1 µl template <br> <br />
<br />
1 µl dNTP 10 µM <br> <br />
<br />
1 µl G1004 (Primer) 10 µM<br> <br />
<br />
1 µl G1005 (Primer) 10 µM<br> <br />
<br />
5 µl 10x Taq-buffer&nbsp; (500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM&nbsp;MgCl<sub>2</sub>)&nbsp; <br />
<br />
0,2 µl Taq-Polymerase (add last) 5,000 U/ml<span style="background-color: rgb(255, 0, 0);"><br />
</span> <br />
<br />
<br> <br />
<br />
40.8 µl Water<br> <br />
<br />
Final volume 50µl <br />
<br />
'''<br>''' <br />
<br />
'''Processing: '''(program saved as '''IGEMPCR ''')'''<br>''' <br />
<br />
*preheating of PCR chamber to 94 °C<br />
<br />
&nbsp;&nbsp; --&gt; insert sample <br />
<br />
*2 min at 94 °C <br />
*loop 35x:<br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp; - 30 sat 94°C (according to IGEM protocols) <br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp; - 30 s at 56 °C <br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp; - 45s at 72°C <br />
<br />
*7 min at 72°C <br />
*stay at 4°C <br><br><br />
<center>'''colony PCR '''</center><br />
*Colony PCR<br />
**pick colonies and resuspend them in 20 µl LB+Antibiotic (each)<br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified), store remaining 18 µl for overnight cultures<br />
**afterwards, mix 15 µl of each PCR product with 3 µl GLPn and load to Gel<br />
**make overnight cultures of positive clones by adding the remaining 18 µl to 5 ml LB+AB<br />
<br />
'''program:colonypcr ''' <br />
<br />
*preheating of PCR chamber to 94 °C<br />
<br />
&nbsp;&nbsp; --&gt; insert sample <br />
<br />
*5 min 30 sec at 94 °C <br />
*loop 35x:<br />
**30 sat 94°C (according to IGEM protocols) <br />
**30 s at 58 °C <br />
**60s at 72°C <br />
*7 min at 72°C <br />
*stay at 4°C <br />
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*DNA Purification<br />
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<center>'''PCR samples '''</center><br />
<br />
''' ZYMO RESEARCH DNA Clean&amp;Concentration Kit '''<br />
<br />
[http://www.zymoresearch.com/zrc/pdf/D4003i.pdf Protocol and Information]<br> <br />
<br />
#In a 1.5 ml microcentrifuge tube, add 2-7 volumes of DNA Binding Buffer to each volume of DNA sample (see table below). Mix briefly by vortexing.<br><br><br />
<br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| Application <br />
| DNA Binding Buffer&nbsp;: Sample <br />
| Example<br />
|-<br />
| Plasmid, genomic DNA (&gt;2 kb) <br />
| 2&nbsp;: 1 <br />
| 200 μl&nbsp;: 100 μl<br />
|-<br />
| PCR, cDNA, DNA fragment <br />
| 5&nbsp;: 1 <br />
| 500 μl&nbsp;: 100 μl<br />
|-<br />
| ssDNA (e.g., M13 phage) <br />
| 7&nbsp;: 1 <br />
| 700 μl&nbsp;: 100 μl<br />
|}<br />
<br />
#Transfer mixture to a provided Zymo-Spin™ Column1 in a Collection Tube.<br> <br />
#Centrifuge at ≥10,000 x g for 30 seconds. Discard the flow-through.<br> <br />
#Add 200 μl Wash Buffer to the column. Centrifuge at ≥10,000 x g for 30 seconds. Repeat wash step.<br> <br />
#Add ≥6 μl water2,3 directly to the column matrix. Transfer the column to a 1.5 ml microcentrifuge tube and centrifuge at ≥10,000 x g for 30 seconds to elute the DNA.<br>Ultra-pure DNA in water is now ready for use.<br><br />
<br />
<br> <br />
<br />
''' QIAquick purification Kit <br> '''<br />
<br />
[http://www1.qiagen.com/literature/render.aspx?id=103715 Handbook] <br />
<br />
Procedure<br> 1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene. For example, add 500 μl of Buffer PB to 100 μl PCR sample (not including oil).<br> 2. If pH indicator I has beein added to Buffer PB, check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.<br> 3. Place a QIAquick spin column in a provided 2 ml collection tube. <br>4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s. '''We changed it to 3 min @ 6000rpm&nbsp;! '''<br>5. Discard flow-through. Place the QIAquick column back into the same tube. Collection tubes are re-used to reduce plastic waste.<br> 6. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.<br> 7. Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 min.'''repeat!'''<br> IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.<br> 8. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.<br> 9. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.<br> IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 μl from 50 μl elution buffer volume, and 28 μl from 30 μl elution buffer. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.<br> 10. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.<br><br />
<center>''' Gel samples<br> '''</center><br />
<br />
''' ZYMO RESEARCH Gel DNA Recovery Kit '''<br />
[http://www.acgtinc.com/PDF_files/Sample%20Prepation_ACGT/Zymoclean%20Gel%20DNA%20Recovery%20Kit_Zymo%20Research.pdf Product informartion]<br />
<br />
'''Protocol'''<br><br />
<br />
#Excise the DNA fragment1 from the agarose gel using a razor blade or scalpel and transfer it to a 1.5 ml microcentrifuge tube.<br />
#Add 3 volumes of ADB to each volume of agarose excised from the gel (e.g. for 100 μl (mg) of agarose gel slice add 300 μl of ADB).<br />
#Incubate at 37-55 °C for 5-10 minutes until the gel slice is completely dissolved2. For DNA fragments &gt;8 kb, following the incubation step, add one additional volume (equal to that of the gel slice) of water to the mixture for better DNA recovery (e.g. 100 μl agarose, 300 μl ADB and 100 μl water).<br />
#Transfer the melted agarose solution to a Zymo-SpinTM I Column in a Collection Tube.<br />
#Centrifuge at ≥10,000 x g for 30-60 seconds. Discard the flow-through.<br />
#Add 200 μl of Wash Buffer to the column and centrifuge at ≥10,000 x g for 30 seconds. Discard the flow-through. Repeat the wash step.<br />
#Add ≥6 μl of water3,4 directly to the column matrix. Place column into a 1.5 ml tube and centrifuge ≥10,000 x g for 30-60 seconds to elute DNA.<br>Ultra-pure DNA in water is now ready for use.<br><br />
<br />
<center>''' Miniprep '''</center><br />
<br />
'''Protocol:'''<br />
<br />
# Add 600 μl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube.<br />
# Add 100 μl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.<br />
# Add 350 μl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.<br />
# Centrifuge at 11,000 – 16,000 x g for 2-4 minutes.<br />
# Transfer the supernatant (~900 μl) into the provided Zymo-Spin™ IIN column. Avoid disturbing the cell debris pellet.<br />
# Place the column into a Collection Tube and centrifuge for 15 seconds.<br />
# Discard the flow-through and place the column back into the same Collection Tube.<br />
# Add 200 μl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube.<br />
# Add 400 μl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds.<br />
# Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 μl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature.<br />
# Centrifuge for 15 seconds to elute the plasmid DNA.<br />
<br />
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*Digestion<br />
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<center>''' Restriction Digest '''</center><br />
<br />
{| width="60%" cellspacing="1" cellpadding="1" border="1"<br />
|-<br />
| Enzyme<br> <br />
| 10 units is sufficient, generally 1µl is used<br />
|-<br />
| DNA <br />
| 1 µg<br />
|-<br />
| 10X NEBuffer<br> <br />
| 5 µl (1X)<br />
|-<br />
| BSA <br />
| Add to a final concentration of 100 µg/ml (1X) if necessary<br />
|-<br />
| Total Reaction Volume <br />
| 50 µl<br />
|-<br />
| Incubation Time <br />
| 1 - 1.5 hour<br><br />
|-<br />
| Incubation Temperature Enzyme dependent <br />
| <br />
XbaI, SpeI, PstI, SpeI&nbsp;: 37 °C <br />
<br />
|}<br />
<br />
[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp activity of restriction enzymes in NEB buffers] <br> <br />
<br />
''' Biobrick standard <br> '''<br />
<br />
[http://openwetware.org/wiki/Restriction_digest Protocols for IGEM standard digestion] <br />
<br><br />
<center>'''Dephosphorylation'''</center><br />
using Antarctic Phosphatase<br />
#Add 1/10 volume of 10X Antarctic Phosphatase Reaction Buffer to 1-5 µg of DNA cut with any restriction endonuclease in any buffer.<br />
#Add 1 µl of Antarctic Phosphatase (5 units) and mix.<br />
#Incubate for 15 minutes at 37°C for 5´ extensions or blunt-ends, 60 minutes for 3´ extensions.<br />
#Heat inactivate (or as required to inactivate the restriction enzyme) for 5 minutes at 65°C.<br />
#Proceed with ligation.<br />
[http://www.neb.com/nebecomm/products/protocol76.asp from NEB]<br />
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*Ligation<br />
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''' Using T4 Ligase, New England Labs '''<br />
*1 µl T4 Ligase <span style="color: rgb(255, 102, 0);">(10.000 U)</span> <br />
*50 ng plasmid <br />
*3x mol(plasmid) insert <br />
*2 µl T4 Ligase 10x buffer <br />
*add H<sub>2</sub>O to reach final volume of 20 µl<br><br />
<br />
*incubation at 22°C for 1 h <br />
*storing at 16 °C for 40 min<br><br />
<br />
<br />
<u>'''Biobrick Standard'''</u><br> <br />
<br />
[http://parts2.mit.edu/wiki/index.php/Standard_Assembly Standard BioBrick assembly]<br> <br />
<br />
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*Transformation<br />
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<br />
'''At Woehlke's S1-Lab !!!'''<br><br />
<br />
#Thaw competent cells on Ice<br />
#Add DNA, pipette gently to mix<br />
#Let sit for 30 minutes on ice<br />
#Incubate cells for 45 seconds at 42°C<br />
#Incubate cells on ice for 2 min<br />
#Add 1 ml LB0<br />
#Incubate for 1 hour at 37oC on shaker<br />
#Spread 100-300 μl onto a plate made with appropriate antibiotic.<br />
#Grow overnight at 37 °C.<br />
#Save the rest of the transformants in liquid culture at 4 °C<br />
<br />
modified from [http://openwetware.org/wiki/Transforming_chemically_competent_cells open wetware]<br />
<br />
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*Gel electrophoresis<br />
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<center>'''Agarose Gels '''</center><br />
usual volume needed: 80 ml<br />
[http://www.promega.com/enotes/faqspeak/fq0065.htm Optimum resolution according to NEB]<br><br />
[http://www.cwcboe.org/19992051412432550/lib/19992051412432550/Molecular%20Biology/Gel%20electrophoresis/Lab_manual_8_gel_elect.pdf further information on optimizing gel electrophoresis, e.g. recommanded voltage per cm2 gel]<br />
<br />
'''stain'''<br />
<br />
*SybrGold ([http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Nucleic-Acid-Detection-and-Genomics-Technology/Nucleic-Acid-Detection-and-Quantitation-in-Electrophoretic-Gels-and-Capillaries.html invitrogen])<br />
**Cover Gel with 1x TAE<br />
**Add SybrGold to a 1:10000 dilution<br />
**cover with aluminium foil (light sensitive)<br />
**shake&incubate 20 min (for 2% Agarose Gels at least 45 min!)<br />
<br><br />
*SybrSafe<br />
**used just like SybrGold<br />
<br />
''' standards<br> '''<br />
<br />
<br />
<br />
<br> <br />
<br />
{| width="600" height="386" cellspacing="1" cellpadding="1" border="1" align="center" style=""<br />
|-<br />
| [[Image:TUM2010_Dna lmw.gif]] <br> <br />
| [[Image:TUM2010_N3232_fig1_v1_000034.gif]]<br> <br />
| [[Image:TUM2010_N3200_fig1_v1_000036.gif]]<br><br />
|-<br />
| low molecular weight (NEB)<br> <br />
| 1 kb standard (NEB)<br> <br />
| 2-log standard (NEB)<br><br />
|}<br />
<br />
<br><br />
<center>'''Polyacrylamide Gels'''</center><br />
'''Preparation of Gels'''<br />
<br />
Recipe for denaturing gels: <br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| Gel type<br />
| 1 big gel <br />
| 2 big gels<br />
| 1 small gel<br />
| 2 small gels<br />
|-<br />
| Urea<br />
| 28.8 g <br />
| 57.6 g<br />
| x <br />
| x<br />
|-<br />
| Acrylamide 40%<br />
| 22.5 ml<br />
| 45 ml<br />
| x <br />
| x<br />
|-<br />
| Buffer 10x<br />
| 6 ml<br />
| 12 ml<br />
| x<br />
| x<br />
|-<br />
| End volume (reach by adding water)<br />
| 60 ml<br />
| 120 ml<br />
| x<br />
| x<br />
|-<br />
| APS<br />
| 600 µl<br />
| 1200 µl<br />
| x<br />
| x<br />
|-<br />
| TEMED<br />
| 60 µl<br />
| 120 µl<br />
| x<br />
| x<br />
|-<br />
|}<br />
<br><br />
*Dissolve Urea in Acrylamide-buffer mixture (use Ultrasound bath), this may take more than an hour!<br />
*Tighten the Gel chamber<br />
*add water to desired end volume <br />
*Add APS, then TEMED, mix<br />
*Pipette mixture into gel chamber<br />
*Add desired comb<br />
*let gel polymerize overnight; add buffer in the evening<br />
<br />
'''Running of Gels'''<br />
<br />
mix samples 1:1 with formamide loading dye (stored @ -20°C)<br />
carefully remove comb<br />
blow air into pockets with a 50 µl syringe<br />
fill samples into pockets<br />
run the gel <br />
(usually about 200 V)<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==''In vivo'' Measurement==<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
===Bacterial Cell Growth===<br />
Bacteria from over night cultures were diluted 1:50 into 20 ml culture in LBamp and incubated at 37 °C. Upon OD<sub>600</sub> of 0.7-0.8 the cultures were induced with 0.4% Arabinose and 0.4% Arabinose + 1mM IPTG, respectively. Subsequently Cultures were incubated at 25°C for at least 12 h.<br />
<br />
===Fluorescence Measurement===<br />
Cell samples for the fluorescence measurement were diluted to OD<sub>600</sub>=0.03 and analyzed in a JASCO fluorimeter. eGFP excitation wavelength was set to 501 nm and mCherry fluorescence was measured with an excitation at 587 nm. Standard parameters for the fluorimeter included scanning speed of 100 nm/ min and data points every 0.2 nm as well as medium detector sensivity. The cuvette holder was temperated to 25 °C.<br />
The resulting spectra were corrected for instrumental wavelength dependencies and quantum yield of the fluorescent proteins. A pure LBamp spectrum was subtracted and the corrected spectra were normalized using eGFP fluorescence as reference.<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==''In vitro'' Translation==<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
The [http://www.promega.com/catalog/catalogproducts.aspx?categoryname=productleaf_335&ckt=1 Promega Kit] is used according to the provided protocols. Further Information about this Kit can be found in the [http://partsregistry.org/Cell-free_chassis/Commercial_E._coli_S30 Parts Registry].<br />
<br />
Fluorescence kinetics are recorded for at least 3 hours, settings are applied as in the in vitro measurement. <br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==''In vitro'' Transcription==<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
===Buffers===<br />
Three different buffers were used for ''in vitro'' transcription experiments:<br />
* For Epicentre E. coli RNA Polymerase the recommended buffer was used<br />
* For T7 RNA Polymerase experiments two buffers were tested: <br />
**T7 RPO buffer as recommended and used by members of the Simmel group <br />
**T7 RPO "paper buffer", as used in the paper of XXX<br />
Of all buffers 2x concentrated stocks were prepared. Malachite green was usually added to the buffer stocks.<br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| <br />
| E. coli RPO buffer <br />
| T7 RPO buffer<br />
| T7 RPO buffer "paper"<br />
|-<br />
| Tris<br />
| 40 mM <br />
| 40 mM<br />
| 40 mM<br />
|-<br />
| pH<br />
| 7.5<br />
| 7.1<br />
| 7.9<br />
|-<br />
| MgCl2<br />
| 10 mM<br />
| 40 mM<br />
| 6 mM<br />
|-<br />
| KCl<br />
| 150 mM<br />
| /<br />
| 100 mM<br />
|-<br />
| Triton X-100<br />
| 0.01%<br />
| /<br />
| /<br />
|-<br />
|}<br />
===Sample Preparation===<br />
Different concentrations were tested for malachite green and DNA templates. Components of a standard experiment are listed in the table below. <br />
<br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| <br />
| E. coli RPO buffer <br />
| T7 RPO buffer<br />
| T7 RPO buffer "paper"<br />
|-<br />
| buffer<br />
| 1x<br />
| 1x<br />
| 1x<br />
|-<br />
| DTT <br />
| 10 mM<br />
| 10 mM<br />
| 10 mM<br />
|-<br />
| Malachite green<br />
| 5-10 µM<br />
| 5-10 µM<br />
| 5-10 µM<br />
|-<br />
| NTP's<br />
| 1 mM<br />
| 4 mM<br />
| 0.8 mM<br />
|-<br />
| RPO<br />
| 2 U<br />
| 125 U<br />
| 125 U<br />
|-<br />
|}<br />
In each run up to 4 samples are measured simultaneously. Components that are the same in each of the 4 samples (buffer, DTT, NTPs, RPO, Water) are prepared as a 4.1x MasterMix in a loBind tube and split to the 4 cuvettes. Final Volume of each sample is 100 µl.<br />
===Cary Eclipse===<br />
For fluorescence measurements, a Cary Eclipse Spectrofluorimeter with a Multicellholder (4 cells) is used. Kinetics are recorded at 37° C. Excitation wavelength is 630 nm, emission is followed at 650 nm and 655 nm. After each kinetics measurement, spectra are to be recorded. <br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
=References=<br />
<html><a name="ref1"></a></html>[1] http://www.promega.com/catalog/catalogproducts.aspx?categoryname=productleaf_335&ckt=1<br />
<html><a name="ref2"></a></html>[2] Zubay, G. (1980) Meth. Enzymol. 65, 856–77, Zubay, G. (1973) Ann. Rev. Genet. 7, 267–87.<br />
<br />
<!-- ############## WIKI-PAGE STOPS HERE ############## --><br />
{{:Team:TU_Munich/Templates/End}}<br />
<br />
<includeonly><br />
= Experiments =<br />
We designed several experiments to test our switches, all of them based on fluorescence measurements. We designed experiment setting for measurements ''in vivo'' as well as ''in vitro''. Our ''in vitro'' measurements relied on two different experiment set-ups. While the first was based on a commercial ''E. coli''-lysate, the latter was reporting on a transcriptional level only, eliminating most of the possible side-effects one could expect in the complex behaviour of a living cell or cell-lysate. [[Experiments_main|Read more]]<br />
<br />
== The Experiments ==<br />
===Fluorescent proteins as reporter===<br />
<div align="justify"><br />
Our initial idea to prove our concept of antitermination was to use flourescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks ''in vivo'' as well as ''in vitro'', the latter using a translation kit based on <i>E. coli</i> lysate. <br>We decided to use the flourescent proteins GFP and RFP, as their spectra do not overlap and we would not measure any FRET. We would use GFP fluorescence as internal control and RFP fluorescence as signal to detect termination/antitermination by our switch we cloned in between the coding sequences of the proteins. Both protein sequences are under the control of one (L-arabinose induced) promoter.<br />
[[Image:TUM2010_gfprfp_schalter_klein.gif|center|our idea]]<br />
<br><br />
When measuring the termination of our BioBricks and the antitermination by their corresponding signal-RNA, we should be able to observe an increasing RFP emission compared to the GFP emission upon induced signal-RNA production in the cells/in the kit:<br><br />
[[Image:TUM2010_Expected_emission_spextra.png|center|our idea]]<br />
Wiith these measurements, it should also be possible to observe differences in efficiency of termination as well as antitermination between our designed switches. <br><br><br />
<br />
===Measurements with the malachite green aptamer as reporter===<br />
A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramaticly (about 3000 times) upon binding of a specific RNA-aptamer.<br><br>#<br />
[[Image:TUM2010_Malachitgruen-2.png|500px|center|our idea]]<br />
---concept to be desribed, as well as literature---<br />
<ref>refs</ref><br />
<br><br><br />
To study the switches on the transcriptional level gives the advantage, that we would have less interferences and possible artefacts. Also, we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems.<br><br />
<br />
[[Image:TUM2010_Malachit_emission.png|200px|thumb|left|Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA]]We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.<br>Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
<br />
= Results =<br />
We ...blablabla<br />
[[Resultss_main|Read more]]<br />
==Flourescent proteins==<br />
Unfortunatly, we had to change the reporter construct two times during our experiments as several problems occured in our measurements:<br><br><br />
<br />
===First Try: based on the measurement plasmid pSB1A10===<br />
At the beginning, we decided to use the reporter plasmid [http://partsregistry.org/Part:pSB1A10 pSB1A10] from the registry. It consists of the fluorescent proteins eGFP and mRFP1. Each sequence includes a ribosome binding site and a stop-codon; the two genes are divided by a cloning side including the BioBrick cleavage sites.[[Image:ScreeningPlasmid1.0.PNG|300px|thumb|right|pSB1A10]]<br> In front of the eGFP sequence, the plasmid includes an arabinose-inducable promoter. The plasmid also contains an ampicilline resistence.<br><br />
We cloned our switches into the cloning site of the measurement plasmid and used an empty cloning site as control; our signal-RNAs we cloned into the [http://partsregistry.org/Part:pSB1K3 pSB1K3] vector, together with the BioBricks R0011 (Lac promoter) and B0014 (double terminator of transcription). Afterwards, we cut pSB1K3 with Aat2 and Pst1 and pSB1A10 with Nsi1 and Aat2 and ligated those fragments of each plasmid that contained our Bricks to get a Monsterplasmid.<br>We had to do so, as both plasmids contain the same ori mechanism. In addition, having both the switch and the signal RNA transcribed from the same plasmid gives us a high local concentration of the signal, once its transcription is induced.<br><br />
[[Image:TUM2010_Messplasmid_nr1.png|left|the measurement plasmid]]<br />
<br />
We transformed BL21(DE3) cells with the plasmid. We set up cultures, induced the arabinose promoter and measured the GFP and mRFP1 excitation/emission spectra within time.<br><br><br />
Unfortunatly, we were ot able to detect any RFP signal, not even in the positive control with no switch in between the GFP/RFP sequences.<br><br><br />
From these experiments, we concluded, that the mRNA of the RFP variant used was instable and rapidly degraded by RNases, so the RFP was not synthesized in the cells. This was also the conclusion from XXX...<br />
<br><br><br />
As solution to this, we decided to design a measurement plasmid ourselves:<br><br />
<hr width="300"><br />
<br><br />
<br />
===Second Try: A measurement plasmid of our own design===<br />
To design our own plasmid to overcome the problems that occurred in our first try gave us tghe possibility to overcome several other problems:<br><br />
#<br />
#<br />
#<br />
[[Image:TUM2010_Construct_no2.png|400px|our construct|center]]<br />
<br />
===Third Try: One promoter for each protein===<br />
We decided to use the measuremnt plasmid we developed in our second try but to clone another L-arabinose induced promoter into the plasmid, in front of our switch followed by the mCherry sequence.<br><br />
<br>[[Image:TUM2010_Construct_no3.png|left|400px|Construct #3]]In this way, we still can use GFP fluorescence as internal control, because both protein transcription is under the control of a promoter of identical design.<br />
Though we are still not able to tell exactly why our previous measurements did not work, but with this construct we measured the first time fluorescence of the mCherry protein in our positive control.<br />
<br><br><br><br><br />
<br />
On this page you can find our protocols for standard molecular biology procedure as well as the full notebook containing lab progress.<br />
<br />
<!-- ############## scheiß teil, wieso ist alles zentriert? ############## --><br />
=Protocols=<br />
==Gels==<br />
Agarose Gels<br />
*1-3 % agarose gels were used<br />
* TAE buffer<br />
** 0.4 M Tris<br />
** 0.01 M EDTA <br />
** 0.01 M acetic acid<br />
** pH=8.0<br />
;Stain<br />
*SybrGold stain<br />
**Cover Gel with 1x TAE<br />
**Add SybrGold to a 1:10000 dilution<br />
**cover with aluminium foil (light sensitive)<br />
**shake&incubate 20 min (for 2% Agarose Gels at least 45 min!)<br />
<br><br />
*SybrSafe<br />
**used just like SybrGold<br />
<br><br />
;Molecular weight marker<br />
*all molecular weight marker were purchased from NEB<br />
*in use: <br />
**low molecular weight<br />
**1 kb<br />
**2-log<br />
<br><br />
;Polyacrylamide Gels<br />
*Preparation of denaturing gels<br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| Gel type<br />
| 1 big gel <br />
| 2 big gels<br />
| 1 small gel<br />
| 2 small gels<br />
|-<br />
| Urea<br />
| 28.8 g <br />
| 57.6 g<br />
| x <br />
| x<br />
|-<br />
| Acrylamide 40%<br />
| 22.5 ml<br />
| 45 ml<br />
| x <br />
| x<br />
|-<br />
| Buffer 10x<br />
| 6 ml<br />
| 12 ml<br />
| x<br />
| x<br />
|-<br />
| End volume (reach by adding water)<br />
| 60 ml<br />
| 120 ml<br />
| x<br />
| x<br />
|-<br />
| APS<br />
| 600 µl<br />
| 1200 µl<br />
| x<br />
| x<br />
|-<br />
| TEMED<br />
| 60 µl<br />
| 120 µl<br />
| x<br />
| x<br />
|-<br />
|}<br />
<br><br />
*Dissolve Urea in Acrylamide-buffer mixture (use Ultrasound bath), this may take more than an hour!<br />
*Tighten the Gel chamber<br />
*add water to desired end volume <br />
*Add APS, then TEMED, mix<br />
*Pipette mixture into gel chamber<br />
*Add desired comb<br />
*let gel polymerize overnight; add buffer in the evening<br />
<br />
'''Running of Gels'''<br />
mix samples 1:1 with formamide loading dye (stored @ -20°C)<br />
carefully remove comb<br />
blow air into pockets with a 50 µl syringe<br />
fill samples into pockets<br />
run the gel <br />
(usually about 200 V)<br />
<br />
== PCR ==<br />
<br> <br />
<br />
=== used protocols<br> ===<br />
<br />
'''a) Taq Polymerase''' ''''Hot Start'''' <br />
<br />
'''PCR Pippeting plan: '''<br> <br />
<br />
1 µl template <br> <br />
<br />
1 µl dNTP 10 µM <br> <br />
<br />
1 µl G1004 (Primer) 10 µM<br> <br />
<br />
1 µl G1005 (Primer) 10 µM<br> <br />
<br />
5 µl 10x Taq-buffer&nbsp; (500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM&nbsp;MgCl<sub>2</sub>)&nbsp; <br />
<br />
0,2 µl Taq-Polymerase (add last) 5,000 U/ml<span style="background-color: rgb(255, 0, 0);"><br />
</span> <br />
<br />
<br> <br />
<br />
40.8 µl Water<br> <br />
<br />
Final volume 50µl <br />
<br />
'''<br>''' <br />
<br />
'''Processing: '''( program saved as '''IGEMPCR ''' )'''<br>''' <br />
<br />
*preheating of PCR chamber to 94 °C<br />
<br />
&nbsp;&nbsp; --&gt; insert sample <br />
<br />
*2 min at 94 °C <br />
*loop 35x:<br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp; - 30 sat 94°C (according to IGEM protocols) <br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp; - 30 s at 56 °C <br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp; - 45s at 72°C <br />
<br />
*7 min at 72°C <br />
*stay at 4°C <br><br />
<br />
<br> <br />
<br />
'''b) colony PCR'''<br />
*Colony PCR<br />
**pick colonies and resuspend them in 20 µl LB+Antibiotic (each)<br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified), store remaining 18 µl for overnight cultures<br />
**afterwards, mix 15 µl of each PCR product with 3 µl GLPn and load to Gel<br />
**make overnight cultures of positive clones by adding the remaining 18 µl to 5 ml LB+AB<br />
<br />
'''program:colonypcr '''' <br />
<br />
*preheating of PCR chamber to 94 °C<br />
<br />
&nbsp;&nbsp; --&gt; insert sample <br />
<br />
*5 min 30 sec at 94 °C <br />
*loop 35x:<br />
**30 sat 94°C (according to IGEM protocols) <br />
**30 s at 58 °C <br />
**60s at 72°C <br />
*7 min at 72°C <br />
*stay at 4°C <br><br />
<br />
<br><br />
<br />
== DNA Purification<br> ==<br />
<br />
=== PCR samples ===<br />
<br />
'''ZYMO RESEARCH DNA Clean&amp;Concentration Kit'''<br />
<br />
[http://www.zymoresearch.com/zrc/pdf/D4003i.pdf Protocol and Information]<br> <br />
<br />
#In a 1.5 ml microcentrifuge tube, add 2-7 volumes of DNA Binding Buffer to each volume of DNA sample (see table below). Mix briefly by vortexing.<br><br><br />
<br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| Application <br />
| DNA Binding Buffer&nbsp;: Sample <br />
| Example<br />
|-<br />
| Plasmid, genomic DNA (&gt;2 kb) <br />
| 2&nbsp;: 1 <br />
| 200 µl&nbsp;: 100 µl<br />
|-<br />
| PCR, cDNA, DNA fragment <br />
| 5&nbsp;: 1 <br />
| 500 µl&nbsp;: 100 µl<br />
|-<br />
| ssDNA (e.g., M13 phage) <br />
| 7&nbsp;: 1 <br />
| 700 µl&nbsp;: 100 µl<br />
|}<br />
<br />
#Transfer mixture to a provided Zymo-Spin™ Column1 in a Collection Tube.<br> <br />
#Centrifuge at =10,000 x g for 30 seconds. Discard the flow-through.<br> <br />
#Add 200 µl Wash Buffer to the column. Centrifuge at =10,000 x g for 30 seconds. Repeat wash step.<br> <br />
#Add =6 µl water2,3 directly to the column matrix. Transfer the column to a 1.5 ml microcentrifuge tube and centrifuge at =10,000 x g for 30 seconds to elute the DNA.<br>Ultra-pure DNA in water is now ready for use.<br><br />
<br />
<br> <br />
<br />
'''QIAquick purification Kit''' <br> <br />
<br />
[http://www1.qiagen.com/literature/render.aspx?id=103715 Handbook] <br />
<br />
Procedure<br> 1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene. For example, add 500 µl of Buffer PB to 100 µl PCR sample (not including oil).<br> 2. If pH indicator I has beein added to Buffer PB, check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.<br> 3. Place a QIAquick spin column in a provided 2 ml collection tube. <br>4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s. '''We changed it to 3 min @ 6000rpm&nbsp;! '''<br>5. Discard flow-through. Place the QIAquick column back into the same tube. Collection tubes are re-used to reduce plastic waste.<br> 6. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.<br> 7. Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 min.'''repeat!'''<br> IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.<br> 8. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.<br> 9. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.<br> IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 µl from 50 µl elution buffer volume, and 28 µl from 30 µl elution buffer. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.<br> 10. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.<br><br />
<br />
=== Gel samples<br> ===<br />
<br />
'''ZYMO RESEARCH Gel DNA Recovery Kit'''<br />
<br />
[http://www.acgtinc.com/PDF_files/Sample%20Prepation_ACGT/Zymoclean%20Gel%20DNA%20Recovery%20Kit_Zymo%20Research.pdf Product informartion]<br />
<br />
'''Protocol'''<br><br />
<br />
#Excise the DNA fragment1 from the agarose gel using a razor blade or scalpel and transfer it to a 1.5 ml microcentrifuge tube.<br />
#Add 3 volumes of ADB to each volume of agarose excised from the gel (e.g. for 100 µl (mg) of agarose gel slice add 300 µl of ADB).<br />
#Incubate at 37-55 °C for 5-10 minutes until the gel slice is completely dissolved2. For DNA fragments &gt;8 kb, following the incubation step, add one additional volume (equal to that of the gel slice) of water to the mixture for better DNA recovery (e.g. 100 µl agarose, 300 µl ADB and 100 µl water).<br />
#Transfer the melted agarose solution to a Zymo-SpinTM I Column in a Collection Tube.<br />
#Centrifuge at =10,000 x g for 30-60 seconds. Discard the flow-through.<br />
#Add 200 µl of Wash Buffer to the column and centrifuge at =10,000 x g for 30 seconds. Discard the flow-through. Repeat the wash step.<br />
#Add =6 µl of water3,4 directly to the column matrix. Place column into a 1.5 ml tube and centrifuge =10,000 x g for 30-60 seconds to elute DNA.<br>Ultra-pure DNA in water is now ready for use.<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
*add other kits here...<br />
<br />
<br><br />
== Restriction <br> ==<br />
<br />
{| width="60%" cellspacing="1" cellpadding="1" border="1"<br />
|-<br />
| Enzyme<br> <br />
| 10 units is sufficient, generally 1µl is used<br />
|-<br />
| DNA <br />
| 1 µg<br />
|-<br />
| 10X NEBuffer<br> <br />
| 5 µl (1X)<br />
|-<br />
| BSA <br />
| Add to a final concentration of 100 µg/ml (1X) if necessary<br />
|-<br />
| Total Reaction Volume <br />
| 50 µl<br />
|-<br />
| Incubation Time <br />
| 1 - 1.5 hour<br><br />
|-<br />
| Incubation Temperature Enzyme dependent <br />
| <br />
XbaI, SpeI, PstI, SpeI&nbsp;: 37 °C <br />
<br />
|}<br />
<br />
[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp activity of restriction enzymes in NEB buffers] <br> <br />
<br />
=== Biobrick standard <br> ===<br />
<br />
[http://openwetware.org/wiki/Restriction_digest Protocols for IGEM standard digestion] <br />
<br />
<br><br />
<br />
==Dephosphorylation==<br />
using Antarctic Phosphatase<br />
#Add 1/10 volume of 10X Antarctic Phosphatase Reaction Buffer to 1-5 µg of DNA cut with any restriction endonuclease in any buffer.<br />
#Add 1 µl of Antarctic Phosphatase (5 units) and mix.<br />
#Incubate for 15 minutes at 37°C for 5´ extensions or blunt-ends, 60 minutes for 3´ extensions.<br />
#Heat inactivate (or as required to inactivate the restriction enzyme) for 5 minutes at 65°C.<br />
#Proceed with ligation.<br />
[http://www.neb.com/nebecomm/products/protocol76.asp from NEB]<br />
<br />
== Ligation<br> ==<br />
<br />
'''Using T4 Ligase, New England Labs''' '''<u></u>'''<br />
<br />
*1 µl T4 Ligase <span style="color: rgb(255, 102, 0);">(10.000 U)</span> <br />
*50 ng plasmid <br />
*3x mol(plasmid) insert <br />
*2 µl T4 Ligase 10x buffer <br />
*add H<sub>2</sub>O to reach final volume of 20 µl<br><br />
<br />
<br> <br />
<br />
*incubation at 22°C for 1 h <br />
*storing at 16 °C for 40 min<br><br />
<br />
<br> <br />
<br />
<u>'''Biobrick Standard'''</u><br> <br />
<br />
[http://parts2.mit.edu/wiki/index.php/Standard_Assembly Standard BioBrick assembly]<br> <br />
<br />
<br> <br />
<br />
<br />
<br />
==Transformation==<br />
'''At Woehlke's S1-Lab !!!'''<br><br />
<br />
#Thaw competent cells on Ice<br />
#Add DNA, pipette gently to mix<br />
#Let sit for 30 minutes on ice<br />
#Incubate cells for 45 seconds at 42°C<br />
#Incubate cells on ice for 2 min<br />
#Add 1 ml LB0<br />
#Incubate for 1 hour at 37oC on shaker<br />
#Spread 100-300 µl onto a plate made with appropriate antibiotic.<br />
#Grow overnight at 37 °C.<br />
#Save the rest of the transformants in liquid culture at 4 °C<br />
<br />
modified from [http://openwetware.org/wiki/Transforming_chemically_competent_cells open wetware]<br />
<br />
==Miniprep==<br />
==Preparation of BioBricks from distribution 2008==<br />
<br />
==Sequencing==<br />
* Monsterplasmids contain GATC-Standard-Primer pBR1 (CGAAAAGTGCCACCTGAC ) directly in front of AATII cleavage site. <br />
* Monsterplasmids contain GATC-Standard-Primer pGFP-FP () approx. 100 bp upstream of Biobrick insert site.<br />
<br> !!! Please always fill in iGEM-Sequencing-YYMMDD (e.g. iGEM-Sequencing-100625 for today´s date) as internal billing number!<br />
<br />
=Notebook=<br />
<br />
Boxes: for example Cloning --> work done in this week<br />
<br />
'''Week 1 (BOX1) (BOX2)'''{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Tag 1 <br><br />
<br />
Tag2<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Week 2==<br />
<br />
<br />
<br />
</includeonly></div>Hartlmuellerhttp://2010.igem.org/Team:TU_Munich/LabTeam:TU Munich/Lab2010-10-28T03:57:57Z<p>Hartlmueller: </p>
<hr />
<div>{{:Team:TU_Munich/Templates/Beginn}}<br />
<!-- Title of this page here--><br />
Lab<br />
{{:Team:TU_Munich/Templates/Middle}}<br />
<!-- ############## WIKI-PAGE STARTS HERE ############## --><br />
=Experiment Design=<br />
In this section we do not only want to present the experiments and results we gained but also to encourage you to evaluate your own switch based on the protocols and general procedure on how to evaluate basic parameters of a switch. In theory, every terminator can be turned into a switch with minor modifications and the right signals which are based on individual applications. While the principle of how to turn a terminator into a switch is explained in detail [https://2010.igem.org/Team:TU_Munich/Project here], experimental setups and protocols are provided in the following. Due to time and equipment limitations we could not perform all the experiments we planned but next to the hope that another iGEM team might proceed with our project we would also like to encourage you to design and test some basic switches on which you can base a complete, tightly regulated network. <br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
The complexity of our experimental setups vary, since we planned to characterize an individual switch with one exemplary signal on all relevant levels: Starting from the most general, complicated but also relevant level, ''in vivo'' measurements we approached to testing different switches on each smaller scale: We developed setups for ''in vitro'' translation which can be done without much effort following the ''in vitro'' measurements and also provide detailed description of ''in vitro'' transcription verification providing an inside to the molecular functionality of our basic idea. We do not see the methods we used here as the gold standard for bioLOGICS evaluation and encourage you to include your own ideas as well as check in our outlook section where we suggest experiments we could not do during the limited iGEM 2010 time. Together with our [https://2010.igem.org/Team:TU_Munich/Parts Biobrick submissions] this year, we offer a complete set for switch evaluation on all cellular levels.<br />
<br><br />
Most measurements are based on fluorescence reporters which provide easy handling, fast output and are well studied. Next to the fluorescent proteins GFP and mCherry we used ''in vivo'', a malachite green binding aptamer serves as a reporter ''in vitro'' providing a reliable fluorescent output upon antitermination. <br />
<br><br />
Most setups up to now were only used to evaluate switches with an default state "off" which are applied for AND/OR devices. In principle the same methods can be used for NOT devices which are based on a switch with an default state "off". Again, time limitation circumvented further tested from our team but we hope that further studies can be done in the future. <br />
<br />
<!-- Our initial idea to prove our concept of antitermination was to use fluorescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks ''in vivo'' as well as ''in vitro'', the latter using a translation kit based on <i>E. coli</i> lysate. Later on, we decided to develop an experiment, that relies only on transcription. In this set-up, we used a fluorescent dye, malachite green, that binds a specific RNA aptamer and thus makes it possible to detect transcription activity. --><br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==''In vivo'' Measurements==<br />
''In vivo'' measurements have the highest complexity compared to any other experimental set-up. Different parameters and circumstances deriving from both the cellular environment as well as technical considerations like scatter have to be taken into account. Nevertheless, the measurements are essential, as our switches should finally work inside cells to fulfill our vision of an intracellular logic network. This year's submitted Biobricks provide you with a basic kit of plasmids which allow a quick beginning of the measurements. <br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
===Design===<br />
For the measurements ''in vivo'' we decided to use an expression cassette consisting of Green Fluorescent Protein (GFP) coding sequence upstream of the switch and another fluorescent protein coding sequence downstream of it. Both protein coding sequence share the same ribosome binding site allowing the usage of the GFP as an internal control in measurements. Since the spectra should not overlap and to avoid FRET as well as an pure overlap of the spectra, we settled on the usage of red fluorescent protein variants, namely mRFP1 in the first try and mCherry in an modified variant of the pSB1A10 vector. <br />
<br><br />
While the GFP fluorescence can be used to normalize the measurements, the RFP fluorescence serves as a reporter to detect and evaluate termination and antitermination. To stimulate the expression of the fluorescent proteins, we took advantage of the pBAD promoter family (sensitive towards arabinose). The signal upon which the antitermination events and therefore switching relies on were under the control of an IPTG inducible promoter. We went with this well-established pair of controllable promoters to deliver an easy setup in the beginning, like described [https://2010.igem.org/Team:TU_Munich/Software here], every sort of input may later be combined with our basic switching units. <br />
<br />
<br><br />
[[Image:invivo3.png|500px|thumb|center|general measurement principle]]<br />
<br><br />
<br />
The GFP internal control carries the advantage that errors in the measurement set can be detected easily. Lack of arabinose or promoter insensitivity can be recognized as well as problems with the fluorescence measurement itself. Plus, it allows normalizing measurements to compare different preparations in relation to each other. <br />
<br><br />
Upon binding of a signal to the terminator switch, termination is circumvented and the reporter protein behind the switch can be translated. In the experimental setup presented here, this will result in an RFP expression, but again, every protein or DNA-encoded element in general may be used as an output. Since the RFP fluorescence spectra do not overlap with GFP it offers an easy possibility to evaluate the effect of signal induction. Next to GFP fluorescence, RFP fluorescence will show up.<br />
<br><br />
[[Image:TUM2010_Expected_emission_spextra.png|375px|thumb|center|schematic estimated fluorescence spectra]]<br />
<br><br />
<br />
===Construction and Cloning===<br />
<br />
In a first try we cloned a measuring construct based on pSB1A10. The resulting plasmid, nicknamed pMonsterplasmid due to its size was tested in the fluorescent measurements described [https://2010.igem.org/Team:TU_Munich/Lab#Switch_evaluation_in_vivo below]. Unfortunately after two months of cloning we had to recognize that the plasmid in use did not work for us (see also [[Team:TU_Munich/Parts#Falsified_Parts|pSB1A10 Falsification]]). <br><br />
So after the first unsuccessful attempts we decided to reclone the system, substituting RFP to mCherry, a dsRED derivative with a spectrum in the far red, and adding arabinose inducible promoters in front of both fluorescent proteins to guarantee stable and comparable expression of both proteins<br />
{|<br />
|[[Image:TUM2010 Plasmid1flo.png|350px|right|first measuring construct]]<br />
|[[Image:TUM2010 Plasmid2flo.png|350px|left|BBa_K494006 cloned in BBa_K494001]]<br />
|}<br />
<br><br />
To control the expression of the switch, the particular DNA sequence itself is under the control of an IPTG dependent promoter. In the future we want our networks to be able to respond to a variety of external signals like small metabolites, ions or whatever can be found in the parts registry. For basic switch evaluation, an established and well-working system like the ''lac''-operon was chosen to avoid side-effects of less well-characterized promoters.<br />
<br><br />
<br />
===Measurements based on submitted Biobricks===<br />
The Biobricks BBa_K494001-BBa_K494006 are constructed for easy design of a switch-evaluation system. Detailed information can be found [https://2010.igem.org/Team:TU_Munich/Parts#Plasmids here].<br />
<br />
===Switch evaluation ''in vivo''===<br />
To evaluate the switching efficiency, output with and without signal needs to be monitored. In this case, GFP fluorescence (internal control) will always appear upon arabinose induction, while RFP/mCherry fluorescence is only present upon binding of a signal and occurring antitermination. <br><br><br />
Upon induction with arabinose a rise of GFP expression can be seen. To monitor changes in gene expression we used a fluorimeter and measured fluorescence of whole living cells. While this approach provides easy handling and monitoring, too much scattering has to be carefully avoided: the cell density should not exceed an OD<sub>600</sub> of 0.05. RFP/mCherry emission should be visible only in case of a working switch or inefficient termination.<br />
<br><br><br />
For evaluation of the measuring plasmid itself we incorporated a positive control in every measurement. A random sequence in between GFP and RFP/mCherry was chosen in a corresponding length instead of a terminator. An increase in both GFP and mCherry was detectable in comparable amounts after quantum yield correction, showing the measuring plasmid to beworking nicely. While the positive control may be the same for all evaluated devices, the negative control has to be specific for every switch and terminator, respectively. <br />
<br><br />
[[image:TUM2010_bacteriaAll.JPG|thumb|375px|center|Bacterial cultures after incubation of 16h]]<br />
<br><br />
The negative control contained the evaluated switch without any possibility for induction of the corresponding signal. Thereby the switch's function is limited to termination, leading to no detectable RFP/mCherry fluorescence. By definition every switch type has to be tested using a negative control without a corresponding signal, since termination efficiency may vary depending on the terminator itself, cell strain and general growth conditions. We recommend to chose your terminator of choice and evaluate it using the provided plasmids. <br><br />
In our experimental part we evaluated terminators based on the regulatory unit of the tryptophan (Trp-Term) and histidine (His-Term) operons. Those synthetic operons are regulated based on the principle of attenuation, a terminator in front of genes involved in amino acid biosynthesis avoids transcription until environmental stimulis suggest a lack of those amino acids. Since both sequences are known to be regulated by changes in secondary structure, those two attenuators became the basis for our designed switches. <br><br />
The terminators we tested can be found in the Parts Registry. With the construction of the backbone [http://partsregistry.org/Part:BBa_K494001 BBa_K494001], potential switches and signals can be easily subcloned in two steps and tested. [http://partsregistry.org/Part:BBa_K494002 BBa_K494002] was constructed as a positive control, producing maximal mCherry fluorescence which may be used to characterize terminator and switch efficiency. [http://partsregistry.org/Part:BBa_K494003 BBa_K494003] and [http://partsregistry.org/Part:BBa_K494004 BBa_K494004] carry the His-Terminator with and without the corresponding signal, [http://partsregistry.org/Part:BBa_K494005 BBa_K494005] and [http://partsregistry.org/Part:BBa_K494006 BBa_K494006] being the same for Trp-Terminator. <br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==''In vitro'' Translation==<br />
To go more into detail, the next complexity level is to study the effect of switches on a translational level. ''In vitro'' measurements with ''E. coli'' lysate make the fluorescence signals independent of cell growth and physical or biological factors like cell density or growth stadium.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
===Design===<br />
Since the same constructs can be used both for ''in vivo'' and ''in vitro'' translation, no additional cloning effort is needed. This implements, that the Biobricks we [https://2010.igem.org/Team:TU_Munich/Plasmids provided this year], can again be used as the groundwork for constructing vectors for measurements. <br><br />
Reporter proteins GFP and mCherry are well expressed ''in vitro'', the limiting factors are mostly the capacity of a kit versus the maturation time of fluorescent proteins. Since we used a fast-folding GFP variant and mCherry, which was characterized with a maturation time of 15 minutes by Tsien and Coworkers, the problem should be minimized. Alternative tags may be considered, a major advantage of measuring translation ''in vitro'' may be the use of non-cell permeable tags for switch evaluation.<br />
<br />
===Measurements===<br />
We used the cell-free ''E. coli'' S30 extract system for circular DNA provided by Promega<sup>[[Team:TU_Munich/Lab#ref1|&#91;1&#93;]]</sup>, which is prepared by modifications of the Method Zubay ''et al.''<sup>[[Team:TU_Munich/Lab#ref2|&#91;2&#93;]]</sup>. The characterization of the kit can be obtained from the [http://partsregistry.org/Cell-free_chassis/Commercial_E._coli_S30 Parts Registry]. <br><br />
Experiments were performed at 37°C with an amount of approximately 1 µg plasmid in a reaction volume of 50 µL. Fluorescence was followed over time in a jasco fluorolog with wavelength corresponding to those used ''in vivo''. <br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==''In vitro'' Transcription==<br />
To monitor transcription termination and antitermination on a the molecular level, ''in vitro'' transcription of individual switches and their response to signals offer an elegant way for fast and easy prove of principle. Most side effects occurring in a complex environment given in a cell or a cell lysate do not arise here. Another major advantage of ''in vitro'' transcription experiments is the possibility to test many signals for one switch to optimize antitermination efficiency and binding specificity without much cloning work. Data gained by ''in vitro'' transcription experiments can be used to improve switches and signals for ''in vivo'' usage. <br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Since we are working on a totally new principle of transcriptional control, we used this approach beside the above mentioned advantages for easy variation of different variables like the length of the core unit and the switch to signal ratio. <br><br />
To study the switches on a transcriptional level offers the advantage, to reduce interference and possible artifacts to a minimum. Since we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems, ''in vitro'' transcription was also used as a minumum system from which more complexity can derive.<br />
<br><br />
Working with in vitro systems also has the advantage that an input is not needed anymore and the output can also be generated easily. We used '''two readouts''' with '''two different transcription systems''' to check and investigate our devices: First, we used an [https://2010.igem.org/Team:TU_Munich/Parts malachitegreen-binding aptamer] for an fluorescence output and second, we simply put our reaction educts on an denaturing acrylamide-gel to check for RNA varying in length. As for two different transcription systems we used on the one hand ''E. coli''-RNA Polymerase (RPO) based transcription since the aim is to apply the so gained results ''in vivo'' and on the other hand T7 based transcription which is well established through literature and delivers good RNA yields.<br />
<br><br />
=== T7 RNA polymerase ===<br />
<br />
<div align="justify"><br />
<br />
The T7 RNA polymerase is known for satisfying RNA yields together with easy handling. In our approach we had PCR amplified, double stranded switches with an malachitegreen binding aptamer following after a T7 terminator which was constructed to function as a switch. Different signals were tested varying in length of the specificity site and the triggering unit. <br />
<br> <br />
For in vitro expression the T7 RNA Polymerase requires a double stranded promotor region at the beginning of the DNA template but is otherwise capable of handling single stranded DNA, so a sense strain corresponding to the T7 promoter region was added. Transcription is more effective with double stranded DNA as template. Apart from that, no more requirements are needed in theory which makes the evaluation of many signals especially easy. Since we ordered the signal sequences we tested we chose the cheaper way in the beginning by using single stranded signals with corresponding sense T7 pieces and switched to double stranded constructs after narrowing down the most promising switch/signal pairs. Later on we also used double stranded signals and switches since transcription rates are higher with those. <br />
<br><br />
As a positive control, the malachite green binding aptamer right behind the T7 promoter was used. Transcription proceeds without termination and the maximal fluorescence intensity should be gained. <br />
<br> Transcription termination can also be estimated by measuring just the switch without interfering signals. Since upon transcription of a signal sequence, less RNA Polymerase is available, the transcription rate of the switch and therefore the fluorescence output is reduced by merely adding the signal. Therefore randomly chosen short sequences in the range of the tested signals were added to the negative control. <br />
<br />
</div><br />
=== ''E. coli'' RNA polymerase ===<br />
In comparison to the T7 RNA Polymerase the ''E. coli'' RNA Polymerase requires slightly more sophisticated proceedings when it comes to the design of switches and handling of the enzyme. The biggest in our case was to store it properly since the only -80°C fridge was in another building, so make sure you have a big supply of dry ice ready if you encounter the same problem. <br><br />
E. coli RPO was ordered saturated with σ70-factor.<br />
<br />
=== Denaturing Polyacrylamide gel electrophoresis ===<br />
<br />
<div align="justify"><br />
Polyacrylamide gel electrophoresis (PAGE) was used for evaluation of termination and switching efficiency. Gels containing 15 % acrylamide and 6 M urea were used for separation of terminated and readthrough RNAs. The same constructs as designed for the malachite green binding aptamer were used. <br />
<br><br />
Polyacrylamide gels separate RNA and DNA according to their size in an electric field. Since the negative charge equals the size of nucleotides in the RNA/DNA, the number of base pairs can be compared between two samples often with one base pair resolution. Since RNA forms three-dimensional structures, the samples are preheated and run in 6 or 7 M urea. The polyacrylamide gel is stained in SybrGold afterwards which binds to both single and double stranded DNA and RNA. A Dnase digestion was applied before running the samples to avoid confusion caused by DNA templates.<br />
<br><br />
Denaturing PAGE is a simple yet elegant way to check for transcription efficiency and termination rates. Since it is a very direct way and it provides a simple yet clear readout, we used it as another method beside the more sophisticated malachitegreen binding assay to evaluate and characterize our switch. Equipment for denaturing PAGE can be found in nearly every biochemical lab, so this method also applies for an easy controlexperiment. <br />
</div><br />
<br />
=== Malachite green assay ===<br />
<br />
<div align="justify"><br />
<br />
[[Image:TUM2010_Malachit_emission.png|200px|thumb|left|Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA]]<br />
In this year's DNA submission we contribute the [[Team:TU_Munich/Parts#Malachite_Green_Binding_Aptamer_-_BBa_K494000 | malachite green binding aptamer]] which can be used as a transcription reporter in ''in vitro'' transcription experiments. <br><br />
Malachite green is a dye with a negligible fluorescence in solution but undergoes a dramatic increase about 3000 times if bound by the RNA aptamer making it an exceptional good marker. Since the binding is very specific, transcription in dependence of a signal can be monitored by measuring the fluorescence of malachite-green over time if the aptamer is located behind the switch. Transcription of the aptamer will only take place after anti-termination by a signal. An increase should be visible over time. Other triphenyldyes are also recognized with weaker effects on the fluorescence but may also serve as reporters if the emission or excitation of malachite green does not fit the experimental setup. <br />
<br><br><br><br />
[[Image:TUM2010_Malachitgruen-2.png|600px|center|thumb|Description of the malachit green assay. Antitermination allows the polymerase to produce the malachite green aptamer ]]<br />
Malachite green binding can be used to follow RNA transcription over time, a rise in the fluorescence is then detectable. Fluorescence marker of specific RNA structures are still rare, so the malachite green binding aptamer provides one of the only possibilities to continuously monitor transcription reactions. In comparison to PAGE, kinetics can be taken, while with PAGE only end point estimations can be made. This makes the malachite green binding aptamer a valuable tool to study ''in vitro'' transcription in general and the principles underlying our switch in principle. <br />
<br />
<br />
<!--For the T7-based measurements we ordered single stranded signals for a first attempt and added matching single strands complementary to the T7 promoter region. The switch was amplified using PCR and consisted of the following elements: Primer-binding site - T7 promotor - switch - malachitegreen binding aptamer. Upon binding of a correct signal to the switch, the stem loop dissolves and transcription is possible. <br />
<br />
<br />
<br><br><br />
<br />
OLD: A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramatically (about 3000 times) upon binding of a specific RNA-aptamer. The RNA-aptamer<br />
<br><br><br />
---concept to be described, as well as literature---<br />
<ref>refs</ref><br />
<br><br><br />
<br><br />
<br />
We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.<br />
<br><br />
Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level. --><br />
</div> <br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
=Lab Book=<br />
<br />
==Explanations==<br />
In the following we present an overview regarding our work in the lab. For easier understanding we summarized the work of each week using colored boxes. To get a better overview we used the following color code for the boxes:<br />
{|<br />
|-<br />
| {{:Team:TU_Munich/Templates/RedBox | text=&nbsp;}} The red box represents general cloning steps that were required for our measurements. See the [[Team:TU_Munich/Lab#Molecular_Biology | protocol section]] for further details.<br />
|-<br />
| {{:Team:TU_Munich/Templates/BlueBox | text=&nbsp;}} The blue box indicates <i>in vivo</i> measurements which are described [[Team:TU_Munich/Lab#In vivo Measurements | here]].<br />
|-<br />
| {{:Team:TU_Munich/Templates/GreenBox | text=&nbsp;}} The green box indicates <i>in vitro</i> measurements relying on <i>in vitro</i> transcription and malachite green measurements. Details can be found [[Team:TU_Munich/Lab#In vitro Transcription | here]].<br />
|-<br />
| {{:Team:TU_Munich/Templates/YellowBox | text=&nbsp;}} The yellow box represents measurements done with an <i>in vitro</i> translation kit and is described in more details [[Team:TU_Munich/Lab#In vitro Translation | here]].<br />
|}<br />
<br><br />
To learn more about the work and results of a specific week, just click on the according week number. You will find detailed notes on our daily lab work. We present these notes in an unedited form as a record of our work, for for processed results please check the [[Team:TU_Munich/Project#Results | results section on our project page]].<br />
<br />
==Chronological Lab Book==<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week01{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===08.04.2010===<br />
==Chronological Lab Book==<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week01{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===08.04.2010===<br />
Flo & Philipp<br />
<br />
'''PCR''' <br />
*samples:<br />
** R0011_His<br />
** R0011_Trp<br />
** Control<br />
*protocol: [[Team:TU_Munich/Lab#Molecular_Biology| protocol]]<br />
**templates: purified PCR products from 5.2.2010<br />
**primer G1004/1005<br />
**polymerase: Taq<br />
**programm: igempcr<br />
<br />
<br />
'''Purification of PCR products with QIAquick PCR purification Kit '''<br />
*protocol followed. exceptions: DNA-binding/unbinding with 3min 6000rpm followed by 60sec full speed<br />
<br />
<br />
'''2% Agarose Gel'''<br />
*in 1x TAE.<br />
<br />
===09.04.2010===<br />
Philipp & Flo<br />
<br />
----<br />
'''Gel of PCR products from 08.04.2010'''<br />
*loaded: 10 µL sample+2 µL 6x GLD, 4/2 µL LMW standard<br />
* 110 V, 90 min<br />
*stained with Sybrgold, 20 min, 1:10.000 dilution in TAE<br />
*Standard - Control - R0011_His - R0011_Trp - Standard(=low molecular weight (see [[Team:TU_Munich/Lab#Molecular_Biology Lab_Protocols]]))<br />
[[Image:TUM2010_100409.JPG]]<br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week02{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
<br />
===15.04.2010===<br />
Philipp & Flo<br />
<br><br>[http://web.e14.physik.tu-muenchen.de/igem/index.php/Team:TU_Munich/Lab#Molecular_Biology PCR PCR] of B0014 and R0011<br />
<br />
===16.04.2010===<br />
Philipp & Flo<br><br><br />
<br />
*'''Purification''' of PCR products from [[15.04.2010]] using [[Team:TU_Munich/Lab#Molecular_Biology QIAquick_purification_Kit|QIA kit]] <br><br><br />
<br />
*'''Concentrations''' measured with nanodrop:<br><br><br />
<br />
{| width="200" cellspacing="1" cellpadding="1" border="1" align="center"<br />
|-<br />
| B0014 <br />
| 2.5 ng/µL<br><br />
|-<br />
| R0011<br> <br />
| 27.5 ng/µL<br><br />
|}<br />
<center><br> --&gt; worked for R0011, not for B0014 <br> </center> <br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|PCR]] '''of B0014<br><br />
**Purification with the [[Team:TU_Munich/Lab#Molecular_Biology ZYMO_RESEARCH_DNA_Clean.26Concentration_Kit|Zymo Kit]], Elution in 20 µL H2O<br />
**Concentration measured with nanodrop, 17.5 ng/µL --> worked<br><br><br />
<br />
*'''Digestions''' of:<br><br><br />
<br />
{| width="618" cellspacing="1" cellpadding="1" border="1" align="center" style=""<br />
|-<br />
| '''template'''<br> <br />
| '''restriction enzymes (biobrick assembly)'''<br><br />
|-<br />
| B0014 (from Christoph, verified PCR products, 21 ng/uL)<br> <br />
| EcoRI, PstI<br><br />
|-<br />
| R0011 (from PCR [15042010], 27.5 ng/µL<br> <br />
| SpeI<br><br />
|-<br />
| HisSig (1:100 dilution)<br> <br />
| XbaI<br><br />
|-<br />
| TrpSig (1:100 dilution)<br> <br />
| XbaI<br><br />
|-<br />
| psB1K3 (with RFP insert, from HiWiPhilipp, 81 ng/µL) <br />
| EcoRI, PstI<br />
|}<br />
<br />
<br> <br />
<center>5 µL template used for each setup. [[Team:TU_Munich/Lab#Molecular_Biology Restriction|protocol]] followed</center> <br />
<br> <br />
<br />
*'''Gel''' for purification of the cleaved plasmid <br />
**2% Agarose in 1x TAE <br />
**120 V, 90 min <br />
**[[Team:TU_Munich/Lab#Molecular_Biology stain|stained]] with SybrGold<br />
**digestion, digestion, [[Team:TU_Munich/Lab#Molecular_Biology standards|1 kb ladder]]<br />
***Digestion worked (partly). band at 2000 bp (backbone) cut<br><br><br />
[[Image:TUM2010_100416.png]]<br><br />
<br />
<br><br><br />
*'''Purification of DNA from Gel'''<br />
**using the [[Team:TU_Munich/Lab#Molecular_Biology ZYMO_RESEARCH_DNA_Clean.26Concentration_Kit|Zymo Kit]]<br />
<br />
<br><br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' of HisSig/TrpSig with R0011in 2 reactions<br><br><br />
<br />
{| width="617" cellspacing="1" cellpadding="1" border="1" align="center" style=""<br />
|-<br />
| <br> <br />
| '''used Volume'''<br> <br />
| '''approx. concentration*'''<br><br />
|-<br />
| HisSig<br> <br />
| 6 µL<br> <br />
| 7 ng/µL<br><br />
|-<br />
| TrpSig<br> <br />
| 6 µL<br> <br />
| 5 ng/µL<br><br />
|-<br />
| R0011<br> <br />
| 3 µL<br> <br />
| 6 ng/µL<br><br />
|}<br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
* approximated from the amount used in the digestion before<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week03{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===19.04.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|PCR]] '''of R0011-TrpSig and R0011-HisSig<br><br />
**Purification with the [[Team:TU_Munich/Lab#Molecular_Biology ZYMO_RESEARCH_DNA_Clean.26Concentration_Kit|Zymo Kit]], Elution in 30 uL H2O<br />
**Concentration measured with nanodrop: c(R0011-TrpSig)=20 ng/µL, c(R0011-HisSig)=12.5 ng/µl --> worked<br><br><br />
<br />
*'''Gel''' for analysis of ligation and PCR <br />
**2% Agarose in 1x TAE <br />
**110 V, 90 min <br />
**[[Team:TU_Munich/Lab#Molecular_Biology stain|stained]] with SybrGold 1:10000 20 min<br />
**pure R0011 PCR product used as control<br />
<br>[[Image:TUM2010_GEL_20100419beschriftet.png]] <br><br />
{| width="617" cellspacing="1" cellpadding="1" border="1" align="center" style=""<br />
|-<br />
| '''LMW'''<br> <br />
| 4 µl<br> <br />
|-<br />
| '''R0011-TrpSig'''<br> <br />
| 5 µL<br> <br />
|-<br />
| '''R0011-HisSig'''<br><br />
| 5 µL<br> <br />
|-<br />
| '''R0011'''<br><br />
| 5 µL<br> <br />
|-<br />
<br />
|}<br />
<br />
<br><br />
Samples seem to have run further than the buffer/dye-Front! But: Ligation Products show bands at shorter lengths than R0011 alone --> Ligation didn't work ?!?<br />
<br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' of HisSig/TrpSig with R0011in 2 reactions<br><br><br />
<br />
{| width="617" cellspacing="1" cellpadding="1" border="1" align="center" style=""<br />
|-<br />
| <br> <br />
| '''used Volume'''<br> <br />
| '''concentration'''<br><br />
|-<br />
| pSB1K3<br> <br />
| 5 µL<br> <br />
| 10 ng/µL (nanodrop)<br><br />
|-<br />
| B0014<br> <br />
| 3 µL<br> <br />
| 5 ng/µL approx.*<br><br />
<br />
|}<br />
<br />
<br />
<br />
* approximated from the amount used in the digestion before<br />
===20.04.2010===<br />
*'''Gel''' for analysis of ligation and PCR (repeat of [[19.04.2010|yesterday's gel]])<br />
**2% Agarose in 1x TAE <br />
**130 V, 75 min <br />
**[[Team:TU_Munich/Lab#Molecular_Biology stain|stained]] with SybrGold 1:10000 60 min<br />
**pure R0011 PCR product used as control<br />
**Excision and purification of marked bands at 200 bp using QIA Kit, elution in 30 µl H2O<br><br />
[[Image:TUM2010_Gel100420marked.png ]]<br />
<br />
<br />
<br><br><br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|PCR]] '''of excised and purified bands of R0011-TrpSig and R0011-HisSig<br><br />
**complete samples (30 µl) used as templates<br />
**Purification with the [[Team:TU_Munich/Lab#Molecular_Biology ZYMO_RESEARCH_DNA_Clean.26Concentration_Kit|Zymo Kit]], Elution in 30 uL H2O<br />
**Concentrations of PCR-products: 0.5-1 ng/µl --> Gel excision or PCR didn't work<br />
<br />
<br><br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]] ''' (Woehlke-Lab)<br />
**8 µl of ligation product pSB1K3-B0014 to 50 µl XL-10 competent cells<br />
**200 µl plated on a Kanamycin-containing Plate<br />
**remaining 800 µl stored @4°C in S1-lab<br />
<br />
===21.04.2010===<br />
*'''Gel''' for analysis of ligation and PCR (repeat of yesterday's gel) <br />
**2% Agarose in 1x TAE <br />
**110 V, 90 min <br />
**[[Team:TU_Munich/Lab#Molecular_Biology stain|stained]] with SybrGold 1:10000 80 min <br />
**pure R0011 PCR product used as control <br />
**Excision and purification of marked bands at 200 bp using Zymo 5 Kit, elution in 20 µl H2O<br><br />
<br />
[[Image:TUM2010_100421beschriftet.gif??]] <br />
<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|PCR]] '''of excised and purified bands of R0011-TrpSig and R0011-HisSig<br> <br />
**complete samples (20 µl) used as templates <br />
**Purification with the [[Team:TU_Munich/Lab#Molecular_Biology ZYMO_RESEARCH_DNA_Clean.26Concentration_Kit|Zymo Kit]], Elution in 25 uL H2O <br />
**Concentrations of PCR-products: <br />
*** R0011-TrpSig: 22.5 ng/µl<br />
*** R0011-HisSig: 9.5 ng/µl<br />
--> worked!!!!!<br />
<br><br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]] '''<br />
**7 Colonies picked and resuspended in 20 µl LB+Kana (each)<br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified)<br />
**15 µl of each sample mixed with 3 µl GLPn and loaded to Gel<br />
<br />
<br />
[[Image:TUM2010_100421colony.png]] <br />
<br><br><br />
*Overnight cultures: <br />
**remaining 18 µl of samples 1, 3, 6, and 7 added to 5 ml LB + kanamycin<br />
**37°C on Shaker<br />
<br />
===22.04.2010===<br />
*'''Gel''' for purification of PCR products R0011-TrpSig and R0011-HisSig ([[21.04.2010|yesterday's result]]) <br />
**2% Agarose in 1x TAE <br />
**110 V, 90 min <br />
**[[Team:TU_Munich/Lab#Molecular_Biology stain|stained]] with SybrGold 1:10000 30 min <br />
**pure R0011 PCR product used as control <br />
**Excision and purification of marked bands at 200bp using Zymo 5 Kit, elution in 20 µl H2O<br><br />
<br />
[[Image:TUM2010_100422beschriftet.png]]<br />
<br />
<br><br><br />
*Miniprep<br />
**Result: about 4 µg Plasmid<br />
<br />
===23.04.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Restriction|Digestion]]''' of:<br><br><br />
<br />
{| width="618" cellspacing="1" cellpadding="1" border="1" align="center" style=""<br />
|-<br />
| '''template'''<br> <br />
| '''template volume'''<br> <br />
| '''restriction enzymes'''<br><br />
| '''Buffer'''<br> <br />
|-<br />
| HisSig (1:100 dilution)<br> <br />
| 5 µl<br><br />
| EcoRI, SpeI<br><br />
| NEB4<br><br />
|-<br />
| TrpSig (1:100 dilution)<br> <br />
| 5 µl<br><br />
| EcoRI, SpeI<br><br />
| NEB4<br><br />
|-<br />
| psB1K3-B0014 from Miniprep (No 7, 35 ng/µl) <br />
| 5 µl<br><br />
| EcoRI, XbaI<br />
| NEB4<br><br />
|}<br />
<br />
<br> <br />
Incubated 90 min @ 37°C<br />
<br><br />
<br />
*'''Gel''' for purification of the cleaved plasmid <br />
**2% Agarose in 1x TAE (leftover from yesterday)<br />
**140 V, 90 min <br />
**[[Team:TU_Munich/Lab#Molecular_Biology stain|stained]] with SybrGold 40 min<br />
**4 µl [[Team:TU_Munich/Lab#Molecular_Biology standards|1 kb ladder]], 10 µl purified digestion + 2 µl GLPn, 10 µl purified digestion + 2 µl GLPn<br />
***Digestion worked (partly). band at 2400 bp cut out<br><br><br />
[[Image:TUM2010_100423beschriftet.png]]<br><br />
<br />
<br><br><br />
*'''Purification of DNA from Gel'''<br />
**using the [[Team:TU_Munich/Lab#Molecular_Biology ZYMO_RESEARCH_Gel_DNA_Recovery_Kit|Zymo Kit]]<br />
**elution in 25 µl H2O<br />
* A260/A230 and A260/A280 values were strange (see labbook)<br />
<br><br><br />
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{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week04{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
<br />
===26.04.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of pSB1K3-B0014 with EcorI and XbaI<br />
** 10 µl template (No1, 50 ng/µl)<br />
** 5 µl BSA, 5 µl Buffer NEB#4<br />
** 1 µl EcoRI, 1 µl XbaI<br />
** 28 µl H2O<br />
** 1.5 h @ 37°C<br />
**Purification with Zymo5 Kit, elution in 20 µl H2O<br />
<br><br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' of Signals and PSB1K3-B0014<br />
**3 µl of each sample, end volume 20 µl<br />
<br><br />
*Preparation of Measurement Plasmid from Folder, Transformation<br />
**Plate 1022, Spots 1E, 1G, 2A: pSB1A10 with different Inserts, all inserts are Zinc-finger constructs with about 1.6 kb<br />
<br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]] ''' of XL10 with Ligation Products (8 µl each) and pSB1A10 (2 µl each)<br />
<br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Preparation of BioBricks from distribution 2008|Preparation]]''' of Measurement Plasmid from Folder, Transformation<br />
**Plate 1022, Spots 1E, 1G, 2A: pSB1A10 with different Inserts, all inserts are Zinc-finger constructs with about 1.6 kb<br />
<br><br />
*growing over night cultures of remaining PSB1K3-B0014-transformed cells<br />
**2x 5 ml, 2x 1 ml<br />
<br />
===27.04.2010===<br />
*Plenty of cultures on both HisSig and TrpSig Ligation plates, but nothing on pSB1A10 plates! --> repeat DNA extraction, ask Prof. Simmel for new Distribution<br />
<br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
**7 Colonies picked and resuspended in 20 µl LB+Kana (each)<br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified)<br />
**10 µl of each sample mixed with 2 µl GLPn and loaded to Gel<br />
<br />
[[Image:TUM2010_100427beschriftet.png]] <br />
<br />
Many colonies with pSB1K3-B0014, not one with pSB1K3-Sig-B0014<br />
<br />
*Miniprep of PSB1K3-B0014<br />
**Samples I and II: 5 ml overnight cultures, centrifuged 10 min @ 3200 g, resuspended in 600 µl of the same culture<br />
**Samples III and IV: 600 µl overnight cultures<br />
**all samples mixed with 100 µl lysis buffer, Miniprep with Zyppy kit, each sample eluted in 50 µl H2O <br />
**Concentration measured (Nanodrop, LP=1mm, Factor 10, 4 µl sample)<br />
***cI=61.5 ng/µl<br />
***cII=33.5 ng/µl<br />
***cIII=103 ng/µl<br />
***cIV=108 ng/µl<br />
<br><br />
--> Better results for 600 µl cultures without centrifuging!!!<br />
<br><br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Preparation of BioBricks from distribution 2008|Preparation]]''' of Measurement Plasmid from Folder, Transformation<br />
**Plate 1022, Spots 1E, 1F, 1G, 1H, 2A: pSB1A10 with different Inserts, all inserts are Zinc-finger constructs with about 1.6 kb<br />
<br><br />
<br />
===28.04.2010===<br />
*No colonies on plates from Yesterday's transformations, but on the older plates (from monday) some colonies appeared<br />
**7 Colonies picked and resuspended in 20 µl LB0 (each)<br />
**1&2 from plate "1E", 3&4 from plate "1G", 5,6&7 from plate "2A", 8 LB0<br />
<br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified)<br />
**15 µl of each sample mixed with 3 µl GLPn and loaded to Gel<br />
**1% Agarose in 1xTAE, 95 V, after 50 minutes changed to 110 V<br />
<br />
[[Image:TUM2010_100428beschriftet.png]] <br />
<br />
<br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of pSB1K3-B0014 with EcorI and XbaI<br />
** 10 µl template (No1, 50 ng/µl)<br />
** 5 µl BSA, 5 µl Buffer NEB#4<br />
** 1 µl EcoRI, 1 µl XbaI<br />
** 28 µl H2O<br />
** 1.5 h @ 37°C<br />
** heat inactivation 5min @60°C <br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Dephosphorylation|Dephosphorylation]]''' of restricted vector<br />
**Purification with Zymo5 Kit, elution in 20 µl H2O<br />
**loaded on gel (with 4 µl GLPn) (Gel shown above)<br />
*Gel excision with Zymo Kit<br />
**<br />
**<br />
<br><br />
<br />
<br />
===29.04.2010===<br />
<br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of pSB1K3-B0014 with EcorI and XbaI<br />
** 10 µl template (NoIV, 108 ng/µl)<br />
** 5 µl BSA, 5 µl Buffer NEB#4<br />
** 1 µl EcoRI, 1 µl XbaI<br />
** 28 µl H2O<br />
** 1.5 h @ 37°C<br />
** heat inactivation 5min @60°C <br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Dephosphorylation|Dephosphorylation]]''' of restricted vector<br />
**Purification with Zymo5 Kit, elution in 20 µl H2O<br />
**loaded on gel (with 4 µl GLPn)<br />
[[Image:x]] <br />
*Gel excision with Zymo Kit<br />
**<br />
**<br />
<br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of R0011 with SpeI<br />
** 10 µl template (R0011, X ng/µl)<br />
** 5 µl BSA, 5 µl Buffer NEB#4<br />
** 1 µl SpeI<br />
** 29 µl H2O<br />
** 1.5 h @ 37°C<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' <br />
** 5 µl R0011 (S-digested) with 12 µl TrpSig or HisSig, respectively (X-digested)<br />
** complete ligation (20 µl) loaded on Gel (with 4 µl GLPn)<br />
[[Image:TUM2010_100429beschriftet.png]] <br />
** Gel excision with Zymo Kit, eluted in 42 µl H2O<br />
<br><br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]]''' <br />
**50 µl XL-10 transformed with 2 µl of pSB1A10 prepared from IGem 2009 Distribution (13 µl left in pink Box @-20°C)<br />
*<br />
<br><br />
<br />
===30.04.2010===<br />
PCR R0011-HisSig and R0011-TrpSig --> 13 ng/µl x 20 µl<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week05{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===04.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of pSB1K3-B0014 with EcorI and XbaI<br />
** 10 µl template (NoIII, 103 ng/µl)<br />
** 5 µl BSA, 5 µl Buffer NEB#4<br />
** 1 µl EcoRI, 1 µl XbaI<br />
** 28 µl H2O<br />
** 1.5 h @ 37°C<br />
**Purification with Zymo5 Kit, elution in 15 µl H2O<br />
**loaded on gel (with 3 µl GLPn) <br />
[[Image:TUM2010_100504beschriftet.png]] <br />
*Gel excision with Zymo Kit<br />
**<br />
**<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of R0011-HisSig and R0011-TrpSig with EcorI and SpeI<br />
** 10 µl template (PCR-product)<br />
** 5 µl BSA, 5 µl Buffer NEB#4<br />
** 1 µl EcoRI, 1 µl SpeI<br />
** 28 µl H2O<br />
** 1.5 h @ 37°C<br />
**Purification with Zymo5 Kit, elution in 20 µl H2O<br />
<br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' <br />
** 4 µl R0011-Signal (E/S-digested) with 4 µl pSB1K3-B0014 (E/X-digested)<br />
**15 min @ RT, 20 min heat inactivation @ 65°C<br />
<br><br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]]''' <br />
**50 µl XL-10 transformed with 10 µl of Ligation mix<br />
<br><br />
<br />
*Overrnight liquid cultures of pSB1A10-RFP made from<br />
**1 and 2: picked clones from original plates from *[[29.04.2010|Do 29.04.2010]]<br />
**3a: picked clone from copy plate from *[[30.04.2010|Fr 30.04.2010]]<br />
**3b: resuspended clone N° 3 from *[[30.04.2010|Fr 30.04.2010]]<br />
--> each in 600 µl LB+Carbenicillin (=Ampicillin) @37°C<br />
===05.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Miniprep|Miniprep]]''' of pSB1A10; 4 samples (1, 2; 3a; 3b)<br />
**eluted in 50 µl H2O each<br />
**Concentrations:<br />
*** c1=37.5 ng/µl<br />
*** c2=56.5 ng/µl<br />
*** c3a=46.5 ng/µl<br />
*** c3b=30 ng/µl<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of pSB1A10 with EcorI and PstI, 4 samples (1, 2; 3a; 3b)<br />
** 15 µl template <br />
** 5 µl BSA, 5 µl Buffer NEB#3<br />
** 1 µl EcoRI, 1 µl PstI<br />
** 23 µl H2O<br />
** 1.5 h @ 37°C<br />
** heat inactivation 5min @60°C <br />
**Purification with Zymo5 Kit, elution in 15 µl H2O<br />
**loaded on gel (with 3 µl GLPn)<br />
[[Image:TUM2010_100505beschriftet.png]]<br />
<br> <br />
Insert @ 1 kb as expected, but vector @ 2 kb and not @ 5 kb as expected!!!!<br />
--> Wrong Plasmid! Comparison to the [http://partsregistry.org/cgi/assembly/plate_egel.cgi?id=615 Gel in the registry] shows: The Distribution contains the wrong plasmid!<br />
<br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of HisTerm and TrpTerm with EcorI and PstI<br />
** 5 µl template <br />
** 5 µl BSA, 5 µl Buffer NEB#3<br />
** 1 µl EcoRI, 1 µl PstI<br />
** 33 µl H2O<br />
** 1.5 h @ 37°C<br />
<br><br><br />
*Clones picked: 7 from each Plate (pSB1K3-R0011-TrpSig-Boo14 and pSB1K3-R0011-HisSig-Boo14)<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR)<br />
**15 µl of each sample mixed with 3 µl GLPn and loaded to Gel<br />
**2% Agarose in 1xTAE, 130 V, 90 min<br />
<br />
[[Image:TUM2010_100505bbeschriftet.png]] <br />
<br />
<br><br />
<br />
===06.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of pSB1K3 with EcorI and XbaI<br />
** 20 µl template (sample III, 103 ng/µl)<br />
** 5 µl BSA, 5 µl Buffer NEB#3<br />
** 1 µl EcoRI, 1 µl XbaI<br />
** 18 µl H2O<br />
** 1.5 h @ 37°C<br />
** heat inactivation 5min @60°C <br />
**loaded on gel (with 10 µl GLPn) in 4 lanes<br />
[[Image:TUM2010_100506beschriftet.png]] <br />
*Gel excision with Zymo Kit (lanes 1&2) and with Qiaquick Kit (lanes 3&4)<br />
** c1=4.5 ng/µl<br />
** c2=3.5 ng/µl<br />
** c3=2 ng/µl<br />
** c1=7 ng/µl<br />
* A260/A230 and A260/A280 values were strange (see labbook)<br />
<br><br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' <br />
** 4 µl R0011-Signal (E/S-digested) with 10 µl pSB1K3-B0014 (E/X-digested, from [[23.04.2010|23.04.]])<br />
**15 min @ RT, 20 min heat inactivation @ 65°C<br />
<br><br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]]''' <br />
**50 µl XL-10 transformed with 7 µl of Ligation mix<br />
<br><br />
<br />
===07.05.2010===<br />
<br><br />
*Clones picked: 7 from each Plate (pSB1K3-R0011-TrpSig-Boo14 and pSB1K3-R0011-HisSig-Boo14)<br />
<br> <br />
---Too damn stupid to do a PCR!!!---<br> <br> <br />
<br><br />
* replated picked clones on new plates, incubated at RT<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week06{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
<br />
===10.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]''' of picked clones from [[07.05.2010|Fr 07.05.2010]]<br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified)<br />
**15 µl of each sample mixed with 3 µl GLPn and loaded to Gel<br />
**2% Agarose in 1xTAE, 120 V, 110 min<br />
**stained in SybrSafe 50 min<br />
[[Image:TUM2010_100510beschriftet.png]] <br />
<br />
<br><br />
Interpretation: <br />
Colonies contain an Insert with Prefix and Suffix, length is 200 bp. This is too short for the desired R0011-Signal-B0014 (245 or 247 bp) construct, but longer than B0014 (136 bp) which was the Insert in the digested vector. <br />
Possible explanation: Ligation worked, but not with R0011-Signal-construct but with R0011 '''or''' Signal. Which?<br />
<br />
<br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| fragment<br />
| length without Prefix/Suffix <br />
| length with Prefix/Suffix<br />
|-<br />
| R0011<br />
| 55 bp <br />
| 96 bp<br />
|-<br />
| B0014<br />
| 95 bp<br />
| 136 bp<br />
|-<br />
| TrpSig/HisSig<br />
| 34 bp/32 bp<br />
| 75 bp/73 bp<br />
|-<br />
| TrpSig-B0014/HisSig-B0014<br />
| 135 bp/ 133 bp<br />
| 176 bp/ 174 bp<br />
|-<br />
| R0011-TrpSig-B0014/R0011-HisSig-B0014<br />
| 206 bp/ 204 bp<br />
| 247 bp/ 245 bp<br />
|-<br />
| R0011-B0014<br />
| 156 bp<br />
| 197 bp<br />
|-<br />
| R0011-TrpSig/R0011-HisSig<br />
| 95 bp/93 bp<br />
| 136 bp/134 bp<br />
|-<br />
|}<br />
<br />
Prefix: 20 bp; Suffix: 21 bp; X-S-scar: 6 bp<br />
<br />
--> it looks as if R0011 is ligated to B0014, which makes the whole construct wothless. The R0011-HisSig control looks more like R0011 alone as well.<br />
<br />
===11.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' <br />
** 4 µl Signal (E/S-digested; from ) with 5 µl pSB1K3-B0014 (E/X-digested; from)<br />
**15 min @ RT<br />
<br><br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]]''' <br />
**50 µl XL-10 transformed with 7 µl of Ligation mix<br />
<br><br />
<br />
===12.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]''' of picked clones from [[11.05.2010|Tu 12.05.2010]]<br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified)<br />
**15 µl of each sample mixed with 3 µl GLPn and loaded to Gel<br />
**2% Agarose in 1xTAE, 120 V, 110 min<br />
**stained in SybrSafe 60 min<br />
[[Image:TUM2010_100512beschriftet.png]] <br />
<br />
<br><br />
<br />
<br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| fragment<br />
| length without Prefix/Suffix <br />
| length with Prefix/Suffix<br />
| length after PCR<br />
|-<br />
| R0011<br />
| 55 bp <br />
| 96 bp <br />
| 104 bp<br />
|-<br />
| B0014<br />
| 95 bp<br />
| 136 bp <br />
| 154 bp<br />
|-<br />
| TrpSig/HisSig<br />
| 34 bp/32 bp<br />
| 75 bp/73 bp <br />
| 93 bp/91 bp<br />
|-<br />
| TrpSig-B0014/HisSig-B0014<br />
| 135 bp/ 133 bp<br />
| 176 bp/ 174 bp <br />
| 194 bp/ 192 bp<br />
|-<br />
| R0011-TrpSig-B0014/<br>R0011-HisSig-B0014<br />
| 206 bp/ 204 bp<br />
| 247 bp/ 245 bp <br />
| 265 bp/ 263 bp<br />
|-<br />
| R0011-B0014<br />
| 156 bp<br />
| 197 bp <br />
| 215 bp<br />
|-<br />
| R0011-TrpSig/R0011-HisSig<br />
| 95 bp/93 bp<br />
| 136 bp/134 bp <br />
| 154 bp/152 bp<br />
|-<br />
|}<br />
<br><br />
Prefix: 20 bp/29 bp after PCR; Suffix: 21 bp/30 bp after PCR; X-S-scar: 6 bp<br />
===14.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of pSB1K3 with EcorI and XbaI<br />
** 10 µl template (sample III, 103 ng/µl)<br />
** 2 µl BSA, 2 µl Buffer NEB#4<br />
** 1 µl EcoRI, 1 µl XbaI<br />
** 4 µl H2O<br />
** 1 h @ 37°C<br />
**loaded on gel (with 4 µl GLPn) in 1 lane<br />
[[Image:TUM2010_100514beschriftet.png]] <br />
*Gel excision with Zymo Kit <br />
<br />
<br><br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' of HisSig and TrpSig with EcorI and SpeI<br />
** 10 µl template ("1:100")<br />
** 2 µl BSA, 2 µl Buffer NEB#3<br />
** 1 µl EcoRI, 1 µl SpeI<br />
** 4 µl H2O<br />
** 1.5 h @ 37°C<br />
** Purification with [[Team:TU_Munich/Lab#Molecular_Biology ZYMO RESEARCH DNA Clean&amp;Concentration Kit|Zymo 5 ]]<br />
** or heat inactivated (20 min @ 80°C)<br />
<br><br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' <br />
**<br />
<br />
<br><br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]]''' <br />
**50 µl XL-10 transformed with 10 µl of Ligation mix<br />
**50 µl untransformed cells plated on Kana-plate as control<br />
<br><br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week07{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===17.05.2010===<br />
*Plates from Friday:<br />
**plenty colonies on control plate --> XL10 cells are impure!<br />
**use DH5a from now on!!!!!<br />
<br />
<br><br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]]''' <br />
**50 µl DH5a transformed with 10 µl of Friday's Ligation mix<br />
**plated on Kana-Plates; Overnight @ 37°C<br />
<br><br />
<br />
*DNA Isolation from BioBrick Distribution 2010<br />
** 10 µl H2O added to Well 1A of plate 1 containing pSB1A10 with RFP-insert<br />
** 2 µl used for [[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]] of 50 µl DH5a-cells<br />
** plated on Carbenicillin (=Amp-analogon)-plates, Overnight @ 37°C<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Preparation of Gels|Polyacrylamide Gel]]''' prepared for tomorrow<br />
** 1 big denaturing Gel with 20 pockets<br />
<br />
===18.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]''' of picked clones <br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR)<br />
**10 µl of each sample mixed with 10 µl Formamide loading buffer and loaded to Polyacrylamide Gel<br />
<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Running of Gels|Polyacrylamide Gel]]'''<br />
Samples: <br />
LMW|R0011|HisSig|TrpSig|HisSig E/S-Dig|TrpSig E/S-Dig|B0014|LMW2|Colony PCR His1|His2|Trp1|Trp2|control|HisTerm|TrpTerm|HisTerm E/P-Dig|TrpTerm E/P-Dig<br />
<br />
*5 µl of each samples mixed with 5 µl formamide loading dye and loaded to gel(except Ladder and colonyPCR)<br />
**LMW: 3 µl LMW (Korbinian) + 3 µl Formamide loading Dye<br />
**LMW2: 5 µl LMW Quickload (with GLP) + 10 µl Formamide loading Dye<br />
*stained in SybrSafe<br />
<br />
<font color=red>Important Mistake! See below Gel! </font><br />
[[Image:TUM2010_100518beschriftet.png|600px?]]<br />
<br><br />
IMPORTANT MISTAKE: DENATURING GELS NOT USEFUL FOR dsDNA!!!<br />
REPEAT WITH NATIVE GEL, IGNORE INTERPRETATION!!!<br />
<br />
<br />
<br />
(*R0011 and B0014 look normal<br />
*ColonyPCR: bands that look like B0014 in all clones (and in control) --> Religation?<br />
*Signals at the wrong size: should be about 75 bp, look like 200 bp!!!<br />
*terminators completely strange: should be around 100 bp!<br />
<br />
--> are all of our sequences just wrong???<br />
What are we going to do? Order everything new?)<br />
===19.05.2010===<br />
* Gel from Korbinian<br />
*5 µl of each samples mixed with 5 µl formamide loading dye and loaded to gel(except Ladder and colonyPCR)<br />
**LMW: 3 µl LMW (Korbinian) + 3 µl Formamide loading Dye<br />
**colony PCR: from Tuesday, 8 µl sample with 8 µl Formamide loading Dye<br />
*stained in SybrSafe 20 min<br />
[[Image:TUM2010_100519paabeschriftet.png]]<br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
** 4 colonies picked from each Plate (Ligations from yesterday; Signal-B0014)<br />
**15 µl of each Sample mixed with 3 µl GLPn and loaded to Gel:<br />
** 3% Agarose in 1x TBE, 130 V<br />
[[Image:TUM2010_100519beschriftet.png600px]]<br />
<br />
===20.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Digestion|Digestion]]''' <br />
** template<br />
** 2 µl BSA<br />
** 2 µl Buffer <br />
** 1 µl of each enzyme<br />
** water to reach 20 µl<br />
<br><br />
{| width="618" cellspacing="1" cellpadding="1" border="1" align="center" style=""<br />
|-<br />
| '''Template'''<br> <br />
| '''Enzymes'''<br><br />
| '''NEB Buffer #'''<br />
|-<br />
| HisTerm & TrpTerm (10 µl)<br><br />
| EcoRI, PstI<br><br />
| 3<br />
|-<br />
| HisSig & TrpSig (10 µl)<br> <br />
| EcoRI, SpeI<br><br />
| 4<br><br />
|-<br />
| B0014 (5 µl)<br> <br />
| XbaI, PstI<br><br />
| 3<br><br />
|-<br />
| pSB1A10_RFP (14 µl)<br><br />
| EcoRI, PstI<br><br />
| 3<br />
|-<br />
| pSB1K3_RFP (14 µl)<br><br />
| EcoRI, PstI<br><br />
| 3<br />
|-<br />
| pSB1K3_B0014 N° 4 (14 µl)<br><br />
| EcoRI, XbaI<br><br />
| 4<br />
|}<br />
<br><br />
** incubated @37°C for 1.5 h<br />
**digested inserts heat inactivated (20 min @ 80°C)<br />
**digested plasmids loaded on gel (with 4 µl GLPn) in 1 lane<br />
[[Image:TUM2010_100520beschriftet.png]] <br />
*Gel excision with Zymo Kit <br />
**c(pSB1A10, I)=4.5 ng/µl<br />
**c(pSB1A10, II)=1.5 ng/µl ?!?!?!<br />
**c(pSB1K3 E/P)=7 ng/µl<br />
**c(pSB1K3_B0014 E/X)=2.5 ng/µl<br />
<br><br><br />
<br />
*Gel of PCR products<br />
**3% Agarose in 1x TBE; 2h @130 V<br />
[[Image:TUM2010_100520bbeschriftet.png]]<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''' <br />
** templates<br />
**2 µl T4-buffer 10x<br />
**1 µl T4-Ligase<br />
**Water to reach 20 µl<br />
<br />
{| width="618" cellspacing="1" cellpadding="1" border="1" align="center" style=""<br />
|-<br />
| '''Vector'''<br> <br />
| '''Insert'''<br><br />
|-<br />
| psB1A10 (E/P; sample I) (10 µl)<br><br />
| TrpTerm (E/P) (4 µl)<br><br />
|-<br />
| psB1A10 (E/P; sample I) (10 µl)<br><br />
| HisTerm (E/P) (4 µl)<br><br />
|-<br />
| psB1K3_B0014 (E/X) (12 µl)<br><br />
| HisSig (E/S) (2 µl)<br><br />
|-<br />
| psB1K3_B0014 (E/X) (12 µl)<br><br />
| TrpSig (E/S) (2 µl)<br><br />
|-<br />
| psB1K3 (E/P) (8 µl)<br><br />
| HisSig (E/S)(2 µl) + B0014 (X/P)(1.5 µl)<br><br />
|-<br />
| psB1K3 (E/P) (8 µl)<br><br />
| TrpSig (E/S)(2 µl) + B0014 (X/P)(1.5 µl)<br><br />
|}<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]]''' <br />
** 50 µl DH5a transformed with 10 µl of Ligation mix<br />
** 50 µl DH5a transformed with 2 µl of pSB1K3_B0014<br />
** 50 µl DH5a transformed with 2 µl of pSB1K3_RFP<br />
** 50 µl DH5a transformed with 2 µl of pSB1A10_RFP<br />
===21.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
** 4 colonies picked from each Plate (pSB1K3_HisSig_B0014, pSB1K3_TrpSig_B0014, pSB1K3_HisSig_B0014 double ligation, pSB1K3_TrpSig_B0014 double ligation)<br />
** each clone resuspended in 20 µl LB0, 3 µl used as template for PCR <br />
** 15 µl of each Sample mixed with 3 µl GLPn and loaded to Gel:<br />
** 3% Agarose in 1x TBE, 130 V<br />
<br />
[[Image:TUM2010_100521beschriftet.png]]<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week08{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
<br />
===25.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
** 4 colonies picked from each Plate <br />
***pSB1K3_HisSig_B0014<br />
***pSB1K3_TrpSig_B0014<br />
***pSB1K3_HisSig_B0014 double ligation<br />
***pSB1K3_TrpSig_B0014 double ligation<br />
***pSB1A10_TrpTerm<br />
***pSB1A10_HisTerm<br />
** each clone resuspended in 20 µl LB0, 2 µl used as template for PCR <br />
** 15 µl of each Sample mixed with 3 µl GLPn and loaded to Gel:<br />
** 3% Agarose in 1x TBE, 220 V (double Gel, 35 cm)<br />
** stained in SybrSafe<br />
<br><br />
[[Image:TUM2010_100525beschriftet.|600px]]<br><br />
[[Image:TUM2010_100525bbeschriftet.png]]<br><br />
*overnight cultures made of<br />
**HisSig 3, DL1, DL4<br />
**TrpSig DL2, DL4<br />
**HisTerm/TrpTerm 1,2,3<br />
<br />
===26.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology #ZYMO RESEARCH DNA Clean&Concentration Kit|Miniprep]]''' of cultures set up [[25.05.2010]]<br />
**HisSig 3, DL1, DL4<br />
**TrpSig DL2, DL4<br />
**HisTerm/TrpTerm 1,2,3<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Restriction|Restriction]]'''<br />
**analytical: E/P HisSig(3, DL2); TrpSig(DL2, DL4): 1.5 h 37 °C<br />
**prep: E/X HisSig(3, DL2); TrpSig(DL2, DL4): 1.5 h 37 °C<br />
**prep: E/S R0011: 1.5 h 37 °C, inactivation 20 min @ 80 °C<br />
***total volume each 20 µl, 10 µL template<br />
*'''Gel''': 1% Agarose, TAE - 1,5 h 110 V<br />
**[[Team:TU_Munich/Lab#Molecular_Biology #ZYMO RESEARCH Gel DNA Recovery Kit|bands excised]]: all E/X cleaved vectors<br />
[[Image:TUM2010_100526beschriftet.png]]<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]''':<br />
**HisSig_3 (E/X) with R0011 (E/S)<br />
**HisSig_DL2 (E/X) with R0011 (E/S)<br />
**TrpSig_DL2 (E/X) with R0011 (E/S)<br />
**TrpSig_DL4 (E/X) with R0011 (E/S)<br />
***'''batches'''<br />
***total volume 20 µL<br />
***2 µL R0011 (E/S)<br />
***2 µL T4 buffer<br />
***1 µL T4 Ligase<br />
***15 µL vector<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]]''' of ligations<br />
** DH5a<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|PCR]]'''<br />
**His and TrpSig<br />
**His and TrpTerm<br />
**R0011<br />
**B0014<br />
***50 µL total volume<br><br />
***1 µL template<br><br />
***1 µl G1004<br><br />
***1 µl G1005<br><br />
***0.2 µL Taq<br><br />
***5 µl Taq standard buffer<br><br />
***rest water<br />
<br />
===27.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
** 3 colonies picked from each Plate <br />
***pSB1K3_R0011_HisSig_B0014 (N° 3 from yesterday)<br />
***pSB1K3_R0011_HisSig_B0014 (N° DL1 from yesterday)<br />
***pSB1K3_R0011_TrpSig_B0014 (N° DL2 from yesterday)<br />
***pSB1K3_R0011_TrpSig_B0014 (N° DL4 from yesterday)<br />
***"N6"<br />
***"N15"<br />
** each clone resuspended in 20 µl LB0, 2 µl used as template for PCR <br />
** 15 µl of each Sample mixed with 3 µl GLPn and loaded to Gel:<br />
** 3% Agarose in 1x TBE, 220 V (double Gel, 35 cm)<br />
** stained in SybrSafe<br />
<br><br />
[[Image:TUM2010_100527beschriftet.png]]<br><br />
<br><br />
<br />
<br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| fragment<br />
| length without Prefix/Suffix <br />
| length after PCR<br />
|-<br />
| R0011<br />
| 55 bp <br />
| 104 bp<br />
|-<br />
| B0014<br />
| 95 bp<br />
| 154 bp<br />
|-<br />
| TrpSig/HisSig<br />
| 34 bp/32 bp<br />
| 93 bp/91 bp<br />
|-<br />
| TrpSig-B0014/HisSig-B0014<br />
| 135 bp/ 133 bp<br />
| 194 bp/ 192 bp<br />
|-<br />
| <font color=red>R0011-TrpSig-B0014/<br>R0011-HisSig-B0014<br />
| 206 bp/ 204 bp <br />
| <font color=red>265 bp/ 263 bp</font><br />
|-<br />
| <br />
| <br />
| <br />
|-<br />
| I712074 ("N6")<br />
| 46 bp<br />
| 105 bp<br />
|-<br />
| I719005 ("N15")<br />
| 23 bp <br />
| 82 bp<br />
|-<br />
|}<br />
<br><br />
Prefix: 29 bp after PCR; Suffix: 30 bp after PCR; X-S-scar: 6 bp<br />
<br />
*overnight cultures made of<br />
**HisSig 3_1, DL1_3<br />
**TrpSig DL4_1, DL4_3<br />
**N15 1&2<br />
**HisTerm/TrpTerm (picked colonies from yesterdays plates #2 each)<br />
<br />
<br />
*Purification of yesterday's PCR<br />
**elution in 50 µl H2O<br />
*** c(HisSig)=5.5 ng/µl<br />
*** c(TrpSig)=10 ng/µl<br />
*** c(HisTerm)=6.5 ng/µl<br />
*** c(TrpTerm)=6.5 ng/µl<br />
*** c(R0011)=13 ng/µl<br />
*** c(B0014)=12.5 ng/µl<br />
** 5 µl loaded on gel with 1 µl GLPn; 3% Agarose in 1x TBE, 220 V (double Gel, 35 cm)<br />
<br />
[[Image:TUM2010_100527bbeschriftet.png]]<br><br />
===28.05.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology ZYMO_RESEARCH_DNA_Clean.26Concentration_Kit|Miniprep]]''' of cultures from [[27.05.2010]], Elution in 50 uL nuclease free water <br />
** (1)HisSig 3_1 <br />
** (2)HisSig DL1_3 <br />
** (3)TrpSig DL4_1 <br />
** (4)TrpSig DL4_3 <br />
** (7)HisTerm#1 <br />
** (8)HisTerm#2 (7 ml culture) <br />
** (9)TrpTerm#1 <br />
** (10)TrpTerm#2 (7ml culture) <br />
** (5)N15-1 (=BBa_I719005) <br />
** (6)N15-2 (=BBa_I719005)<br />
*** ()=numbers on gel<br />
<br><br />
{| cellspacing="1" cellpadding="1" border="1" style="width: 367px; height: 271px;"<br />
|-<br />
| sample<br> <br />
| DNA concentration (ng/uL)<br><br />
|-<br />
| HisSig 3_1 <br> <br />
| 6<br><br />
|-<br />
| HisSig DL1_3<br> <br />
| 11<br><br />
|-<br />
| TrpSig DL4_1 <br> <br />
| 16<br><br />
|-<br />
| TrpSig DL4_3 <br> <br />
| 21.5<br><br />
|-<br />
| HisTerm#1<br> <br />
| 27<br><br />
|-<br />
| HisTerm#2<br> <br />
| 66<br><br />
|-<br />
| TrpTerm#1<br> <br />
| 34<br><br />
|-<br />
| TrpTerm#2<br> <br />
| 29.5<br><br />
|-<br />
| N15-1 (=BBa_I719005)<br> <br />
| 19.5<br><br />
|-<br />
| N15-2 (=BBa_I719005)<br> <br />
| 18.5<br><br />
|}<br />
<br />
<br> <br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Restriction|analytical digest]]''' (E/P)<br><br />
**of all samples. total volume 20 uL, 5 uL template used for Term-constructs, 10 uL teplate for all others<br />
<br />
*'''Agarose Gel''' <br />
**3% broad range agarose in TBE. Run in TBE, 140 V, 1.50 h <br />
**stained with SybrGold, 45 min<br />
**signals look fine<br />
**terminators also (without pre/suffix 97/104 bp)<br />
**T7 promoter without pre/suffix has a length of 23 bp + cut pre/szffix ca at 60 bp -->buffer?<br />
[[Image:TUM2010_100528_beschriftet.png]]<br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week09{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===31.05.2010===<br />
* Digestions<br />
**15N-1 (BBa_I719005, 19.5 ng/uL) with S/P<br />
**HisSig/TrpSig (6.5/10 ng/uL) with X/P<br />
::2 h 37 °C<br />
:heat inactivation of insert-digestions<br />
<br />
*Gel: 1% Agarose in 1x TAE<br />
:1 h 25 min, 115 V<br />
:stained with SybrGold, 40 min<br />
:[[Image:TUM2010_100531.png]]<br />
<br />
:Band at ~2100b cut und purified using the [[Team:TU_Munich/Lab#Molecular_Biology ZYMO RESEARCH Gel DNA Recovery Kit |zymo kit]]<br />
<br />
* [[Team:TU_Munich/Lab#Molecular_Biology Ligation|ligation]]<br />
** HisSig (4 µL of digest) with purified plasmid (with BBa_I719005)<br />
** TrpSig (2 µL of digest) with purified plasmid -=-<br />
::reason: concentration of His Sig before digest was 1/2 of TrpSig<br />
<br />
* [[Team:TU_Munich/Lab#Molecular_Biology Transformation|transformation]]<br />
: of DH5a with Ligation batches, HisSig1-3, HisSig3-1, TrpSig DL4-1, TrpSig 4-3<br />
===01.06.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
<br />
:of Ligations transformed into DH5a yesterday <br />
:4 Colonies of each Ligation<br />
<br />
**T7-HisSig <br />
**T7-TrpSig<br />
<br />
<br> <br />
<br />
*'''Gel: 3% broad range Agarose in 1xTBE'''<br />
<br />
:1.5 h 140 V <br />
:stained with SybrGold<br />
<br />
[[Image:TUM2010_100601.png]] <br />
<br />
:calculation for the expected size of the fragments<br />
<br />
:{| cellspacing="1" cellpadding="1" border="1" width="200"<br />
|-<br />
| part<br />
| size (bp)<br />
|-<br />
| HisSig<br />
| 32<br />
|-<br />
| TrpSig<br />
| 34<br />
|-<br />
| T7 promoter<br />
| 23<br />
|-<br />
| prefix<br />
| 20<br />
|-<br />
| suffix<br />
| 21<br />
|-<br />
| X/S scar<br />
| 6<br />
|}<br />
<br />
:in PCR we get additional bp due to the primers - +9 at pre/suffix=+18 bp<br> <br />
<br />
:overall size of the fragments expected to come out of the PCR: T7_HisSig: 120 bp, T7_TrpSig: 122 bp<br />
<br />
*'''ON cultures'''<br />
<br />
:5 ml cultures of pSB1K3_R0011_HisSig_B0014 (1_3 &amp; 3_1) and pSB1K3_R0011_TrpSig_B0014 (DL4_1 &amp; DL4_3) <br />
:1 ml cultures of each colony monitored in Colony PCR<br />
<br />
===02.06.2010===<br />
*Miniprep of yesterdays cultures using Zymokit, elution by nuclease-free water <br />
*Concentration determination <br />
*analytic digestion <br />
*results on gel:<br />
<br />
<br> <br />
<br />
*Sequencing <br><br />
<br />
JobNr. Barcode Last change Date/Time Last message / Files 6549287 AE2739 02.06.2010 / 13:51:12 HisSig 1-3-forward G1004 <br />
<br />
We just received your order. Many thanks.<br />
<br />
6549288 AE2738 02.06.2010 / 13:51:12 HisSig 3-1-forward G1004 <br />
<br />
We just received your order. Many thanks.<br />
<br />
6549289 AE2737 02.06.2010 / 13:51:12 TrpSig DL4-1-forward G1004 <br />
<br />
We just received your order. Many thanks.<br />
<br />
6549290 AE2736 02.06.2010 / 13:51:12 TrpSig DL4-3-forward G1004 <br />
<br />
We just received your order. Many thanks.<br />
<br />
6549291 AE2735 02.06.2010 / 13:51:12 HisTerm-forward G1004 <br />
<br />
We just received your order. Many thanks.<br />
<br />
6549292 AE2734 02.06.2010 / 13:51:12 TrpTerm-forward G1004 <br />
<br />
We just received your order. Many thanks.<br />
<br />
*Gel 3% broad range agarose in 1x TBE<br />
: [[Image:TUM2010_100602_beschr.png]]<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week10{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===07.06.2010===<br />
*'''Sequenbcing results from GATC''' <br />
**HisSig DL1-3 is ok <br />
**HisSig 3-1 is ok <br />
**TrpSig DL4-1 is ok <br />
**TrpSig DL4-3 is ok <br />
**TrpTerm + HisTerm bad runs... --&gt; new sequencing order with Primer 100 bp upstream (within GFP)<br />
<br />
:Files can be found stored in our [[Zugangsdaten für GATC|GATC account]]<br />
<br />
<br><br />
<br />
*'''Sequencing@GATC:''' both Term-constructs with primer pGFP-FP provided by GATC<br />
<br />
<br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Restriction|Restrictions]] '''<br />
**psB1A10_TrpTerm/HisTerm with Nsi1, Aat2 <br />
**pSB1K3_R0011_HisSig/TrpSig_B0014 with Pst1, Aat2 <br />
**T7 bb with Spe1, Pst1 <br />
**PCRProducts: HisSig/TrpSig with Pst1, Xba1, 2 h @37°C<br />
<br />
:all plasmid digests done''' sequential''' as enzymes do not have 100% activity in the same buffer, each reaction 1.5 h@37°C <br />
:psB1A10_TrpTerm/HisTerm and T7 bb dephosphorylated the last 30 min <br><br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]] ''' <br><br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Transformation|Transformation]] '''<br />
<br />
<br> liquid culture (10 ml) of pSB1K3_R0011_HisSig/TrpSig_B0014<br />
===08.06.2010===<br />
*'''Sequencing results from GATC''': His/TrpTerm with pGFP-FP primer<br />
**HisTerm worked<br />
**TrpTerm worked<br />
: checked with blast2seq<br />
<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology PCR|Colony PCR]]'''<br />
: 8 colonies from each plate of T7His, T7Trp, MonsterHis, MonsterTrp as ligations resulted in many colonies<br />
<br />
*'''3% Agarose Gel in 1xTBE'''<br />
: for all colony PCR reactions<br />
[[Image:TUM2010_100608_beschriftet.png|600px]]<br />
[[Image:TUM2010_100608-2_beschriftet.png|600px]]<br />
<br />
<br> Interpretation/Info: The R0011_Sig_B0014 construct was cut with PstI, but not ligated into a PstI site but instead into the NsiI site --> even if sticky end are compatible, the bases in the 3` direction are different --> primer lags 7 bp compared to standard procedure --> we didn´t expect to find the signal construct by colony PCR --> control digestion tomorrow<br />
: T7-Signal constructs seem to have worked, expected size was 23 bp (T7)+ 32 (HisSig)/34 (TrpSig) bp + 30+29 (PCRPre+Suf)=114/116 bp<br />
*over night cultures<br />
<br />
===09.06.2010===<br />
* Miniprep of 4 Monster_His, 4 Monster_Trp, 3 T7_His and 3 T7_Trp cultures<br />
* Analytical digestion of plasmids mentioned above<br />
* gel of Monster_Plasmid digestion<br />
[[Image:TUM2010_100609_Monster_inverse.jpg|600px]]<br />
<br><br />
gel didn´t work at all --> even after > 2 h, bands were not separated correctly, even the 1kb ladder was "stacked" in the gel-pockets, the 100 bp ladder should show EQUAL distances between the lines [http://www.neb.com/nebecomm/productfiles/778/images/N3231_fig1_v1_000034.gif see here], it looks like the gel was "more dense" at the pockets---> no idea what happened --> repeat Monster-digestion tomorrow?<br />
<br><br />
* gel of T7_Plasmid digestion<br />
[[Image:TUM2010_100609_T7_sig_invers.jpg|600px]]<br />
<br><br />
T7_Trp E + S digestion 107 bp and T7_His 105 bp --> worked for all picked colonies. (regard that there is an excess of plasmid DNA-basepairs of factor >30 --> thats why the inserts are much weaker than the plasmid signals.<br />
<br><br />
occured trouble: <br />
** Ladders and loading dye´s empty --> i used those of eike, BUT: eikes 1 kb ladder is different --> compare [http://www.neb.com/nebecomm/products/productN3272.asp here] and his loading dye was much more diluted, even if there was also 6x Sac GLP written on it -> i hope this won´t cause any trouble<br />
<br />
===10.06.2010===<br />
*'''ordered'''<br />
:Promega E.coli S30 in vitro transcription/translation kit<br />
:Spe1, Aat2 from NEB, 500 U each<br />
<br><br />
* '''[[Team:TU_Munich/Lab#Molecular_Biology Restriction|analytical Digestion]]<br />
:of Ligation colonies from MonsterHis/trp 1-3 and T7His/Trp 1,2,5/1,2,3<br />
:2h digestion<br />
:: Monster: 6 µL DNA template with Aat2/Spe1 in Buffer 4/Bsa<br />
:: T7-Signal: 6 µL DNA template with E/P in Buffer 3<br />
<br><br />
* '''Agarose Gels'''<br />
:used standards: lmw, 2-log [[Team:TU_Munich/Lab#Molecular_Biology standards|click here]]<br />
: Gel1: 1% Agarose in 1xTBE for Digestions of Monsterplasmid<br />
:: run in big chamber @ 200 V for 1 h 20 min<br />
:[[Image:TUM2010_100610_t7sig.png]]<br />
:Gel2: 3% Agarose (broad range) in 1xTBE for Digestions of T7-Signal<br />
:: run in small chamber @140 V for 1 h 35 min<br />
:[[Image:TUM2010_100610_monster.png]]<br />
<br><br><br />
:Conclusions:<br />
# all T7-Signal ligations loaded on the gel worked<br />
# monsterplasmid didn't work? bands at 800 bp, 900 bp, 1.3 kbp, 2.2 kbp, 3 kbp, we SHOULD expect to see our Insert, wich is Prefix+R0011_Signal_B0014_small Suffix, which should run around 300-400 bp...<br />
<br />
===11.06.2010===<br />
*'''Gel''': large 1% Agarose in TAE. Load: The rest of N/A cut Messplasmids from [[07.06.2010]]. Run @220 V for 3.5 h<br />
: fragments expected are 5087 and 176. original size of plasmid is 5263. This is a Try to differ between 5087 and 5263 bp<br />
:[[Image:TUM2010_100611.png]]<br />
: Band @ 5000 bp of Trp_Term purified, obviously digestion was 100%. Bad point is that HisTerm includes an Nsi1 cleavage site...<br />
<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week11{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }}{{:Team:TU_Munich/Templates/ClearBox }}{{:Team:TU_Munich/Templates/ClearBox }} {{:Team:TU_Munich/Templates/YellowBox | text=Promega Kit }}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
<br />
===14.06.2010===<br />
*'''[[Team:TU_Munich/Lab#Molecular_Biology Ligation|Ligation]]'''<br />
: 10 µL pSB1A10_TrpTerm Aat2/Nsi1 0.5 ng/uL<br />
: 1 µL R0011_TrpSig_B0014 Aat2/Psb1 11 ng/uL<br />
: 2 µL T4 ligase buffer<br />
: 1 µL T4 Ligase<br />
: 6 µL H2O<br />
::10 min RT<br />
<br><br />
*'''Transformation''' of DH5a with<br />
: Ligation<br />
: T7His#1<br />
: T7Trp#1<br />
<br><br />
*'''over night cultures''' of<br />
: pSB1K3_R0011_TrpSig_B0014<br />
: pSB1K3_R0011_HisSig_B0014<br />
: pSB1A10_TrpTerm<br />
: pSB1A10_HisTerm<br />
<br />
===15.06.2010===<br />
*Over night cultures <br />
*Aliquots of the Promega in vitro expressions kit from ''E. coli'' S30 extract:<br />
: 40 µL with aa mix including all aa.<br />
<br />
===16.06.2010===<br />
'''Fluoresence measurements using in vitro kit'''<br />
* in vitro kit sample <br />
* adding psBA1A10 Trp_Term --> constant over time, no significant changes compared to kit alone --> high efficiency of AraC<br />
* adding L-(+)Arabinose (final concentration 2%) --> after approx. 10 min significant GFP production --> measuring for xxx min --> RFP is slightly increased (to proof if correlated to GFP peak --> crossdetection)<br />
* adding psB1K3 R0011_TrpSig_B0014<br />
'''Cell culture'''<br />
5 ml culture for<br />
* psBA1A10 Trp_Term/HisTerm<br />
* psB1K3 R0011_TrpSig/Hissig_B0014<br />
<br />
===17.06.2010===<br />
''' Cloning '''<br />
<br><br />
Digestion of Trp-Sig with E/P and psB1A10 Trp_Term with E/P<br />
* Gel purification of psB1A10 Trp_Term E/P cut<br />
[[Image:TUM2010_100617_pSB1A10_EPcut_dunkler.jpg|700px]]<br />
* Heat inactivation of Trp-Sig E/P cut<br />
* Ligation for 10 min @ RT and Transformation in DH5-a cells<br />
<br />
===18.06.2010===<br />
'''cloning'''<br />
* Transformation (about 20 colonies) --> picking 5 colonies<br />
* colony PCR<br />
* Gel <br><br />
2 % broad range agarose, 1 h 120 V [[Image:TUM2010_100618_psb1A10-Trp_sig_colonypcr.jpg|600px]]<br />
Sample 2, 4, 5 shows probably Trp-Signal + Pre/Suffix --> send sample 2 for sequencing!<br />
<br />
<br><br><br />
* control digestion of all 10 picked psB1A10-TrpSig in 1% broad range agarose, > 3 h, 120 V<br />
[[Image:TUM2010_100618_psb1A10-Trp_sig_controlverdau.jpg|600px]]<br />
--> digestions worked, but again, no insert can be found, despite gel was at maximum resolution ( 3h 120 V, see LMW)<br />
<br />
''' in vitro measurements '''<br />
f$%&&§ s%§$! Again, nothing worked! Although we saw an increasing "GFP signal" comparable to 16.06.10, taking spectra suggested we DON'T see significant GFP-production! We used new water for preparing the samples, cleaned cuvettes with "new water", used other DNA-samples etc. Somehow, it seems as if we don't express GFP (we compared Christoph's results! We should see a really significant spectrum! <br>Next steps:<br />
* Try in vivo measurements, just using psb1A10_xTerm without Signal (thus just measuring plasmid) to proof if kit or measuring plasmid causes this problem!<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week12{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} {{:Team:TU_Munich/Templates/BlueBox | text=First steps }} {{:Team:TU_Munich/Templates/ClearBox }} {{:Team:TU_Munich/Templates/YellowBox | text=Promega Kit }}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
<br />
===22.06.2010===<br />
<br />
'''Transformation'''<br />
* psb1A10_HisTerm/TrpTerm into BL21 (DE3) RIL<br />
* psb1A10_Monster-Trp No. 7, 8, 10 (Positiv control) into DH5-a<br />
<br />
'''Liquid culture'''<br />
* psb1A10_TrpSig (Positiv control) No. 2<br />
<br />
===23.06.2010===<br />
<br />
*Miniprep<br />
: pSBN1A10_TrpSig - 40 µL, 12.5 ng/µL -->very low amount of DNA...<br />
: culture is slightly red? -> strange because there cannot be any rfp-insert with constitutive promoter as the construct was built up from pSB1A10_TrpTerm (digested) and TrpSignal (PCR product)<br />
<br><br />
*cultures<br />
**5 ml cultures of DH5a <br />
::with MonsterTrp, #7,8,10 (Carbamp)<br />
:*50 ml cultures of BL21 (DE3) RIL<br />
::pSB1A10_HisTerm<br />
::pSB1A10_TrpTerm<br />
<br><br />
*Arabinose Stock<br />
0.2%<br />
<br />
===24.06.2010===<br />
<br />
* Miniprep<br />
: MonsterTrp #7,8,10<br />
: positive Control<br />
:: concentrations are too low for sequencing --> again we have to set up 5 ml cultures for tomorrow<br />
<br><br />
* measurements<br />
<br />
===25.06.2010===<br />
'''Plasmid purification and sequencing'''<br />
* Monster_Trp 7, 8, 10 and psB1A1ß_TrpSig plasmids are isolated (concentrations up to 110 ng/ul) and sent for sequencing<br />
* psB1A1ß_TrpSig liquid culture was completely pink! still not clear what happend (wrong labeling of digested psB1A10_Trpterm?) --> wait for sequencing details<br />
'''Fluorescence Measurements'''<br />
* Induction by putting Arabinose directly into cuvettes with cells IS NOT WORKING at all! Expression of GFP increases, but marginally. probably, despite stirring, oxygen is lacking?<br />
* Induction on shaker work perfectly --> both Trp and His showed strong GFP-signals, BUT: Probably, too high OD results in not exciting all GFP within the sample (incident beam is already scatterd enormously on the edge of the cuvette --> only small volume is excited correctly). For instance, a sample showing OD of 0.7 shows a signal of 30 a.u., diluted to OD 0.35 signal falls only to 19 a.u.! Thus dilution did not result in a linear decrease of flourescence! a.u. !!! We diluted down to OD 0.1; the result: OD´s smaller than 0.4 show linear change of fluorescence signal --> using OD´s up to 0.4 results in meaningful measurements!!!<br />
<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week13{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} {{:Team:TU_Munich/Templates/BlueBox | text=Testing pSB1A10 }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===28.06.2010===<br />
* Sequencing results<br />
:psB1A10_TrpSig (Positive control) worked: Trp-Sig is inside, directly in front of RFP (without the promotor of the measurement plasmid insert [http://partsregistry.org/Part:BBa_J04450 BBa_J04450], so it seems like everything worked. Furthermore, i performed a promotor prediction with the following tool [http://linux1.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb bacteria promotor prediction tool] to proof if our Trp-Sig in combination with the flanking regions is not forming a promotor,by mischance. According to this, there are two promotors, BUT: <br> one in and after the suffix (so it should be in each of our constructs), but the tools says theres is no known sigma-factor for this promotor! the second one is within the RFP and there is a sigma-factor for this one ( rpoD16). So i don´t see a explanation, why our colonies were pink in contrast to the other "Messplasmids". <br><br> None of the Monsterplasmids contains the signal construct. Probably, the problem is there is no selection methods which allowed us to distinguish uncut plasmids.... --> we should discuss at our next meeting, one possiblity would be connecting our construct to a resistance marker. I summed up all sequences in this document: [[File:25.06.-sequenzierung.doc]] <br><br><br />
<br />
*Induction in cuvette and measuring fluorescence at the same time IS NOT WORKING! (probably cells are not growing and expressing very well, maybe lacking oxygen despite stirring. Bleaching is more unlikely) <br> In vivo, measuring plasmid (at least GFP) works! we optimized the paramters for fluorescence measurment! We tried different OD´s and found out that only measurments below OD 0.4 result in meaningful measurements. <br> as a result, in vitro expression did somehow not work, reasons are unclear, maybe too low DNA-concentrations. <br> positvie control psB1A10_TrpSig was pink again, we have to wait the results from GATC<br />
<br />
===29.06.2010===<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week14{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} {{:Team:TU_Munich/Templates/ClearBox }} {{:Team:TU_Munich/Templates/ClearBox }} {{:Team:TU_Munich/Templates/YellowBox | text=Invitrogen Kit }}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
<br />
===01.07.2010===<br />
*'''in vitro measurement'''<br />
:using Invitrogen kit<br />
:measuring kinetics for 3 h @ 37°C<br />
:40 µL + 5 µL pSB1A10_TrpSig (126 ng/µL) + 0.5 µL 100x L-Arabinose (=0.2%) + 4.5 µL H2O<br />
::observations: GFP signal grows, after 30 min it crashes. RFP grows<br />
::emission spectra for GFP and RFP result in no spectrum<br />
::looks strange, a problem might be evaporation of liquid and hence scattering of light which produces artefacts<br />
<br><br />
*'''over night cultures'''<br />
:pSB1A10_TrpSig (DH5a), 5 ml for miniprep<br />
:pSB1A10 XS (DH5a), 5 ml for miniprep<br />
<br />
===02.07.2010===<br />
*Miniprep<br />
:pSB1A10_XS: 30 µL 10 ng/µL<br />
:pSB1A10_TrpSig: 30 µL 10 ng/µL<br />
<br><br />
*Transformation<br />
:BL21 with pSB1A10_XS (positive control without insert and no without any bio brick site left)<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week15{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/ClearBox }} {{:Team:TU_Munich/Templates/BlueBox | text=Testing pSB1A10 }} {{:Team:TU_Munich/Templates/ClearBox }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===05.07.2010===<br />
*'''over night cultures'''<br />
:250 ml of DH5a pSB1A10_XS<br />
:20 ml BL21 pSB1A10_XS<br />
<br><br />
*'''in vitro transcription measurement planned'''<br />
:check [[In_vitro_Measurements]]<br />
<br />
===06.07.2010===<br />
*'''In vivo measurement'''<br />
: BL21 pSB1A10_XS - positive control (to check the measurement plasmid...)<br />
: GFP, RFP Fluorescence<br />
:induced with 0.2% arabinose in (1), uninduced (2)<br />
::at OD 0.15: GFP/RFP emissions spectra /100706/spectra/gfp10 and rfp10<br />
::2.5 h kinetic measurement GFP/RFP /100706/kinetics/<br />
::OD 0.7 (1) and 0.64 (2) after 2.5 h --> GFP/RFP emissions spectra /100706/spectra/gfp11,rfp11,gfp21,rfp21<br />
::in addition for 4 h a culture at OD 0.8 induced (with 0.2% Arab), spectra taken afterwards at 1:15 dilution (OD 0.39) gpf_ku and rfp_ku<br />
:observation: measurement plasmid is totale verarsche. RFP is not expressed at all, or this protein is not rfp. whatever.<br />
<br><br />
<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week16{{:Team:TU Munich/Templates/ToggleBoxStart2}}The Era of Exams<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
No Lab work this week, everybody is busy studying for their exams...<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week19{{:Team:TU Munich/Templates/ToggleBoxStart2}} The Era of Exams<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week20{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=Construction of new Measurement Plasmid }} <br />
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===09.08.2010===<br />
*overnight cultures inoculated from Glycerolstock J06702(mCherry generator) in pSB1A2 from Christoph.<br />
===10.08.2010===<br />
*MiniPrep of pSB1A2-mCherry using ZymoKit<br />
*Digestion<br />
**pSB1A2-mCherry E/P<br />
**pSB1A2-mCherry X/P<br />
**pSB1A10-HisTerm S/P<br />
**pSB1A10-TrpTerm S/P<br />
**pSB1A10-HisTerm E/P<br />
*Purified with agarose gel (1%)<br />
**Gel Doc broken => no picture<br />
**Description: mCherry cut was ok, Plasmid was cut at least once (linear DNA), generally contaminated with genomic DNA<br />
*Ligation<br />
**50 ng Plasmid and 34 ng Insert<br />
**ca. 30min @ RT<br />
*Transformation of DH5a cells with ligation samples<br />
(=> no colonies the next day)<br />
<br />
*overnight cultures<br />
**pSB1A2-mCherry from Christoph`s stock<br />
**pSB1A10-HisTerm from earlier plate<br />
**pSB1A10-TrpTerm from earlier plate<br />
(=> pSB1A10-TrpTerm and pSB1A10-HisTerm did not grow until next day)<br />
<br />
<br />
===11.08.2010===<br />
*MiniPrep of pSB1A2-mCherry using ZymoKit<br />
*analytic gel of Mini preps and ligation of the previous day<br />
**preps still hold genomic DNA<br />
**mCherry Plasmid runs at ca. 2400 bp<br />
<br />
*Digestion<br />
**pSB1A2-mCherry E/P<br />
**pSB1A2-mCherry X/P<br />
*Purified with agarose gel (1%)<br />
**Gel Doc broken => no picture<br />
**Description: mCherry cut was ok, stil contaminated with genomic DNA<br />
*Ligation<br />
**Plasmid (from previous day) and mCherry-Insert<br />
**ca. 30 min @ RT<br />
*Transformation of DH5a cells with<br />
**ligation samples (=> no colonies the next day)<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
<br />
*overnight cultures<br />
**pSB1A2-mCherry from Christoph`s stock<br />
<br />
<br />
===12.08.2010===<br />
*MiniPrep of pSB1A2-mCherry using ZymoKit<br />
*analytic gel of Mini preps and ligation of the previous day<br />
**preps still hold genomic DNA<br />
**mCherry Plasmid runs at ca. 2400 bp<br />
**Gel (1%)<br />
[[Image:TUM2010_100812 ligation100811 mChPrep.jpg]] <br />
<br />
<br />
<br />
*Digestion<br />
**pSB1A2-mCherry X/P<br />
**pSB1A10-HisTerm S/P<br />
**pSB1A10-TrpTerm S/P<br />
*Purified with agarose gel (1%)<br />
[[Image:TUM2010_100812 DigestionmCh pSB1A10.jpg|600px]] <br />
<br />
*Ligation<br />
**Plasmid and mCherry-Insert<br />
**ca. 30 min @ RT<br />
*Transformation of DH5a cells with<br />
**ligation samples (=> no colonies the next day)<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
<br />
*overnight cultures<br />
**pSB1A2-mCherry from Christoph`s stock<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
<br />
'''Caution: ran out of gas => not steril?'''<br />
<br />
===13.08.2010===<br />
*MiniPrep using ZymoKit<br />
**pSB1A2-mCherry<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
*analytic gel of Mini preps and ligation of the previous day<br />
**low concentration<br />
**Gel (1%)<br />
[[Image:TUM2010_100813 Prep-mChHisTrp Ligation100812.jpg|600px]]<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week21{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=Construction of new Measurement Plasmid }}<br />
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<br />
===16.08.2010===<br />
*Concentrating MiniPrep-Samples using ZymoKit<br />
**pSB1A2-mCherry<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
<br />
*Digestion<br />
**pSB1A2-mCherry E/P<br />
**pSB1A2-mCherry X/P<br />
*Purified with agarose gel (1%)<br />
[[Image:TUM2010_100816 Digestion-mCherry.jpg]]<br />
=> no mCherry band!!!<br />
<br />
*Purification using Zymo Concentrator Kit<br />
**pSB1A10-HisTerm S/P<br />
**pSB1A10-TrpTerm S/P<br />
<br />
*Ligation<br />
**Plasmid (from earlier date) and mCherry-Insert ('''from 11.08''')<br />
**ca. 30 min @ RT<br />
**'''used new ligase and new ligase buffer'''<br />
<br />
*Transformation of DH5a cells with<br />
**ligation samples (=> '''colonies found the next day''')<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
**pSB1A10-RFP (BioBrick Standard)<br />
<br />
*overnight cultures<br />
**pSB1A2-mCherry from Christoph`s stock<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
<br />
===17.08.2010===<br />
*MiniPrep using ZymoKit<br />
**pSB1A2-mCherry<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
<br />
*Digestion<br />
**pSB1A2-mCherry X/P<br />
**pSB1A10-HisTerm S/P<br />
**pSB1A10-TrpTerm S/P<br />
: => heating block went up to 50°C<br />
*Purified "digestion" samples with ZymoKit => stored for next day<br />
<br />
<br />
<br />
<br />
*Picked 12 colonies from previous day's ligation<br />
: => Colony PCR => Gel (2%)<br />
[[Image:TUM2010_100817 colonyPCR pSB1A10-mCherry.jpg|600px]]<br />
<br />
<br />
*Purified with agarose gel (1%)<br />
**Gel Doc broken => no picture<br />
**Description: mCherry cut was ok, stil contaminated with genomic DNA<br />
*Ligation<br />
**Plasmid (from previous day) and mCherry-Insert<br />
**ca. 30 min @ RT<br />
*Transformation of DH5a cells with<br />
**ligation samples (=> no colonies the next day)<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
<br />
*overnight cultures<br />
**pSB1A10-RFP (plate from previous day)<br />
**pSB1A2-mCherry<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
<br />
<br />
<br />
'''PROBLEM:<br />
mCherry has a SgrA1-cleavage site! These constructs cannot be used. Starting all over, cloning the linker sequence first...'''<br />
<br />
===18.08.2010===<br />
*MiniPrep using ZymoKit<br />
**pSB1A2-mCherry<br />
**pSB1A10-HisTerm<br />
**pSB1A10-TrpTerm<br />
**pSB1A10-RFP<br />
<br />
*Analytical agarose gel (1%):<br />
[[Image:TUM2010_100818 MiniPreps HisTrpCherryRFP.jpg|600px]]<br />
<br />
*Digestion<br />
**pSB1A10-HisTerm SgrAI/PstI<br />
**pSB1A10-TrpTerm SgrAI/PstI<br />
**pSB1A10-RFP SgrAI/PstI<br />
: =>RFP has a SrgAI cleaving site. Discarded RFP digestion.<br />
*preparativ agarose gel (1%):<br />
[[Image:TUM2010_100818 Digestion HisTrp2.jpg|600px]]<br />
<br />
*Soubilization of SrgAI-PstI Linker<br />
<br />
*Ligation<br />
**Plasmid His-Term(Trp-Term) and Linker<br />
**ca. 30 min @ RT<br />
*Transformation of DH5a cells with<br />
**ligation samples<br />
<br />
*overnight cultures<br />
**pSB1A2-mCherry<br />
**pSB1A10-TrpTerm<br />
<br />
===19.08.2010===<br />
*MiniPrep using ZymoKit<br />
**pSB1A2-mCherry<br />
*analytic agarose gel (1%) from various mCherry Preps<br />
[[Image:TUM2010_100819 MiniPreps mCherry2.jpg]]<br />
<br />
<br />
*Picked 6 colonies from pSB1A10-HisTerm-linker and pSB1A10-TrpTerm-linker each (previous day's ligation)<br />
: => Colony PCR => Gel (1.5%)<br />
[[Image:TUM2010_100819 ColonyPCRlinker2.jpg|600px]]<br />
<br />
<br />
*overnight cultures (600µl)<br />
**pSB1A2-mCherry<br />
**pSB1A2-R0011<br />
**pSB1A10-HisTerm-linker (#7, 11, 12)<br />
**pSB1A10-TrpTerm-linker (#1, 2, 4)<br />
<br />
===20.08.2010===<br />
*MiniPrep using ZymoKit<br />
**pSB1A2-mCherry<br />
**pSB1A2-R0011<br />
**pSB1A10-TrpLinker (picked Colonies)<br />
**pSB1A10-HisLinker (picked Colonies)<br />
<br />
*analytical Digestion<br />
**pSB1A10-HisLinker SgrAI /EcoRI<br />
**pSB1A10-HisLinker NsiI<br />
**pSB1A10-TrpLinker SgrAI /EcoRI<br />
<br />
*analytical agarose gel (1.5%)<br />
[[Image:TUM2010_100820gel1verdau.jpg]]<br />
<br />
*preparativ Digestions<br />
**pSB1A2-mCherry EcoRI /PstI<br />
**pSB1A2-mCherry XbaI /PstI<br />
**pSB1A2-R0011 SpeI /PstI<br />
**pSB1A10-HisLinker SpeI /PstI<br />
**pSB1A10-HisLinker EcoRI /PstI<br />
**pSB1A10-TrpLinker SpeI /PstI<br />
**pSB1A10-TrpLinker EcoRI /PstI<br />
**PCR_BB1006 XbaI /PstI<br />
<br />
*agarose gel (1.5%):<br />
[[Image:TUM2010_100820gel1verdau2.jpg]]<br />
<br />
*agarose gel (1.0%):<br />
[[Image:TUM2010_100820gel3verdau.jpg]]<br />
<br />
*agarose gel (1.0%):<br />
[[Image:TUM2010_100820gel2verdau.jpg|600px]]<br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week22{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=Construction of new Measurement Plasmid }}<br />
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<br />
===23.08.2010===<br />
*Ligation<br />
**pSB1A10-HisLinker SpeI /PstI + mCherry XbaI /PstI<br />
**pSB1A10-HisLinker EcoRI /PstI + mCherry EcoRI /PstI<br />
**pSB1A10-TrpLinker SpeI /PstI + mCherry XbaI /PstI<br />
**pSB1A10-TrpLinker EcoRI /PstI + mCherry EcoRI /PstI<br />
**pSB1A2-R0011 SpeI /PstI + PCR_BB1006 XbaI /PstI<br />
<br />
*Transformation of DH5a cells<br />
<br />
===24.08.2010===<br />
*Picked 2 colonies per plate (previous day's ligation)<br />
**R0011_B1006Sig<br />
**Trp1_mCherry<br />
**Trp2_mCherry<br />
**His7_mCherry<br />
**His11_mCherry<br />
**pSB1A10_mCherry<br />
<br />
*Colony PCR of picked colonies with prefix/suffix primers<br />
<br />
*analytical agarose gel 1 (1.5%)<br />
[[Image:TUM2010_100824ColonyPCRgel1.jpg|600px]]<br />
<br />
*analytical agarose gel 1 (1.5%) <br />
[[Image:TUM2010_100824ColonyPCRgel2.jpg|600px]] <br><br />
Trp=R0011, R0011= Trp :)<br><br />
<br />
Faint bands at the correct length can be guessed. <br />
*overnight cultures (5 ml)<br><br />
** pSB1A10_TrpTerm_mCherry_linker<br />
** pSB1A10_HisTerm_mCherry_linker<br />
** pSB1A10_mCherry_linker<br />
<br />
===25.08.2010===<br />
*Miniprep using Zymo Miniprep-Classic Kit:<br />
** pSB1A10_TrpTerm_mCherry_linker<br />
** pSB1A10_HisTerm_mCherry_linker<br />
** pSB1A10_mCherry_linker<br />
<br />
*analytical digestions<br />
** pSB1A10_TrpTerm_mCherry_linker EcoRI /PstI<br />
** pSB1A10_HisTerm_mCherry_linker EcoRI /PstI<br />
** pSB1A10_mCherry_linker EcoRI /PstI<br />
<br />
*analytical agarose gel 1 (1.0%)<br />
[[Image:TUM2010_100825verdaugel1.jpg|600px]]<br />
<br />
*Picked 2 colonies per plate (day before yesterday's ligation)<br />
**R0011_B1006Sig<br />
**Trp1_mCherry<br />
**Trp2_mCherry<br />
**His7_mCherry<br />
**His11_mCherry<br />
**pSB1A10_mCherry<br />
<br />
*Colony PCR of picked colonies with prefix/suffix primers<br />
** Program: colonypcr<br />
<br />
*analytical agarose gel 1 (1.5%)<br />
[[Image:TUM2010_100825colonypcrGEL1.jpg|600px]]<br />
<br />
*analytical agarose gel 1 (1.5%) <br />
[[Image:TUM2010_100825colonypcrGEL2.jpg|600px]]<br />
<br />
<br />
*overnight cultures (5 ml)<br><br />
** pSB1A10_TrpTerm_mCherry_linker<br />
** pSB1A10_HisTerm_mCherry_linker<br />
** pSB1A10_mCherry_linker<br />
===26.08.2010===<br />
*Miniprep using Zymo Miniprep-Classic Kit:<br />
** pSB1A10_TrpTerm_mCherry_linker<br />
** pSB1A10_HisTerm_mCherry_linker<br />
** pSB1A10_mCherry_linker<br />
**pSB1A2_R0011_B1006<br />
<br />
*PCR<br />
** Trp-Signal (R0011_Sig_B0014)<br />
<br />
** His-Signal (R0011_Sig_B0014)<br />
** Terminator B0014<br />
<br />
*analytical digestions<br />
** pSB1A10_mCherry_linker EcoRI /PstI<br />
*preparative digestion<br />
** pSB1A10_TrpTerm_mCherry_linker SpeI/PstI<br />
** pSB1A10_HisTerm_mCherry_linker SpeI/PstI<br />
**pSB1A2_R0011_B1006 SpeI/PstI<br />
** PCR Trp-Sig XbaI/PstI<br />
** PCR His-Sig XbaI/PstI<br />
** B0014 XbaI/PstI<br />
<br />
*preparative agarose gel 1 (1.0%)<br />
[[Image:TUM2010_100826 prep Verdau.jpg|600px]]<br />
last lane: pSB1A10_His11_mCherry SpeI/PstI<br />
<br />
*analytical agarose gel (1.0 %)<br />
[[Image:TUM2010_100826anaVerdauPCRControl.jpg|600px]]<br />
*Ligations<br />
** pSB1A10_TrpTerm_mCherry_linker + Trp-Signal (R0011_Sig_B0014)<br />
** pSB1A10_HisTerm_mCherry_linker + His-Signal (R0011_Sig_B0014)<br />
**pSB1A2_R0011_B1006 + Terminator B0014<br />
*Transformation of Ligation product in DH5alpha cells<br />
*Transformation of pSB1A10_mCherry_linker in BL21<br />
===27.08.2010===<br />
*Mini-Prep<br />
** pSB1A2_R0011_B1006 <br><br />
for sequencing<br />
*Colony PCR<br />
**2 colonies per plate<br />
<br />
[[Image:TUM2010_100827 coloypcr 2.jpg|600px]]<br />
R0011=R0011_B1006!!!<br />
*Sequencing<br />
** pSB1A2_R0011_B1006 4b with primer Biobrick VR<br />
** pSB1A10mod_mCherry 27b with primer GFP_FP and Biobrick VR<br />
** pSB1A10mod_mCherry 32a with primer GFP_FP and Biobrick VR<br />
<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week23{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=Construction of new Measurement Plasmid }}{{:Team:TU_Munich/Templates/BlueBox | text=Testing new Measurement Plasmid }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===30.08.2010===<br />
*Colony PCR <br />
**picked two colonies per plate from 26.08' ligation <br />
**Program: colonypcr, modified elongation time: 1.15 instead of 1.00<br />
<br />
*analytical agarose gel (1.5%)<br><br />
<br />
<u>'''Gel 1:'''</u><br> <br />
<br />
[[Image:TUM2010_100830colonyPCR gel1.jpg|600px]] <br />
<br />
=&gt; <span style="color: rgb(255, 0, 0);">samples named "1x" to "8x" for pSB1A2-R0011-BB1006Sig-B0014 colonies</span><br> <br />
<br />
=&gt; <span style="color: rgb(255, 0, 0);">samples named "9x" to "12x" for pSB1A10mod-TrpTerm-mCherry-TrpSig colonies</span><br> <br />
<br />
<u>'''Gel 2:'''</u> <br />
<br />
[[Image:TUM2010_100830colonyPCR gel2.jpg|600px]] <br />
<br />
=&gt; <span style="color: rgb(255, 0, 0);">samples named "13x" to "16x" for pSB1A10mod-TrpTerm-mCherry-TrpSig colonies</span><br> <br />
<br />
=&gt; <span style="color: rgb(255, 0, 0);">samples named "17x" to "24x" for pSB1A10mod-HisTerm-mCherry-HisSig colonies</span> <br />
<br />
'''Interpretation for Gel1 and Gel2''': <br />
<br />
Ligation worked for the samples 1x-6x (pSB1A2-R0011-BB1006Sig-B0014), 9x-16x (pSB1A10mod-TrpTerm-mCherry-TrpSig), 18x-24x (pSB1A10mod-HisTerm-mCherry-HisSig) <br />
<br />
<br> <br />
<br />
<br> <br />
<br />
*over night cultures: <br />
**pSB1A10mod_HisTerm_mCherry_HisSignal <br />
**pSB1A10mod_TrpTerm_mCherry_TrpSignal <br />
**PSB1A2_R0011_BB1006_B0014<br />
<br />
<br />
*Received sequencing results from GATC. All Sequences are okay: <br />
**pSB1A10mod_mCherry (27b) and (32a) <br />
**pSB1A2_R0011_BB1006 (4b)<br />
<br />
===31.08.2010===<br />
*'''Fluorescence measurement (positive control experiment):'''<br />
**Settings: GFP-Excitation: 501 nm; mCherry-Excitation: 587 nm;<br />
**endpoint measurements of:<br />
***Timepoints of measurement: 3 h after induction and 9 h after induction (1.5 h and 7 h for 15x-sample)<br />
***Samples:<br />
****pSB1A10mod-mCherry (27b) in BL21 cells, induced with ca. 0.4% L-Arabinose<br />
****pSB1A10mod-mCherry (27b) in BL21 cells, not induced<br />
**kinetic measurement of induced (0.4% L-Arabinose) BL21 cells carrying pSB1A10mod-mCherry (27b)<br />
<br />
*'''Results:'''<br />
**NO mCherry signal detected at all: The GFP signal shows a nice and strong increase; the RFP channel did not change at all.<br />
**GFP signal looks perfect: strong if induced, neglectable if not!<br />
::=> System seems not capable of serving as a testing system for our switches! <br />
<br />
*'''Glycerol stocks'''<br />
**in DH5a cells:<br />
***pSB1A10mod-TrpTerm-mCherry-TrpSig (9x)<br />
***pSB1A10mod-TrpTerm-mCherry-TrpSig (15x)<br />
***pSB1A10mod-TrpTerm-mCherry-TrpSig (10x) (sequence verified)<br />
***pSB1A10mod-HisTerm-mCherry-HisSig (15x)<br />
***pSB1A10mod-HisTerm-mCherry-HisSig (18x)<br />
***pSB1A10mod-HisTerm-mCherry-HisSig (23x) (sequence verified)<br />
***pSB1A10mod-mCherry (32a) (sequence verified)<br />
***pSB1A10mod-mCherry (27b) (sequence verified)<br />
***pSB1A2mod R0011-BB1006Sig-B0014 (2x) (sequence verified)<br />
***pSB1A2mod R0011-BB1006Sig-B0014 (3x)<br />
***pSB1A2mod R0011-BB1006Sig (4b) (sequence verified)<br />
**in BL21 cells:<br />
***pSB1A10mod-mCherry (27b) (sequence verified)<br />
<br />
:"x" refers to Colony-PCR of 30.08.2010<br />
<br />
*5ml '''Over night cultures'''<br />
**pSB1A10mod-TrpTerm-mCherry-TrpSig (9x, 15x, 10x)<br />
**pSB1A10mod-HisTerm-mCherry-HisSig (18x, 23x, 15x)<br />
**pSB1A2mod R0011-BB1006Sig-B0014 (2x, 3x)<br />
<br />
===01.09.2010===<br />
*'''MiniPrep''' using Zymo classical kit. Samples:<br />
**pSB1A10mod-TrpTerm-mCherry-TrpSig (9x, 15x, 10x)<br />
**pSB1A10mod-HisTerm-mCherry-HisSig (18x, 23x, 15x)<br />
**pSB1A2mod R0011-BB1006Sig-B0014 (2x, 3x)<br />
<br />
*'''Fluorescence measurement (positive control experiment):'''<br />
**endpoint measurements:<br />
***Timepoints of measurement: 3 h after induction and 9 h after induction (1.5 h and 7 h for 15x-smaple)<br />
***Settings: GFP-Excitation: 501 nm; mCherry-Excitation: 587 nm; RFP-Excitation: 584 nm<br />
***Samples:<br />
****pSB1A10mod-mCherry (32a) in DH5a cells, induced with ca. 0.4% L-Arabinose<br />
****pSB1A10mod-mCherry (32a) in DH5a cells, not induced<br />
****pSB1A10mod-mCherry (32a) in BL21 DE3 cells, induced with ca. 0.4% L-Arabinose<br />
****pSB1A10mod-mCherry (32a) in BL21 DE3 cells, not induced<br />
****pSB1A10-RFP, in DH5a cells, induced with ca. 0.4% L-Arabinose<br />
****pSB1A10-RFP, in DH5a cells, not induced<br />
****pSB1A10mod-TrpTerm-mCherry-TrpSig (15x), in DH5a cells, induced with ca. 0.4% L-Arabinose<br />
****pSB1A10mod-TrpTerm-mCherry-TrpSig (15x), in DH5a cells, not induced<br />
**'''Results:'''<br />
***Very strong RFP signal in pSB1A10-RFP, induced and not induced<br />
***For the first time we saw a weak but easily-detectable mCherry signal in positive control samples (pSB1A10mod-mCherry) 3 hours after induction! There was hardly no difference between the uninduced and the induced control samples for mCherry. The GFP signals was strong for induced control experiments and very weak for not induced samples! The pSB1A10mod-TrpTerm-mCherry-TrpSig sample also showd a small mCHerry signal.<br />
***After 9 hours the mCherry signals were generally reduced, whereas the GFP signals were still high in all induced samples and low in all uninduced samples.<br />
:::=> Although we saw mCherry for the first time (!), the signal is to weak not reproducable! As a consequence the system can not be used to serve as a measure for our switches! Furthermore the settings of the fluorometer are ok, since we saw strong RFP signal.<br />
===02.09.2010===<br />
'''Starting the cloning of pBAD (BioBrick I13453) downstream of GFP'''<br />
<br />
*'''Amplifing''' the Arabinose-inducable promotor pBAD<br />
**Resuspending the BioBrick I13453 with 10 µl in well 1F in the 2010 Distribution<br />
**PCR using 1 µl template (programm "igempcr")<br />
**Purified using DNA Concentrator (ZymoKit)<br />
<br />
*Digestion (EcoRI and SpeI) of PCR product and heat inactivation (20 min @ 80°C)<br />
<br />
*Digestion of the target vectors using EcoRI and XbaI<br />
**Samples:<br />
***pSB1A10mod-TrpTerm-mCherry-TrpSig (9x, 10x)<br />
***pSB1A10mod-HisTerm-mCherry-HisSig (23x, 24x)<br />
***pSB1A10mod-mCherry (32a, 27b)<br />
***pSB1A10mod-TrpTerm-mCherry (10b, 13b)<br />
***pSB1A10mod-HisTerm-mCherry (18b, 24b)<br />
**Purified using 1% agarose gel:<br />
<br />
[[Image:TUM2010_100902 Digestion 1.jpg|700px]]<br />
<br />
:=> '''Interpretation:''' Gel is overloaded! However digestion seemed to work since the bands show correct masses.<br />
<br />
*Extraction of bands at ca. 6000 bp<br />
<br />
*10 µl '''ligation''' of 50 ng of each digested vector with 8ng insert<br />
<br />
*'''Transformation''' of DH5a cells using 8 µl ligation sample<br />
===03.09.2010===<br />
*Colony PCR <br />
**picked two colonies per plate from 02.09' ligation <br />
**Note: no PCR because Thermocycler was occupied<br />
<br />
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{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week24{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=''in vivo'' constructs }} {{:Team:TU_Munich/Templates/BlueBox | text=Testing HisTrp }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===06.09.2010===<br />
*Colony PCR <br />
**using colonies picked on 03.09.<br />
<br />
*analytical agarose gel (1.5%)<br />
[[Image:TUM2010_100906ColonyPCR 1.jpg|600px]]<br />
<br />
*K1= pSB1A10mod_HisTerm_mCherry_HisSig<br />
*K2 = pSB1A10mod_TrpTerm_mCherry_TrpSig<br />
<br />
*5ml over night cultures:<br />
**positive control pSB1A10mod_Pbad_mCherry<br />
**negative His control pSB1A10mod_Pbad_HisTerm_mCherry<br />
**His-Switch pSB1A10mod_Pbad_HisTerm_mCherry_HisSig<br />
**Trp-Switch pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSig<br />
<br />
===07.09.2010===<br />
*MiniPrep using Zymo Classical kit<br />
**pSB1A10mod_Pbad_mCherry<br />
**pSB1A10mod_Pbad_HisTerm_mCherry<br />
**pSB1A10mod_Pbad_HisTerm_mCherry_HisSig<br />
**pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSig<br />
<br />
<br />
*Fluorescence measurements<br />
**Induction with 0.2% L-arabinose<br />
**Measurement of OD600<br />
**Fluorescence measurement at 30 min, 150 min (only Pos.Control) and 4.5 h<br />
**Samples:<br />
::pSB1A10mod_Pbad_mCherry<br />
::pSB1A10mod_Pbad_HisTerm_mCherry<br />
::pSB1A10mod_Pbad_HisTerm_mCherry_HisSig<br />
::pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSig<br />
<br />
*Transformation of BL21 (DE3)<br />
**pSB1A10mod_Pbad_mCherry<br />
**pSB1A10mod_Pbad_HisTerm_mCherry<br />
**pSB1A10mod_Pbad_HisTerm_mCherry_HisSig<br />
**pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSig<br />
<br />
===08.09.2010===<br />
*Glycerolstocks<br />
:30%Glycerol in LB_Carb<br />
**pSB1A10mod_Pbad_mCherry<br />
**pSB1A10mod_Pbad_HisTerm_mCherry<br />
**pSB1A10mod_Pbad_HisTerm_mCherry_HisSig<br />
**pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSig<br />
<br />
*Fluorescence measurements<br />
**Induction with 0.2% L-arabinose<br />
**Measurement of OD600<br />
**Fluorescence measurement at 24 h and different OD600<br />
**Samples:<br />
::pSB1A10mod_Pbad_mCherry<br />
-->OD600 0.05 reasonable for our cell measurements. Positive control works fine after 24 h induction.<br />
<br />
[[Image:TUM2010_Graph2.jpg|600px]]<br />
[[Image:TUM2010_Graph3.jpg|600px]]<br />
*5ml cultures of BL21 cells<br />
**pSB1A10mod_Pbad_mCherry<br />
**pSB1A10mod_Pbad_HisTerm_mCherry<br />
**pSB1A10mod_Pbad_HisTerm_mCherry_HisSig<br />
**pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSig<br />
<br />
*ColonyPCR<br />
**picked 4 Colonies per Neg.TrpControl from 02.09' ligation plates<br />
**PCR using Program 'colonyPCR'. Elongation time modified to 1:20min<br />
<br />
===09.09.2010===<br />
*Fluoresence measurement<br />
**using BL21 cells<br />
**samples:<br />
***Positive control (pSB1A10mod_pBAD_mCherry)<br />
***Negative control (pSB1A10mod_pBAD_HisTerm_mCherry)<br />
***pSB1A10mod_pBAD_HisTerm_mCherry_HisSignal<br />
***pSB1A10mod_pBAD_TrpTerm_mCherry_TrpSignal<br />
**Induction with 0.4% L-arabinose and 1mM IPTG<br />
**Timepoints: 1 h, 2 h, 4 h, 10 h, 16 h (induced the day before)<br />
::(OD checked seperately each time; average ODs=0.02-0.06)<br />
<br />
*30% glycerol stocks of BL21 cells carrying:<br />
**Positive control (pSB1A10mod_pBAD_mCherry)<br />
**Negative control (pSB1A10mod_pBAD_HisTerm_mCherry)<br />
**pSB1A10mod_pBAD_HisTerm_mCherry_HisSignal<br />
**pSB1A10mod_pBAD_TrpTerm_mCherry_TrpSignal<br />
<br />
===10.09.2010===<br />
*Fluorescence measurements<br />
**Induction with 0.4% L-arabinose and 1 mM IPTG<br />
**Measurement of OD600<br />
**Fluorescence measurement at 15 h and 26 h <br />
*Samples:<br />
::pSB1A10mod_Pbad_mCherry - Positive Control<br />
::pSB1A10mod_Pbad_HisTerm_mCherry - HisNeg. Control<br />
::pSB1A10mod_Pbad_HisTerm_mCherry_HisSignal<br />
::pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSignal<br />
[[Image:TUM2010_Graph100910 2.jpg|600px]]<br />
[[Image:TUM2010_Graph100910 3.jpg|600px]]<br />
[[Image:TUM2010_Graph100910 1.jpg|600px]]<br />
[[Image:TUM2010_Graph100910 4.jpg|600px]]<br />
<br />
*Conclusion:<br />
His-Switch DOES NOT seem to work!!! Measurements of Trp-Switch look weird, since Ara-induced culture showed strong mCherry signal than Ara+IPTG-induced culture. Maybe growing condition were not sufficient. We will use 50 ml flask next time!<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week25{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=Cloning }} {{:Team:TU_Munich/Templates/BlueBox | text=HisTerm/TrpTerm }} {{:Team:TU_Munich/Templates/GreenBox | text=Ordering Aptamer }} {{:Team:TU_Munich/Templates/YellowBox | text=New Measurement Plasmid }}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===13.09.2010===<br />
*<i>In vitro</i> Kit measurement:<br />
**Test of <i>In vitro</i> kit with positive control<br />
**1 h on 37°C<br />
::--> no significant mCherry signal detectable. <i>In vitro</i> Kit measurements aborted. <br />
<br />
*20ml cultures for Fluorescence measurements<br />
**Samples:<br />
::pSB1A10mod_Pbad_mCherry - Positive Control<br />
::pSB1A10mod_Pbad_HisTerm_mCherry - HisNeg. Control<br />
::pSB1A10mod_Pbad_TrpTerm_mCherry - TrpNeg. Control<br />
::pSB1A10mod_Pbad_HisTerm_mCherry_HisSignal<br />
::pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSignal<br />
<br />
:*Growth on 37°C until OD600 = 0.6, then induction and growth on 25°C over night<br />
:*each sample uininduced, induced with 0.4% L-arabinose and 0.4% L-arabinose + 1 mM IPTG,respectively<br />
<br />
* 30% Glycerol stock in LB<br />
**pSB1A10mod_Pbad_TrpTerm_mCherry - TrpNeg. Control<br />
<br />
===14.09.2010===<br />
*Fluorescence measurements<br />
**Induction with 0.4% L-arabinose and 1mM IPTG<br />
**Measurement of OD600<br />
**Fluorescence measurement at 16 h<br />
*Samples:<br />
::pSB1A10mod_Pbad_mCherry - Positive Control<br />
::pSB1A10mod_Pbad_HisTerm_mCherry - HisNeg. Control<br />
::pSB1A10mod_Pbad_TrpTerm_mCherry - TrpNeg. Control<br />
::pSB1A10mod_Pbad_HisTerm_mCherry_HisSignal<br />
::pSB1A10mod_Pbad_TrpTerm_mCherry_TrpSignal<br />
<br />
[[Image:TUM2010_PosControl140910.jpg|500px|inline]][[Image:TUM2010_PosControlklein.JPG|200px|inline]]<br />
[[Image:TUM2010_TrpNegativeControl140910.jpg|500px]][[Image:TUM2010_TrpNegativeControlklein.JPG|200px]]<br />
[[Image:TUM2010_HisNegativeControl140910.jpg|500px]][[Image:TUM2010_HisNegativeControlklein.JPG|200px]]<br />
[[Image:TUM2010_TrpSwitch140910.jpg|500px]][[Image:TUM2010_TrpSwitchklein.JPG|200px]]<br />
[[Image:TUM2010_HisSwitch140910.jpg|500px]][[Image:TUM2010_HisSwitchklein.JPG|200px]]<br />
===15.09.2010===<br />
*Waiting for Mr.Gene...<br />
<br />
===16.09.2010===<br />
*Searching for Mr.Gene package...<br />
::--> our new Testsystem pMalachitApt_BB1006<br />
*PCR and PCR purification with Zymo DNA Clean&Concentrator<br />
**pMalachitApt_BB1006 with terminator (Primer: Apt_For and AptFull_wT_Rev)<br />
**pMalachitApt_BB1006 without terminator (Primer: Apt_For and AptPart_woT_Rev)<br />
**Switches<br />
::Trp-Switch<br />
::His-Switch<br />
:*Signals<br />
::His-Sig with Term (template: pSB1A2_R0011_HisSig_B0014)<br />
::Trp-Sig with Term (template: pSB1A2_R0011_TrpSig_B0014)<br />
::BB1006Sig with Term (template: pSB1A2_R0011_BB1006Sig_B0014)<br />
::BB1006Sig without Term (template: pSB1A2_R0011_BB1006Sig)<br />
::HisSig (ssDNA) (template: original biomers order)<br />
::TrpSig (ssDNA) (template: original biomers order)<br />
<br />
===17.09.2010===<br />
*Digestion<br />
**pMalachitApt_BB1006 with EcoRI/PstI<br />
**pMalachitApt_BB1006 with XbaI/SpeI<br />
**Switches<br />
::Trp-Switch with EcoRI/PstI<br />
::His-Switch with EcoRI/PstI<br />
:*Signals<br />
::His-Sig with XbaI/PstI<br />
::Trp-Sig with XbaI/PstI<br />
<br />
*Purification using Qiagen Kit<br />
::--> Cut off size of Qiagen kit too high. Lost all Switches and Signals<br />
<br />
*Purification pMalachitApt using preparative agarose gel<br />
<br />
*new PCR:<br />
**Switches<br />
::Trp-Switch<br />
::His-Switch<br />
:*Signals<br />
::HisSig (ssDNA) (template: original biomers order)<br />
::TrpSig (ssDNA) (template: original biomers order)<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week26{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=Cloning of Malachit green constructs }} {{:Team:TU_Munich/Templates/ClearBox | text=Measurements }} {{:Team:TU_Munich/Templates/GreenBox | text=HisTerm/TrpTerm }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===20.09.2010===<br />
*PCR Purification using Qiagen MinElute Kit<br />
**Trp-Signal<br />
**His-Signal<br />
**Trp-Switch<br />
**His-Switch<br />
<br />
*Digestions 37°C, 2h<br />
**Trp-Signal XbaI/PstI<br />
**His-Signal XbaI/PstI<br />
**Trp-Switch EcoRI/PstI<br />
**His-Switch EcoRI/PstI<br />
::--> Heat inactivation 20 min, 80°C<br />
<br />
*Ligation<br />
**pSB1A2_R0011 SpeI/PstI + Trp-Signal XbaI/PstI<br />
**pSB1A2_R0011 SpeI/PstI + His-Signal XbaI/PstI<br />
**pMalachit EcoRI/PstI + Trp-Switch EcoRI/PstI<br />
**pMalachit EcoRI/PstI + His-Switch EcoRI/PstI<br />
**pMalachit XbaI/SpeI<br />
<br />
*Transformation of DH5a cells with ligation<br />
<br />
===21.09.2010===<br />
*Picked 2 colonies per plate of yesterday's transformation => we call them z-Series<br />
*ColonyPCR of z-Series<br />
*analytical agarose gel (2.5%)<br />
:=> ColonyPCR did not work. Probably not enough template.<br />
<br />
*Picked futher colonies<br />
*2nd ColonyPCR of z-Series<br />
<br />
===22.09.2010===<br />
*analytical agarose gel (2.5%) of 2nd colonyPCR of z-Series<br />
<br />
:pSB1A2-R0011-TrpSignal looks good. We removed all pSB1A2-R0011-HisSignal samples since there is a contamination on the gel and colonies turned redish. No conclusion regarding the other ligations of z_series can be drawn. LB control produces similar bands as to what we expected => Choose new Primers for PCR.<br />
<br />
*3rd colonyPCR of z-Series (samples 1-16z)<br />
**used Primers Apt_For and Apt_Part_woT instead of BioBrick Primers (G1005 and G1004). Bands should be about 100bp longer.<br />
<br />
*analytical agarose gel (2%)<br />
<br />
===23.09.2010===<br />
*PCR to obtain DNA for malachite green <i>in vitro</i> measurements<br />
**samples from z-Series (see [[21.09.2010]])<br />
::2z (XbaI/SpeI religated positive control)<br />
::16z (pMalachitApt_HisSig positive control)<br />
:*each samples was amplificated with two sets of primers (including and excluding the terminator BB0014):<br />
::Apt_For and AptFull_wT_Rev<br />
::Apt_For and AptPart_woT_Rev<br />
<br />
*Purified with Qiagen MinElute Kit<br />
<br />
*analytical digestion of z-series ligation:<br />
**EcoRI/PstI:<br />
::2z (XbaI/SpeI religated positive control)<br />
::5z (pMalachitApt_TrpTerm)<br />
::12z (pMalachitApt_HisTerm)<br />
::16z (pMalachitApt_HisSig positive control)<br />
::28z (pSB1A2_R0011_TrpSig)<br />
:*NsiI only (HisTerm contains one cutting site for NsiI, so does pMalachiteApt => we expect 2 fragments):<br />
::12z (pMalachitApt_HisTerm)<br />
<br />
*analytical agarose gel:<br />
<br />
<br />
*10 µl ligations using digested samples from [[21.09.2010]]<br />
**pSB1A2_R0011 (SpeI/PstI cut) + HisSig (XbaI/PstI cut)<br />
**pMalachiteApt (EcoRI/PstI cut) + TrpSwitch (EcoRI/PstI cut)<br />
**pMalachiteApt (EcoRI/PstI cut) + HisSwitch (EcoRI/PstI cut)<br />
<br />
*Transformed DH5a cell with 8µl of the above ligation samples<br />
===24.09.2010===<br />
*Fluorescence measurement: Malachite Green Assay<br />
**Samples<br />
::2z positive control (X/S religated: tac_MalachitApt) (PCR-amplified without BB0014 Terminator)<br />
::2z positive control (X/S religated: tac_MalachitApt_BB0014) (PCR-amplified with BB0014 Terminator)<br />
::negative control (tac_BB1006Switch_MalachitApt_BB0014) (PCR-amplified with BB0014 Terminator)<br />
::tac_BB1006Switch_MalachitApt + tac_BB1006Signal (both PCR-amplified without BB0014 Terminator)<br />
:*Sample Mix:<br />
::2 µl sigma70 satured RNA <i>E.Coli</i> Polymerase (epicenter Biozyme) (2U)<br />
::ca. 1µg DNA template<br />
::5 µM Malachite Green<br />
::10 µM DTT<br />
::4 µM NTPs<br />
::40 mM Tris-HCl pH = 7.1 @ 37°C; 7.5 @ 22°C<br />
::10 mM MgCl2<br />
::150 mM KCl<br />
::added H20 to total volume of 100 µl<br />
:*Kinetics measurement @ 37°C<br />
::*Excitation 630 nm<br />
::*Emission 655 nm<br />
[[Image:TUM2010_100924 MalachitKineticsPosControl BB1006.jpg|600px]]<br />
::*Results: <br />
:::no apatmer formation observed => positive controls did not work at all => assay did not work!!<br />
<br />
:*Scanning measurements<br />
::*Exitation: 630 nm<br />
::*Results: <br />
:::no detactable peaks between 640nm and 800nm in any sample at any measured time (Settings were checked by detecting scattering peaks).<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week27{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/ClearBox | text=Cloning }} {{:Team:TU_Munich/Templates/ClearBox | text=Measurements }} {{:Team:TU_Munich/Templates/GreenBox | text=T7 Switch }} <br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
<br />
===27.09.2010===<br />
*Fluoresence measurements<br />
**Cary Spectrometer<br />
**samples:<br />
***with T7 RNA Polymerase (NEB)<br />
:::T7 positive control (T7Promotor_MalachitAptamer)<br />
::*with Epicenter E coli RNA Polymerase<br />
:::2z positive control (X/S religated: tac_MalachitApt) (PCR-amplified without BB0014 Terminator)<br />
:::2z positive control (X/S religated: tac_MalachitApt_BB0014) (PCR-amplified with BB0014 Terminator) (NO KINETICS RECORDED)<br />
::*with in vitro kit (cell lysate)<br />
:::2z positive control (X/S religated: tac_MalachitApt) (PCR-amplified without BB0014 Terminator)<br />
:::2z positive control (X/S religated: tac_MalachitApt_BB0014) (PCR-amplified with BB0014 Terminator)<br />
:*Scanning measurements: excitation at 630nm<br />
:*Kinetics measurements: excitation at 630nm; Emission at 655nm<br />
<br />
[[Image:TUM2010_100927Beginn.jpg]]<br />
<br />
<br />
[[Image:TUM2010_100927kinetik2.jpg]]<br />
<br />
<br />
'''Results:'''<br />
T7 Polymerase works perfectly. E coli Polymerase also produced RNA but much less (however enzyme might show reduced activity due to storage problems). In vitro (Cell lysate) kits do not work at all.<br />
<br />
===01.10.2010===<br />
'''T7-Measurments'''<br />
* PCR of switch_phi_T7-construct to obtain dsDNA (conditions: igemPCR and 30s elongation time). Samples were purified using Qiagen MinElute.<br />
<br />
* 1th measurment: <br> Maxi´s NEB buffer conditions: 40 mM Tris pH 7.4 @ RT, 40mM Mg2Cl, 5 µM Malachit-green, 4 mM NTPs, 2,5 U RNA-Polymerase <br>All DNA templates were all added to a final concentration of 200nM.<br />
**sample 1: Positive control<br />
**sample 2: negative control (= switch without any signal) ('''new DTT''' used for this sample)<br />
**sample 3: switch + SigA1a<br />
**sample 4: switch + SigA1c<br />
*Results<br />
**kinetic results <br> [[Image:TUM2010_kinetik-T7.JPG|600px]]<br />
**Spectra <br> [[Image:TUM2010_spektren.JPG|Spektren|600px]]<br />
<br />
=> Switches do look quite good. However DTT was not the same in all samples.<br />
<br />
<br />
* 2th measurment:<br />
**"Paper´s" buffer conditions: 40 mM Tris pH 7.9 @ RT, 6mM Mg2Cl, 5 µM Malachit-green, 100 mM KCl, 0.8 mM NTPs, 2,5 U RNA-Polymerase<br />
***sample 1: Positive control<br />
***sample 2: negative control (= switch without signal)<br />
**Maxi´s NEB buffer conditions: 40 mM Tris, 40 mM Mg2Cl, 10 µM Malachit-green, ph 7.4 @ RT<br />
***sample 3: Positive control ('''new DTT''' used for this sample)<br />
***sample 4: Negative control (= switch without signal)<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week28{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/ClearBox | text=Cloning }} {{:Team:TU_Munich/Templates/ClearBox | text=Measurements }} {{:Team:TU_Munich/Templates/GreenBox | text=T7 Switch }}<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===04.10.2010===<br />
*1st fluorescence measurement<br />
:*Samples:<br />
::*200 nM T7-Switch (100 nM each strand) + SignalA_1a<br />
::*200 nM T7-Switch (100 nM each strand) + SignalA_2a<br />
::*200 nM T7-Switch (100 nM each strand) + SignalA_2b<br />
::*200 nM T7-Switch (100 nM each strand) + SignalA_2c<br />
:*Buffer:<br />
::40 mM Tris pH 7.1<br />
::40 mM MgCl2<br />
::10 mM DTT<br />
::1.6 mM NTPs<br />
::5 µM Malachite green<br />
::2.5 U T7 RNA Polymerase<br />
:*Results:<br />
:[[Image:TUM2010_10104_Kinetik.jpg|600px]]<br />
:Fluorescence increased in all samples in a very similar way, suggesting all signal to work equally well.<br />
<br />
*2nd fluorescence measurement<br />
:*Samples:<br />
::*200 nM T7-Switch only (Buffer A)<br />
::*200 nM T7-Switch only (Buffer B)<br />
::*positiv control (T7Promoter_MalchitAptamer) (Buffer A)<br />
::*positiv control (T7Promoter_MalchitAptamer) (Buffer B)<br />
:*Buffer A:<br />
::40 mM Tris pH 7.1 @ RT<br />
::40 mM MgCl2<br />
::10 mM DTT<br />
::1.6 mM NTPs<br />
::'''10 µM''' Malachite green<br />
::2.5 U T7 RNA Polymerase<br />
:*Buffer B:<br />
::40 mM Tris pH 7.9 @ RT<br />
::6 mM MgCl2<br />
::100 mM KCl<br />
::10 mM DTT<br />
::1.6 mM NTPs<br />
::'''10 µM''' Malachite green<br />
::2.5 U T7 RNA Polymerase<br />
:*Results:<br />
:[[Image:TUM2010_10104_Kinetik2.jpg|600px]]<br />
::*Very strange results!! Both negative controls increased strongly compared to the positive controls that only slightly increased (buffer A seems to be better for transcription than buffer B).<br />
:::=> Maybe something went very wrong. However, last Friday we observed a similar, strange behavior. Maybe the positive control is no good choice. It would make sence to use T7-Switch+SignalA_1a as a reference.<br />
::*Signals were generally stronger. 10 µM of malachite green seems to be quite good.<br />
<br />
===05.10.2010===<br />
* T7 ''in vitro measurements''<br />
<br />
** First measurement of the day: Signal, 1d, 2c, 3b<br />
*** Master Mix:<br />
*** 204 µl 2x Buffer<br />
*** 40.8 µl DTT<br />
*** 8.16 µl NTPs<br />
*** 21.05 µl Switch (1.94 µM)<br />
*** signals: # volume µl<br />
*** negative control - 1.213<br />
*** 1d - 1.290<br />
*** 2c - 1.450<br />
*** 3b - 1.376<br />
[[Image:TUM2010_10105_Kinetik1.jpg|600px]]<br />
<br />
** PCR with positive control and T7 switch<br />
*** Mastermix:<br />
PCR T7 switch/positive signal<br />
** 32 x Mastermix: 1600 µl total volume<br />
*** 32 µl dNTPs<br />
*** 32 µl T7 forward primer<br />
*** 32 µl T7 reverser primer<br />
*** 160 µl 10x buffer<br />
*** 96 µl MgCl2<br />
*** 6.4 µl Taq Polymerase<br />
*** 10 µl Template (some older PCR)<br />
*** 1209.6 µl H2O<br />
<br />
** Second measurement of the day: Signal negative control, nonsense signal, 1a (with 400 µM signal), 1a (with 2 µM signal)<br />
*** *** Master Mix:<br />
*** 204 µl 2x Buffer<br />
*** 40.8 µl DTT<br />
*** 8.16 µl NTPs<br />
*** 21.05 µl Switch (1.94 µM)<br />
*** signals: # volume µl<br />
*** negative control - 30.34<br />
*** 1d - 1.213<br />
*** 2c - 2.672<br />
*** 3b - 13.36<br />
<br />
[[Image:TUM2010_10105_Kinetik2.jpg|600px]]<br />
<br />
** Evaluation of different malachite green concentrations<br />
*** 5 µM, 10 µM, 15 µM, 25 µM chosen<br />
*** high temperature dependency<br />
*** Equilibration of samples important<br />
<br />
===06.10.2010===<br />
* PCR<br />
** yesterday's PCR Purification using Zymo Clear and Concentrated<br />
*** yields<br />
*** Switch: 164 ng/µl<br />
*** positive control: 28 ng/µl<br />
*** problems with the temperature? Yield too low!<br />
<br />
** PCR of positive control<br />
*** annealing temperature set to 48°C<br />
*** 20 x Mastermix:<br />
*** 20 µl dNTPs<br />
*** 20 µl T7 forward primer<br />
*** 20 µl T7 reverser primer<br />
*** 100 µl 10x buffer<br />
*** 60 µl MgCl2<br />
*** 4 µl Taq Polymerase<br />
*** 1 µl Template (some older PCR)<br />
*** 775 µl H2O<br />
<br />
===07.10.2010===<br />
* T7 Trancription<br />
** new buffer:<br />
*** Stocks, volume for 20 ml, endconcentration in 1x<br />
*** 1M Tris/HCl, 1.6 ml, 40 mM<br />
*** 500 mM MgCl2, 3.2 ml, 40 mM<br />
*** 250 mM malachite green, 1.6 ml, 20 µM<br />
*** H20<br />
** Measurement<br />
*** 4.1 x, switch, switch + nonsense, switch + 1a, switch + 1c<br />
*** 21.16 µl switch, 1.93 µM<br />
*** 10.25 µl RPO<br />
*** 205 µl buffer<br />
*** 41 µl DTT 100 µM<br />
*** 20.5 µl rNTPs<br />
*** H2O<br />
<br />
[[Image:TUM2010_10107_Kinetik1.jpg|600px]]<br />
<br />
**second measurement<br />
** Measurement<br />
*** 4.1 x, switch, switch + nonsense, positive control + 1b, positive control + 1b<br />
*** 21.16 µl switch, 1.93 µM<br />
*** 10.25 µl RPO<br />
*** 205 µl buffer<br />
*** 41 µl DTT 100 µM<br />
*** 20.5 µl rNTPs<br />
*** H2O<br />
<br />
<br />
[[Image:TUM2010_10107_Kinetik2.jpg|600px]]<br />
<br />
===08.10.2010===<br />
<br />
<br />
[[Image:TUM2010_101008_Kinetik2.jpg|600px]]<br />
<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week29{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/ClearBox | text=Cloning }} {{:Team:TU_Munich/Templates/ClearBox | text=Measurements }} {{:Team:TU_Munich/Templates/GreenBox | text=T7 Switch }}<br />
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<br />
===11.10.2010===<br />
* PCR T7 switch<br />
** 32 x Mastermix: 1600 µl total volume<br />
*** 32 µl dNTPs<br />
*** 32 µl T7 forward primer<br />
*** 32 µl T7 reverser primer<br />
*** 160 µl 10x buffer<br />
*** 96 µl MgCl2<br />
*** 6.4 µl Taq Polymerase<br />
*** 10 µl Template (some older PCR)<br />
*** 1209.6 µl H2O<br />
<br />
** Standart iGEM PCR program:<br />
*** 95°C, 2' <br />
*** 1) 95°C, 0,5'<br />
*** 2) 58°C, 0.5'<br />
*** 3) 71°C, 1'<br />
*** 71°C, 7'<br />
<br />
** repeat 1)-3) 35 times<br />
** yield after purification: 80 µl, 1.14 µM<br />
<br />
*Malchitegreen measurement<br />
** for 4.1 x 100 µl<br />
*** 35.88 µl PCR product<br />
*** 20.05 dNTPs<br />
*** 41 µl DTT<br />
*** 10.25 µl T7 RPO<br />
*** 8.75 µl switch<br />
*** 2 µl signal per 100 µl<br />
<br />
** measured signals: (1) nonsense, (2) 1a, (3) 1b, (4) 2a<br />
** Voltage set to 1000 (maximum)<br />
** 38°C, every 15 s measurement<br />
**[[Image:TUM2010_101011_Kinetik.jpg|600px]]<br />
** start at 5 au, end at 25 au<br />
** rise visible over time in all samples, nonsense signal with the fastest rise, 1a similiar, end signal similiar in all measurements<br />
<br />
===12.10.2010===<br />
* PCR T7 switch<br />
** 32 x Mastermix: 1600 µl total volume<br />
*** 32 µl dNTPs<br />
*** 32 µl T7 forward primer<br />
*** 32 µl T7 reverser primer<br />
*** 160 µl 10x buffer<br />
*** 96 µl MgCl2<br />
*** 6.4 µl Taq Polymerase<br />
*** 10 µl Template (some older PCR)<br />
*** 1209.6 µl H2O<br />
<br />
** '''changed''' iGEM PCR program:<br />
*** 95°C, 2' <br />
*** 1) 95°C, 0,5'<br />
*** 2) 50°C, 0.5'<br />
*** 3) 71°C, 1'<br />
*** 71°C, 10'<br />
*** repeat 1) - 3) 35 times<br />
<br />
** --> melting temperature primers: 55/54°C!!!<br />
** yield after purification: 300 ng/µl in 60 µl, 3.66 µM<br />
<br />
* Malachitegreen measurement with preincubation of transcription stuff with signals<br />
<br />
** for 4.1 x 100 µl<br />
*** 35.88 µl PCR product<br />
*** 20.05 dNTPs<br />
*** 41 µl DTT<br />
*** 10.25 µl T7 RPO<br />
*** 2 µl signal per 100 µl<br />
<br />
** preincubation of signal with RPO for one hour, 37°C <br />
** addition of signal after one hour (2.735 µl)<br />
** once done in eppis (low bind), once in cuvettes<br />
** no rise in both<br />
** incubation and measurement of cuvette-incubated mix over night: no rise visible<br />
<br />
* 15 % denaturing acrylamide gels<br />
** for 50 ml<br />
*** 15 % acrylamide: 18.75 ml 40 % acrylamide<br />
*** 6M urea: 18 g<br />
*** 1x TBE: 5 ml 10x TBE<br />
*** 500 µl APS<br />
*** 50 µl TEMED<br />
*** H20 till 50 ml<br />
<br />
** only 30 ml needed for two gels<br />
** big combs for a lot of sample :)<br />
** glass plates cleaned with RNAseZip before pouring the gel<br />
===13.10.2010===<br />
* in vitro T7 transcription, check by polyacrylamide gel electrophoresis <br />
** 10 ml 5 x paper buffer<br />
*** 200 mM Tris/HCl, pH=7.85: 2 ml 1M Tris/HCl, pH=7.85<br />
*** 30 mM MgCl2: 600 µl 0.5 M MgCl2<br />
*** 500 mM KCl: 5 ml 1 M KCl<br />
*** 2.4 ml H2O<br />
<br />
** for in vitro transcription<br />
*** 0.5 µl T7 RPO: 25 U<br />
*** 0.5 µl rNTPs : 20 mM<br />
*** 1.36 µl switch: 250 µM<br />
*** 1 µl signal: 250 µM<br />
*** 2 µl DTT: 10 µM<br />
*** 0.2 µl RNase inhibitor<br />
*** 4 µl 5x buffer<br />
*** 10.44 µl H2O<br />
<br />
**5.1 times<br />
** no signal, nonsense, 1a, 1c, 2a<br />
** in low bind tubes<br />
** 2 hour, 37°C<br />
** actually 2 hours and half a hour pocket cleaning time<br />
<br />
** for PAGE:<br />
*** don't try to run yourgel in the pouring device - if you do so: feel very stupid and embarrassed (I guess I've ran over 100 gels in my life yet... still too stupid...) <br />
*** don't feel tempted to use the Dietz' group's 0.5 x TBE buffer - if you do so: feel very stupid and embarassed, discard buffer carefully and use 1 x TBE<br />
*** don't use a comb which is thinner than your spacers - if you do so: scrap gel pieces out of the pockets for half an hour<br />
*** cook your sample for 5 minutes, 95°C - if you do not so, feel stupid, embarassed and hope that it won't matter so much<br />
*** I did not cool the samples<br />
*** gel runs at 100 V (~ 10V/cm)<br />
*** 20 µl sample + 20 µl ambion loading buffer (1-2 x loading buffer - who does that?) --> 40 µl fits nicely<br />
*** 2 µl low molecular weight marker + 20 µl loading buffer + 18 µ H2O<br />
*** in 1 x TBE<br />
<br />
** M, control (same amounts switch and signals as in the samples), no signal, random, 1a, 2a, 1c, random overnight, 1a overnight, 1c overnight<br />
** overnight samples: measurement with malachitegreen sample over night: no rise visible<br />
** Xylene Cyanol: Comigrating with 60, Bromphenol BLue: comigrating with 15 (http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=845) - Maxi: bei Hälfte ungefähr<br />
** run for 1:45 hours, Bromphenoleblue at about half of the gel<br />
<br />
[[Image:TUM2010_101013 PAGE1.png|600px]]<br />
<br><br />
:--> c=control, ns=no signal, r=random<br />
: --> weird smear everywhere: degraded RNA?<br />
: --> no switch visible on the gel: 133 bp!<br />
: --> signal length: 29-43 bp visible<br />
: --> 25 bp: signal sense, between 50-25 bp: signals: 1a and 1c with approximately the same length, 2a is longer<br />
: --> Wie Sie sehen, sehen Sie nichts.<br />
<br />
*PCR of switch: Biomers original used as template<br />
* PCR T7 switch<br />
** 32 x Mastermix: 1600 µl total volume<br />
*** 32 µl dNTPs<br />
*** 32 µl T7 forward primer<br />
*** 32 µl T7 reverser primer<br />
*** 160 µl 10x buffer<br />
*** 96 µl MgCl2<br />
*** 6.4 µl Taq Polymerase<br />
*** 5 µl Template (some older PCR)<br />
*** 1214.6 µl H2O<br />
<br />
** '''changed''' iGEM PCR program:<br />
*** 95°C, 2' <br />
*** 1) 95°C, 0,5'<br />
*** 2) 50°C, 0.5'<br />
*** 3) 71°C, 1'<br />
*** 71°C, 10'<br />
*** repeat 1) - 3) 35 times<br />
===14.10.2010===<br />
* yesterday's measurement<br />
**[[Image:TUM2010_101014_Kinetik.jpg|600px]]<br />
** rise visible, comparable to tuesday measurement<br />
** samples frozen, put on 15 % PAGE<br />
<br />
* purification of yesterday's PCR<br />
** yield: <br />
<br />
* in vitro T7 transcription, check by polyacrylamide gel electrophoresis <br />
** 10 ml 5 x paper buffer<br />
*** 200 mM Tris/HCl, pH=7.85: 2 ml 1M Tris/HCl, pH=7.85<br />
*** 30 mM MgCl2: 600 µl 0.5 M MgCl2<br />
*** 500 mM KCl: 5 ml 1 M KCl<br />
*** 2.4 ml H2O<br />
<br />
** for in vitro transcription<br />
*** 0.5 µl T7 RPO: 25 U<br />
*** 0.5 µl rNTPs : 20 mM<br />
*** 1.36 µl switch: 250 µM<br />
*** 1 µl signal: 250 µM<br />
*** 2 µl DTT: 10 µM<br />
*** 0.2 µl RNase inhibitor<br />
*** 4 µl 5x buffer<br />
*** 10.44 µl H2O<br />
<br />
**5.1 times<br />
** no signal, nonsense, 1a, 1c, 2a<br />
** in low bind tubes<br />
** 2 hour, 37°C<br />
** actually 2 hours and half a hour pocket cleaning time<br />
<br />
* 2 % agarose gel to check previous PCR products<br />
[[Image:TUM2010_101014 .png]]<br />
:--> yesterdays results bad because no switch :) <br />
<br />
*15 % 6 M urea PAGE<br />
<br />
[[Image:TUM2010 PAGE 101014.png|600px]]<br />
<br />
:--> M = low molecular weight marker (NEB)<br />
:--> c=control=all DNAs mixed together in used concentrations: switch and all signals, ns=no signal, r=random=nonsense<br />
:--> r, 1a, 2a on the left: overnight incubation with malchitegreen<br />
:--> r, 1a, 1c, 2a on the right: two hour in vitro transcription without malachitegreen<br />
<br><br />
: --> switch: 133 bp! <br />
: --> signal length: 29-43 bp<br />
: --> T7 promoter sense: ca. 20 bp (I can't look it up right now) <br />
<br><br />
:--> extra bands visible after overnight transcription (rise in malchitegreen fluorescence visible)<br />
:--> switch runs at a strange height: WHY? looks normal on 2 % agarose gel<br />
:--> upper band: RPO bound to DNA? Compare EMSA<br />
:--> DNA ladder, RNA bands: How to compare?<br />
<br><br />
: Okay, let's try to interpret this:<br />
: Denaturing conditions, everything precooked: No guarantee for double stranded DNA/RNA<br />
: control: switch and signals from tubes on gel - otherwise treated the same<br />
: --> internal standart: switch at about 80 bp (low molecular weight standart) equals 133 bp<br />
: --> band seen in all samples at the height of random/1c --> termination product of switch??? (expected size: about 90 bp? does not fit at all?!)<br />
: --> lower band visible, what is it?<br />
: --> What is left: NO Differences Between Random control (=nonsense) and Designed Switches...<br />
<br />
<br />
* New malachitegreen assay (overnight in vitro transcription) with new PCR product<br />
<br />
** for 4.1 x 100 µl<br />
*** 35.88 µl PCR product<br />
*** 20.05 dNTPs<br />
*** 41 µl DTT<br />
*** 10.25 µl T7 RPO<br />
*** 2 µl signal per 100 µl<br />
<br />
** preincubation of signal with RPO at 37°C<br />
** no rise during preincubation (makes sense)<br />
** addition of signal after about one and a half hour<br />
<br />
===15.10.2010===<br />
* in vitro transcription - malachite green<br />
<br />
** slight rise visible after overnight incubation after preincubation of signal<br />
** only 1/4 of the intensity measured yesterday<br />
** spectra fit but very weak signal, scattered spectra<br />
<br />
*Next step: addition of DNaseI (RNase free) to overnight transcription products<br />
<br />
*Felt ill, went home soon<br />
<br />
===17.10.2010===<br />
*Malachite-green measuring assay<br />
<br />
** 1x:<br />
*** 2 µl signal, 5 µM<br />
*** 4.66 µl switch, 176 µM<br />
*** 2.5 µl RPO<br />
*** 50 µl Paperbuffer<br />
*** 10 µl DTT 100 µM<br />
*** 5 µl rNTPs<br />
*** 25.84 µl H2O<br />
<br><br><br />
<br />
** 4.1 x<br />
*** 19.11 µl switch, 176 µM<br />
*** 10.25 µl RPO<br />
*** 205 µl Paperbuffer<br />
*** 41 µl DTT 100 µM<br />
*** 20.5 µl rNTPs<br />
*** 105.94 µl H2O<br />
<br><br><br />
** Signals: random, 1a, 2a, 1c<br />
<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week30{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=Cloning of Parts into pSB1C3}} {{:Team:TU_Munich/Templates/ClearBox | text=Measurements }} {{:Team:TU_Munich/Templates/GreenBox | text=T7 & E. coli }} <br />
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===18.10.2010===<br />
*Yesterday's malachitegreen-binding assay<br />
**Kinetics<br />
[[Image:TUM2010_101018_Kinetics.png|600px]]<br />
<br />
--> Favourite one ever so far!<br />
<br />
** Emission and Excitation: Exc=530 nm, Em=552 nm<br />
<br />
[[Image:TUM2010_101018_Scan.png|600px]]<br />
<br />
*Biomers order arrived<br />
** DNA resolved according to manual, 100 µM endconcentration<br />
<br />
* PCR T7 switch/positive signal<br />
** 32 x Mastermix: 1600 µl total volume<br />
*** 32 µl dNTPs<br />
*** 32 µl T7 forward primer<br />
*** 32 µl T7 reverser primer<br />
*** 160 µl 10x buffer<br />
*** 96 µl MgCl2<br />
*** 6.4 µl Taq Polymerase<br />
*** 10 µl Template (some older PCR)<br />
*** 1209.6 µl H2O<br />
<br />
** '''changed''' iGEM PCR program:<br />
*** 95°C, 2' <br />
*** 1) 95°C, 0,5'<br />
*** 2) 50°C, 0.5'<br />
*** 3) 71°C, 1'<br />
*** 71°C, 10'<br />
*** repeat 1) - 3) 35 times<br />
<br />
** yields positive control:<br />
*** Mr=67234,6 g/mol <br />
***230 ng/µl --> 3.42 µM<br />
*** 198 ng/µl --> 2.94 µM<br />
*** 209 ng/µl --> used for cloning<br />
*** 178 ng/µl --> 2.65 µM<br />
<br />
** yields switch:<br />
*** Mr=82059 g/mol<br />
*** 170 ng/µl --> 2.07 µM<br />
*** 204 ng/µl --> 2.49 µM<br />
*** 179 ng/µl --> 2.18 µM<br />
*** 160 ng/µl --> 1.95 µM<br />
<br />
* 6 M urea 15 % acrylamid PAGE<br />
** DNase I digestion<br />
*** 1 µl 10 x DNase buffer<br />
*** 1 µl DNaseI <br />
*** 20 µl reaction product from malachitegreen assay, 17.10.10<br />
*** 37°C, 90 minutes<br />
<br />
** Gel:<br />
*** 20 µl loading buffer and 20 µl sample<br />
*** <br />
<br />
* Malachitegreen assay with double stranded signals<br />
<br />
** Concentration verification of sense and antisense in ng/µl<br />
*** diluted 1:8 (5+35 µl)<br />
*** signal - sense ng/µl - antisense ng/µl<br />
*** 1a - 1391 - 1584<br />
*** 2b - 1812 - 1823<br />
*** 1c - 2208 - 1575<br />
*** random2=nonsense2 - 2480 - 1903<br />
<br><br />
** Concentration verification of sense and antisense in µM<br />
*** signal - sense µM - antisense µM<br />
*** 1a - 15.47 - 17.99<br />
*** 2b - 15.43 - 15.70<br />
*** 1c - 16.95 - 12.31<br />
*** r2 - 21.27 - 16.28<br />
<br />
<br><br />
** for 10 µM of both sense and antisense and to put it together to equal 5 µM in total...<br />
*** signal - sense µl - antisense µl - water µl<br />
*** 1a - 32.32 - 27.79 - 38.89<br />
*** 2b - 32.40 - 31.85 - 35.75<br />
*** 1c - 29.50 - 40.62 - 29.88<br />
*** r2 - 23.51 - 30.71 - 45.78<br />
<br />
** Measurement<br />
** 1x switch<br />
<br />
*** 2 µl signal, 5 µM, double stranded!<br />
*** 4.17 µl switch, 2.49 µM<br />
*** 2.5 µl RPO<br />
*** 50 µl Paperbuffer<br />
*** 10 µl DTT 100 µM<br />
*** 5 µl rNTPs<br />
*** 26.3 µl H2O<br />
<br />
<br />
** 3.1 x<br />
*** 12.93 µl switch, 176 µM<br />
*** 7.75 µl RPO<br />
*** 155 µl Paperbuffer<br />
*** 31 µl DTT 100 µM<br />
*** 15.5 µl rNTPs<br />
*** 81.53 µl H2O<br />
*** signals: r2, 1a, 1c<br />
** 1x positive control<br />
*** 2 µl signal, 5 µM, double stranded!<br />
*** 2.94 µl switch, 2.94 µM<br />
*** 2.5 µl RPO<br />
*** 50 µl Paperbuffer<br />
*** 10 µl DTT 100 µM<br />
*** 5 µl rNTPs<br />
*** 27.57 µl H2O<br />
*** signal: r2<br />
<br />
* Cloning Malachitegreen-binding aptamer into pB1C3<br />
** E/P digestion<br />
*** 2 µl 10x NEB 4<br />
*** 2 µl 10x BSA<br />
*** 0.5 µl EcoRI<br />
*** 0.5 µl PstI<br />
*** 15 µl linearized pB1C3 (50 ng/µl)/malachitegreen binding aptamer (203 ng/µl)<br />
*** 37°C, 1 h<br />
*** purification afterwards using DNA clean and concentrated (or something)<br />
<br />
** Concentrations<br />
*** backbone: 11 ng/µl<br />
*** insert: <br />
<br />
** Ligation<br />
*** 5 µl digested plasmid<br />
*** 2 µl malachitegreen binding aptamer, 1:10 diluted<br />
*** 2 µl T4 ligase buffer<br />
*** 1 µl T4 ligase<br />
*** 10 µl water<br />
<br />
** no transformation not possible: no chloramphenicol plates in physic's department...<br />
<br />
===19.10.2010===<br />
* Yesterday's malachitegreen assay<br />
** Kinetics:<br />
[[Image:TUM2010_101019_Kinetik.png|600px]]<br />
** Emission spectra: Exc=530 nm<br />
[[Image:TUM2010_101019_Em.png|600px]]<br />
<br />
** Excitation spectra: Em=552 nm<br />
[[Image:TUM2010_101019_Exc.png|600px]]<br />
<br />
* 7 M urea, 15 % PAGE<br />
<br />
** DnaseI testdigestion<br />
*** plasmid digestion to test DnaseI activity: <br />
*** 10 µl paper buffer<br />
*** 0.2 µl DTT<br />
*** 2 µl 10x DnaseI buffer<br />
*** 1 µl Dnase<br />
*** 1 µl plasmid (some random thing from Wuschel)<br />
*** 5.8 µl water<br />
*** 37°C, 2h<br />
<br />
** samples for DNaseI digestion<br />
*** 2 µl 10x DnaseI Buffer<br />
*** 1 µl DnaseI<br />
*** 17 µl reaction product from malachitegreen assay, 18.10.10<br />
*** 37°C, 2 h<br />
<br />
** 7M urea, 15 % PAGE<br />
*** 30 ml<br />
*** 11.25 ml acrylamide<br />
*** 12.6 g urea<br />
*** 3 ml 10x TBE<br />
*** 300 µl 10 % APS<br />
*** 30 µl TEMED<br />
*** H20 to 30 ml<br />
*** heat a bit and sonificate<br />
** gel could not be run: pockets not solid...<br />
*** most likely reason: too hot when radical starter were added: instant polymerization were they hit the mixture...<br />
<br />
* malachitegreen assay with more malachitegreen this time!<br />
** --> 3x more than usual<br />
** 30 mM 2x malachitegreen paper buffer<br />
*** 1 ml 5x paper buffer without malachitegreen<br />
*** 0.6 ml 250 mM malachitegreen<br />
*** 0.9 ml Water<br />
<br />
** 1x switch<br />
*** 2 µl signal, 5 µM, double stranded!<br />
*** 4.17 µl switch, 2.49 µM<br />
*** 2.5 µl RPO<br />
*** 50 µl Paperbuffer<br />
*** 10 µl DTT 100 µM<br />
*** 5 µl rNTPs<br />
*** 26.3 µl H2O<br />
*** signals: 1a, r2<br />
** 1x positive control<br />
*** 2 µl signal, 5 µM, double stranded!<br />
*** 2.94 µl switch, 2.94 µM<br />
*** 2.5 µl RPO<br />
*** 50 µl Paperbuffer<br />
*** 10 µl DTT 100 µM<br />
*** 5 µl rNTPs<br />
*** 27.57 µl H2O<br />
*** signal: r2, none<br />
<br />
* Cloning Malachitegreen-binding aptamer into pB1C3<br />
<br />
** Ligation<br />
*** 5 µl digested plasmid<br />
*** 2 µl malachitegreen binding aptamer, 1:10 diluted<br />
*** 2 µl T4 ligase buffer<br />
*** 1 µl T4 ligase<br />
*** 10 µl water<br />
<br />
** Transformation<br />
*** borrowed plates from the Prof. Groll's department<br />
*** and from Prof. Becker's<br />
*** the one from Prof. Becker's once contained tetracyclin...<br />
*** in DH5alpha cells<br />
*** 200 µl plated<br />
*** overnight, 37°C<br />
<br />
===20.10.2010===<br />
* Yesterday's malachite green binding assay<br />
** Kinetics: <br />
[[Image:TUM2010_101020 kinetics.png|600px]]<br />
<br />
** Emission spectra: Exc=530 nm<br />
[[Image:TUM2010_101020 em.png|600px]]<br />
<br />
** Excitation spectra: Em=552 nm<br />
[[Image:TUM2010_101020_exc.png|600px]]<br />
<br />
* Cloning Malachitegreen-binding aptamer into pB1C3<br />
** many colonies grown<br />
** Well done, Flo!<br />
** Colony PCRl<br />
** 10 x Mastermix: 500 µl total volume<br />
*** 10 µl dNTPs<br />
*** 10 µl G1004<br />
*** 10 µl G1005<br />
*** 50 µl 10x buffer<br />
*** 30 µl MgCl2<br />
*** 2 µl Taq Polymerase<br />
*** 2 µl Template per reaction<br />
*** H2O<br />
<br><br />
** PCR program<br />
*** iGEM program!<br />
*** 58°C annealing<br />
<br />
<br />
** 2.5 % agarose gel<br />
[[Image:TUM2010_101020_ColonyPCR.png|600px]]<br />
*** lmw: low molecular weight (ladder)<br />
*** 100: 100 kb (ladder)<br />
*** 7 clones chosen to be checked by Colony PCR<br />
*** LB=negative control=LB-Chloramphenicol<br />
*** positive control: PCR with Biomer product<br />
** also on the gel:<br />
*** S=switch=PCR of switch used for measurement<br />
*** P=positive control=PCR product used for measurement and cloning<br />
<br><br />
** Interpretation<br />
*** stupid negative control looks just the same like everything else<br />
*** two bands at approximately the right height, but exactly above and below positive control band<br />
*** shit.<br />
<br />
** Further proceedings<br />
*** 5 ml cultures of clones 3, 4, 5, 7<br />
*** 5 ml cultures of 6 new colonies<br />
*** minipreps and analytical digestions tomorrow<br />
<br />
<br />
<br />
* 7 M urea 15% PAGE<br />
<br />
** samples from 19.10.10<br />
[[Image:TUM2010_101020 page.png|600px]]<br />
<br />
** controls: <br />
*** switch: PCR of switch, used in measurements<br />
*** P: PCR of positive control, used in measurements<br />
*** Plas: random test plasmid used for evaluation of DnaseI activity<br />
** DNase digested samples: 20 µl<br />
*** Plas: DnaseI digested random plasmid, for conditions check previous day<br />
*** P+r2: Positive control together with double stranded nonsense 2/random 2 signal after overnight transcription in paper buffer (check malachitegreen assay, previous day), DnaseI digested<br />
*** S+r2: Switch together with double stranded nonsense 2/random 2 signal after overnight transcription in paper buffer, DnaseI digested<br />
*** S+1a: Switch together with double stranded 1a signal after overnight transcription in paper buffer, DnaseI digested<br />
*** S+1c: Switch together with double stranded 1c signal after overnight transcription in paper buffer, DnaseI digested<br />
** other: 15 µl<br />
*** P+r2: Positive control together with double stranded nonsense 2/random 2 signal after overnight transcription in paper buffer (check malachitegreen assay, previous day)<br />
*** S+r2: Switch together with double stranded nonsense 2/random 2 signal after overnight transcription in paper buffer<br />
*** S+1a: Switch together with double stranded 1a signal after overnight transcription in paper buffer<br />
** no marker this time! Only 12 lanes :)<br />
<br><br />
** Interpretation:<br />
*** no real differences between DnaseI digested and not<br />
*** It is rather stupid to use a plasmid to check for DnaseI activity if you want to run the result on a 15 % gel. A plasmid with some kb does not seperate... But: DNA visible, without Dnase I, no DNA visible with DnaseI (stuck in the upper lane, 1)<br />
*** Next try with PCR product maybe<br />
*** weird lane in all PCR reactions at 2 in both cases and in all other gels and a bit also in agarose gels visible<br />
*** positive control runs totally elsewhere than switch even though they do not really vary in length (3)<br />
<br />
<br />
* Malachitegreen binding assay<br />
** Measurement<br />
** 1x switch<br />
*** 2 µl signal, 5 µM, double stranded!<br />
*** 4.17 µl switch, 2.49 µM<br />
*** 2.5 µl RPO<br />
*** 50 µl Paperbuffer<br />
*** 10 µl DTT 100 µM<br />
*** 5 µl rNTPs<br />
*** 26.3 µl H2O<br />
<br />
** measured: signals all doublestranded, everything in paper buffer, 10 µM malachitegreen<br />
*** positive control<br />
*** positive control with nonsense2/random2<br />
*** switch with nonsense2/random2<br />
*** switch with 1a<br />
<br />
===21.10.2010===<br />
* Yesterday's measurement<br />
[[Image:TUM2010_101021_Kinetik.png|600px]]<br />
<br />
** Fluorescence spetrum, Exc=530 <br />
[[Image:TUM2010_101021_Em.png|600px]]<br />
<br />
** Fluorescence excitation spectrum, Em= 552<br />
[[Image:TUM2010_101021_Exc.png|600px]]<br />
<br />
<br />
* Amplifying in vivo positive control to send for PartsRegistry<br />
** Miniprep of 5 ml overnight control<br />
*** P1 185 ng/µl <br />
*** P2 416 ng/µl<br />
*** check below for gel picture and further proceeding<br />
<br />
*Cloning Malachitegreen-binding aptamer into pSB1C3 <br />
** Mini-prep of 5 ml overnight cultures<br />
*** # - concentration in ng/µl<br />
*** 3 - 272 <br />
*** 4 - 59<br />
*** 5 - 100<br />
*** 7 - 95.56<br />
*** a - 86<br />
*** b - 165<br />
*** c - 114<br />
*** d - 131<br />
*** e - 232<br />
*** f - 144<br />
*** P1 - 185<br />
*** P2 - 416<br />
<br />
** Control digestion<br />
*** 4 µl plasmid<br />
*** 2 µl NEB 3<br />
*** 0.5 µl PstI<br />
*** 0.5 µl EcoRI-HF<br />
*** 13 µl H2O<br />
*** 1 h, 37°C<br />
<br />
** 2 % agarose gel<br />
[[Image:TUM2010_101021_control_digestion.png|600px]]<br />
*** lmw: low molecular weight ladder (NEB)<br />
*** 100 bp: 100 bp ladder (NEB)<br />
*** 3,4,5,7: yesterday's clones, checked and falsified by colony PCR<br />
*** a-g: yesterday picked clones<br />
*** P1, P2: minpreps of in vivo measurement positive control, to be sent to partsregistry<br />
<br />
** Interpreation<br />
*** well, something went seriously wrong while cloning<br />
*** asked at physic's department: DH5alpha cells in use were pretty old: cell from old -80°C are baaaad!<br />
*** showed us new stock<br />
*** further possibilities: EcoRI nearly empty --> not cutting properly anymore<br />
*** T4 ligation buffer: went through 4 tubes until I found one which was not totally full of precipitates<br />
*** Chloramphenicol-plates: got plates from Groll group, plates from 2008<br />
<br />
** Further cloning procedure<br />
*** digestion of linearized pSB1C3 (from parts registry) and positive control with EcoRI-HF (full enzyme)<br />
*** digestion of T7 positive control<br />
*** 15 µl linearized pSB1C3 (25 ng/µl, damn it, I thought there are 50 ng/µl while preparing the ligation...)/5 µl of positive control, PCR product (c=173 ng/µl) + 10 µl H2O<br />
*** 2 µl 10x NEB 3<br />
*** 2 µl 10x BSA<br />
*** 0.5 µl EcoRI<br />
*** 0.5 µl PstI<br />
*** 37°C, 1 h<br />
<br />
** heat inactivation<br />
*** 80°C, 20 minutes<br />
<br />
** ligation<br />
*** 1 µl T4 ligase<br />
*** 2 µl T4 ligase buffer: tested three buffers, one was okay, made aliquots: what happened to all the buffers???<br />
*** 1.33 µl backbone (I thought 50 ng, actually 25 ng)<br />
*** 0.23 µl insert, 1:10 diluted<br />
*** 17.37 µl H2O<br />
*** incubated till transformation<br />
<br />
** Extracting pSB1C3 from iGEM 2010 distribution<br />
*** 10 µl H20 in A3<br />
*** 3 µl used for transformation<br />
<br />
** transformation<br />
*** in DH5alpha cells from new -80°C fridge...<br />
*** also in XL-1 blue: Tetracycline resistance<br />
*** 15 minutes in ice with ligation product<br />
*** 1 minute heat shock, 42°C<br />
*** 2 minutes on ice<br />
*** 500 µl LB0 added<br />
*** 1.5 hours at 37°C, shaking<br />
*** control: digested, linearized pSB1C3 into XL-1 blue<br />
*** reason: DpnI digestion recommended, we don't have any DpnI <br />
*** on chloramphenicol agar plates from Prof. Buchner's lab --> new and shiny<br />
*** XL-1 blue plated on Tetracycline/Chloramphenicol plates from Becker's lab: not used in Becker's lab because Tetracycline broken, does not matter for us...<br />
<br />
* E. coli RPO malachitegreen assay<br />
** E. coli RPO finally arrived<br />
*** called Biozym: RPO was supposed to arrive yesterday, delivery cimpany signed for yesterday<br />
*** called Materialausgabe two times everyday for the last week --> In the end they already knew, when they heard E14<br />
*** nevertheless when the enzyme arrived, somebody forgot to put it into the large book of incoming stuff<br />
*** only one person knew, that the enzyme already arrived <br />
*** stored at -20 °C meantime<br />
*** dry ice already vaporated...<br />
*** people were first mad at me, because I insisted that the package arrived although it was not written in the main book<br />
*** then mad at each other because somebody did not put it into the main book<br />
*** then I was slightly mad because we called two times a day for seven days, argued about -80°C storage and still...<br />
*** bought dry ice, transport into ZNN, stored at -20°C covered in dry ice (-80°C fridge is already standing in the lab but not installed yet) until measurements<br />
*** now stored at old -80°C in physic's department in iGEM box<br />
<br />
** preperation of measuring constructs - without terminator<br />
*** PCR of 12z (His-Terminator in plasmid)<br />
*** 5z (TrpTerm in plasmid)<br />
*** 28z (pSB1A2-R0011-TrpSig)<br />
*** pSB1K3-R0011-HisSig-B0014<br />
*** 4x 8x PCR mix<br />
*** 8 µl dNTPs<br />
*** 8 µl Apt forward<br />
*** 8 µl Apt reverse - without terminator!<br />
*** 40 µl 10x buffer<br />
*** 24 µl MgCl2<br />
*** 1.6 µl Taq Polymerase<br />
*** 302.4 µl Template per reaction<br />
*** H2O<br />
<br><br />
*** recognized that wrong primers were used for the signals, thrown away after PCR<br />
*** protocol iGEM PCR, 52°C annealing temp.<br />
*** purified using Clean and Concentrator<br />
*** yields: 220 ng/µl TrpTerm, 178 ng/µl (or something like that) HisTerm<br />
<br />
** Buffer preparation for E. coli RPO<br />
*** Instructions from Epicentre Biotechnologies Datasheet (included in package)<br />
*** 5x transcription buffer<br />
*** 2 ml Tris, pH=7.49<br />
*** 1 ml 500 mM MgCl2<br />
*** 5 µl Triton X-100<br />
*** 3.75 ml 2 M KCl<br />
*** water to 10 ml<br />
*** stored in fridge<br />
<br><br />
*** 2x transcription buffer with malachitegreen<br />
*** 4 ml 5x buffer<br />
*** 800 µl 250 mM malachitegreen stock<br />
*** water to 10 ml<br />
*** wrapped in aluminium foil, stored in fridge<br />
<br />
** measurement with E. coli RNA Polymerase<br />
*** measured: 12z (TrpTerm - negative control), 5z (HisTerm, negative control), (positive control)<br />
*** 1x measurement<br />
*** 2.5 µl RPO (2.5 U)<br />
*** 10 µl DTT (100 mM stock, 10 mM end)<br />
*** 50 µl 2x buffer <br />
*** 2.5 µl rNTPs (80 mM stock, 2 mM end)<br />
*** 1 µg DNA template<br />
*** water to 100 µl<br />
<br><br />
*** 3.1 x<br />
*** 7.75 µl RPO<br />
*** 31 µl DTT<br />
*** 7.75 µl rNTPs<br />
*** 155 µl 2x buffer<br />
*** 77.5µl H20<br />
*** 4.5 µl 5z/5.5 µl H2O<br />
*** 5.35 µl 12z/4.65 µl H2O<br />
*** 5.88 µl ??? /4.12 µl H20<br />
<br><br />
*** 38°C, Exc=630 nm, Em=650/655 nm<br />
<br />
** Cloning of R0011 and B1006 in pSB1K3<br />
*** digestion of R0011 with EcoRi and SpeI (check above, same procedure)<br />
*** ligation<br />
*** 0.3 µl R0011<br />
*** 0.2 µl B1006 (signal)<br />
*** 5 µl pSB1K3<br />
*** 2 µl T4 ligation buffer<br />
*** 1 µl T4 ligase<br />
*** 11.5 µl H2O<br />
*** Transformation into DH5alpha cells<br />
*** put on Kana-Plates<br />
<br><br />
** Cloning of signal B1006 in pSB1A2_R0011<br />
*** 1.82 µl pSB1A2_R0011<br />
*** 0.2 µl Signal B1006<br />
*** 2 µl T4 DNA ligation buffer<br />
*** 1 µl T4 DNA ligase<br />
*** 15 µl H2O<br />
*** Transformation into DH5alpha cells<br />
** put on Amp-plates<br />
*** check above<br />
<br />
===22.10.2010===<br />
* Yesterday's malachitegreen assay using E. coli RPO<br />
<br />
[[Image:TUM2010_101022_Kinetik.png|600px]]<br />
<br />
** Fluorescence spetrum, Exc=630 <br />
[[Image:TUM2010_101022_Em_skal.png|600px]]<br />
<br />
** Fluorescence excitation spectrum, Em= 652<br />
[[Image:TUM2010_101022_Exc.png|600px]]<br />
<br />
* 7 M 15 % PAGE<br />
** DnaseI digestion of E. coli RPO transcription<br />
*** control: T7 positive control<br />
*** 17 µl Transcription product<br />
*** 2 µl 10 x DnaseI buffer<br />
*** 1 µl Dnase<br />
<br />
[[Image:TUM2010_101022_page.png|600px]]<br />
<br />
*** lmw: low molecular weight ladder, NEB<br />
*** H: His-switch<br />
*** W: Trp-Switch<br />
*** 16z: Positive control (with TrpSignal); using Primer Apt_For+AptPart_woT_Rev<br />
<br />
*** Ladies and Gentleman, we definetly see RNA this time<br />
*** positive control completely transcriped, terminators terminate (finally some terminator terminate...)<br />
*** DnaseI digestion works. DnaseI control completely clean.<br />
*** I think termination products are visible<br />
*** I also think, that RPO/DnaseI/protein in general bound RNA is stuck in pockets<br />
*** 1h. 37°C, Dnase digestion conditions: RNA suffers a bit, paradise for RNases?<br />
<br />
<br />
* Cloning Malachitegreen-binding aptamer into pSB1C3 <br />
** no clones on plates<br />
*** clones expected to show up on A3 transformed cells --> part from partsregistry!<br />
*** weird clones on Tetracycline/Chloramphenicol plates from Becker's department --> maybe both antibiotics not working anymore<br />
*** talked to Moni from E 22 <br />
*** still old cells in new -80°C --> cells from 2008, maybe dead, certainly not competent...<br />
<br />
** repeat transformation (see yesterday's labbook)<br />
<br />
<br />
===23.10.2010===<br />
*''In vitro'' transcription using ''E. coli'' RPO <br />
** PCR of HisSig, TrpSig, HisTerm, TrpTerm, 16z (positive control)<br />
*** 100 µl per template, 800 µl total<br />
*** 16 µl dNTPs<br />
*** 16 µl forward primer<br />
*** 16 µl reverse primer<br />
*** 80 µl Taq-buffer Mg-free<br />
*** 3.2 µl Taq Polymerase<br />
*** 48 µl MgCl2<br />
*** 5 µl template per 100 µl<br />
*** 615.8 µl H2O<br />
** PCR Purification using DNA Quick and Clean<br />
*** yield: Template - # - concentration [ng/µl]<br />
*** 16z - 1 - 146<br />
*** 16z - 2 - 138<br />
*** 16z - 3 - 138<br />
*** 16z - 4 - 128<br />
*** HisTerm - 1 - 96<br />
*** HisTerm - 2 - 160<br />
*** TrpTerm - 1 - 204<br />
*** TrpTerm - 2 - 190<br />
*** HisSig - 1 - 160<br />
*** HisSig - 2 - 165<br />
*** TrpSig - 1 - 83.5<br />
*** TrpSig - 2 - 166<br />
<br />
**Measurement<br />
*** 16z positive control, TrpTerm, both with and without signal<br />
*** 4.1 x Master Mix<br />
*** 105 µl Buffer<br />
*** 41 µl DTT<br />
*** 10.25 µl RPO<br />
*** 10.25 µl rNTPs<br />
*** 77.9 µl H2O<br />
*** take 84 µl Master Mix<br />
*** add to: <br />
*** 10.85 µl 16 z + 6.1 µl H20<br />
*** 10.85 µl 16 z + 6.1 µl TrpSig<br />
*** 10.92 µl TrpTerm + 6.1 µl H20<br />
*** 10.92 µl TrpTerm + 6.1 µl TrpSig<br />
[[Image:TUM2010_101023_kinetik.png|600px]]<br />
Only the positive control without Signal increases!<br />
<br />
<br />
* Cloning of MPA into pSB1C3<br />
** Checked plates, no clones<br />
** being sad<br />
** said stupid plates <br />
** Repetition of ligation<br />
*** for 50 ng plasmid backbone<br />
*** 2.6 µl pBS1C3 E/P digested<br />
*** 0.23 µl MPA, E/P digested<br />
*** 2 µl T4 ligase buffer - I checked four buffers, all were precipitated, took a completely new one and aliquoted it!<br />
*** 1 µl T4 Ligase<br />
*** for 100 ng plasmid backbone<br />
*** 5.2 µl pBS1C3 E/P digested<br />
*** 0.46 µl MPA, E/P digested<br />
*** 2 µl T4 ligase buffer - I checked four buffers, all were precipitated, took a completely new one and aliquoted it!<br />
*** 1 µl T4 Ligase<br />
*** transformation into pSB1K3 (just in case...)<br />
*** 4.5 µl pSB1K3 E/P digested<br />
*** 0.23 µl MPA, E/P digested<br />
*** 2 µl T4 ligase buffer - I checked four buffers, all were precipitated, took a completely new one and aliquoted it!<br />
*** 1 µl T4 Ligase<br />
** found some pSB1C3_RFP clones in the evening, 5 ml cultures<br />
** stroke over the plate with a pipette tip, 5 ml culture<br />
<br />
===24.10.2010===<br />
*Cloning of MPA into pSB1C3<br />
** Miniprep of pSB1C3_RFP and something random from the plate of no clones<br />
*** mixed up samples: yields were good, but I guess they don't matter<br />
** very tiny clones found in the morning<br />
** slightly bigger ones by noon<br />
** pickable clones by evening<br />
** picked 10 clones, 5 ml cultures<br />
<br />
* BBa_K494001-BBa_K494006<br />
** 2x 5 ml LB_Amp from glycerin stocks<br />
<br />
* Measurements with T7 RPO and ''E. coli'' constructs<br />
** 16z positive control, TrpTerm, both with and without signal<br />
*** 4.1 x Master Mix<br />
*** 205 µl Buffer<br />
*** 41 µl DTT<br />
*** 10.25 µl RPO<br />
*** 10.25 µl rNTPs<br />
*** 77.9 µl H2O<br />
<br />
*** take 76.2 µl Master Mix<br />
*** add to: <br />
*** 10.07 µl 16 z + 13.7 µl H20<br />
*** 10.07 µl 16 z + 9.61 µl TrpSig + 4 µl H2O<br />
*** 23.67 µl HisTerm (1)<br />
*** 14.2 µl HisTerm (2) + 9.61 µl TrpSig<br />
<br />
[[Image:TUM2010_101024_kinetik.JPG|600px]]<br />
It looks like an increase in 3 of 4 traces, but spectr show no sign of malachite green binding:<br />
[[Image:TUM2010_101024_scan.JPG|600px]]<br />
<br />
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<br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}Week31{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU_Munich/Templates/RedBox | text=Cloning of Parts into pSB1C3}} {{:Team:TU_Munich/Templates/ClearBox | text=Measurements }} {{:Team:TU_Munich/Templates/GreenBox | text=T7 & E. coli }} <br />
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The Week of Wiki freeze!<br />
<br />
===25.10.2010===<br />
* Submitting Parts<br />
** Miniprep of yesterday's 5 ml cultures using Zymo Classic<br />
** MPA into pSB1C3<br />
*** # - concentration (ng/µl)<br />
*** 1 - 45.5<br />
*** 2 - 96.5<br />
*** 3 - 19.5<br />
*** 4 - 31<br />
*** 5 - 122<br />
*** 6 - 164<br />
*** 7 - 30<br />
*** 8 - 39.5<br />
*** 9 - 20.5<br />
*** 10 - 113<br />
<br />
** BBa_K494001-BBa_K494006<br />
*** BioBrick - # - concentration in ng/µl<br />
*** BBa_K494001 - 1 - 179<br />
*** BBa_K494001 - 2 - 182<br />
*** BBa_K494002 - 1 - 214<br />
*** BBa_K494002 - 2 - 288<br />
*** BBa_K494003 - 1 - 160<br />
*** BBa_K494003 - 2 - 202<br />
*** BBa_K494004 - 1 - 308<br />
*** BBa_K494004 - 2 - 322<br />
*** BBa_K494005 - 1 - 290<br />
*** BBa_K494005 - 2 - 126<br />
*** BBa_K494006 - 1 - 290<br />
*** BBa_K494006 - 2 - 232<br />
<br />
** control digestion of everything<br />
*** EcoRI/PstI<br />
*** MPA clones 1, 3, 4, 7, 8, 9: 8 µl<br />
*** all other: 4 µl<br />
*** 7 x for 4 µl<br />
*** 14 µl 10 x BSA<br />
*** 14 µl 10 NEB3<br />
*** 2.1 µl EcoRI HF<br />
*** 2.1 µl PstI<br />
*** water as needed<br />
<br />
*** 19 x for 4 µl<br />
*** 38 µl 10x BSA<br />
*** 38 µl 10x NEB3<br />
*** 5.7 µl EcoRI HF<br />
*** 5.7 µl PstI<br />
*** water as needed<br />
<br />
*** 37°C, 1 hour, fitted just perfectly into the heat block<br />
<br />
*** recognized later that MPA 2, 5, 6, 10 were digested without DNA<br />
*** NotI Digestion with --> PstI empty... Time to finish :)<br />
*** 10 µl NEB<br />
*** 10 µl 10x BSA<br />
*** NotI<br />
<br />
** pSB1C3_MPA (BBa_K494000) run on 2 % agarose gel<br />
[[Image:TUM2010 101025 MPA.PNG|center|500 px]]<br />
<br />
**--> clone 1 picked for submission: Looked better in reality!<br />
<br />
** BBa_K494001-BBa_K494006 run on 1.5 % agarose gel<br />
[[Image:TUM2010 101025 backbones.PNG|center|500 px]]<br />
<br />
** pSB1C3_MPA (BBa_K494000) missed preps run on 2 % agarose gel<br />
<br />
===26.10.2010===<br />
* Waited for the FedEx man from 8:00-15:00. Called at 15:15. Said, oh, they forgot to pick it up! Sent somebody immediately who arrived at 15:45. Happyness --> Parts submitted, not our fault anymore :) Track: 871353522440<br />
<br />
===27.10.2010===<br />
* Ran around to shoot weird ''E. coli'' pics<br />
<br />
* Not in the lab anymore... FedEx delivered our parts! 9.14 am EDT, we recognized it at 16:22 MEST! More Happyness, now back to serious working maybe...<br />
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<br />
<br><br />
<br />
=Protocols=<br />
<br />
==Molecular Biology==<br />
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*PCR <br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
<br />
<center>'''Taq Polymerase Hot Start '''</center><br />
<br />
'''PCR Pippeting plan: '''<br> <br />
<br />
1 µl template <br> <br />
<br />
1 µl dNTP 10 µM <br> <br />
<br />
1 µl G1004 (Primer) 10 µM<br> <br />
<br />
1 µl G1005 (Primer) 10 µM<br> <br />
<br />
5 µl 10x Taq-buffer&nbsp; (500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM&nbsp;MgCl<sub>2</sub>)&nbsp; <br />
<br />
0,2 µl Taq-Polymerase (add last) 5,000 U/ml<span style="background-color: rgb(255, 0, 0);"><br />
</span> <br />
<br />
<br> <br />
<br />
40.8 µl Water<br> <br />
<br />
Final volume 50µl <br />
<br />
'''<br>''' <br />
<br />
'''Processing: '''(program saved as '''IGEMPCR ''')'''<br>''' <br />
<br />
*preheating of PCR chamber to 94 °C<br />
<br />
&nbsp;&nbsp; --&gt; insert sample <br />
<br />
*2 min at 94 °C <br />
*loop 35x:<br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp; - 30 sat 94°C (according to IGEM protocols) <br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp; - 30 s at 56 °C <br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp; - 45s at 72°C <br />
<br />
*7 min at 72°C <br />
*stay at 4°C <br><br><br />
<center>'''colony PCR '''</center><br />
*Colony PCR<br />
**pick colonies and resuspend them in 20 µl LB+Antibiotic (each)<br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified), store remaining 18 µl for overnight cultures<br />
**afterwards, mix 15 µl of each PCR product with 3 µl GLPn and load to Gel<br />
**make overnight cultures of positive clones by adding the remaining 18 µl to 5 ml LB+AB<br />
<br />
'''program:colonypcr ''' <br />
<br />
*preheating of PCR chamber to 94 °C<br />
<br />
&nbsp;&nbsp; --&gt; insert sample <br />
<br />
*5 min 30 sec at 94 °C <br />
*loop 35x:<br />
**30 sat 94°C (according to IGEM protocols) <br />
**30 s at 58 °C <br />
**60s at 72°C <br />
*7 min at 72°C <br />
*stay at 4°C <br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
*DNA Purification<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
<center>'''PCR samples '''</center><br />
<br />
''' ZYMO RESEARCH DNA Clean&amp;Concentration Kit '''<br />
<br />
[http://www.zymoresearch.com/zrc/pdf/D4003i.pdf Protocol and Information]<br> <br />
<br />
#In a 1.5 ml microcentrifuge tube, add 2-7 volumes of DNA Binding Buffer to each volume of DNA sample (see table below). Mix briefly by vortexing.<br><br><br />
<br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| Application <br />
| DNA Binding Buffer&nbsp;: Sample <br />
| Example<br />
|-<br />
| Plasmid, genomic DNA (&gt;2 kb) <br />
| 2&nbsp;: 1 <br />
| 200 μl&nbsp;: 100 μl<br />
|-<br />
| PCR, cDNA, DNA fragment <br />
| 5&nbsp;: 1 <br />
| 500 μl&nbsp;: 100 μl<br />
|-<br />
| ssDNA (e.g., M13 phage) <br />
| 7&nbsp;: 1 <br />
| 700 μl&nbsp;: 100 μl<br />
|}<br />
<br />
#Transfer mixture to a provided Zymo-Spin™ Column1 in a Collection Tube.<br> <br />
#Centrifuge at ≥10,000 x g for 30 seconds. Discard the flow-through.<br> <br />
#Add 200 μl Wash Buffer to the column. Centrifuge at ≥10,000 x g for 30 seconds. Repeat wash step.<br> <br />
#Add ≥6 μl water2,3 directly to the column matrix. Transfer the column to a 1.5 ml microcentrifuge tube and centrifuge at ≥10,000 x g for 30 seconds to elute the DNA.<br>Ultra-pure DNA in water is now ready for use.<br><br />
<br />
<br> <br />
<br />
''' QIAquick purification Kit <br> '''<br />
<br />
[http://www1.qiagen.com/literature/render.aspx?id=103715 Handbook] <br />
<br />
Procedure<br> 1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene. For example, add 500 μl of Buffer PB to 100 μl PCR sample (not including oil).<br> 2. If pH indicator I has beein added to Buffer PB, check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.<br> 3. Place a QIAquick spin column in a provided 2 ml collection tube. <br>4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s. '''We changed it to 3 min @ 6000rpm&nbsp;! '''<br>5. Discard flow-through. Place the QIAquick column back into the same tube. Collection tubes are re-used to reduce plastic waste.<br> 6. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.<br> 7. Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 min.'''repeat!'''<br> IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.<br> 8. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.<br> 9. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.<br> IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 μl from 50 μl elution buffer volume, and 28 μl from 30 μl elution buffer. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.<br> 10. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.<br><br />
<center>''' Gel samples<br> '''</center><br />
<br />
''' ZYMO RESEARCH Gel DNA Recovery Kit '''<br />
[http://www.acgtinc.com/PDF_files/Sample%20Prepation_ACGT/Zymoclean%20Gel%20DNA%20Recovery%20Kit_Zymo%20Research.pdf Product informartion]<br />
<br />
'''Protocol'''<br><br />
<br />
#Excise the DNA fragment1 from the agarose gel using a razor blade or scalpel and transfer it to a 1.5 ml microcentrifuge tube.<br />
#Add 3 volumes of ADB to each volume of agarose excised from the gel (e.g. for 100 μl (mg) of agarose gel slice add 300 μl of ADB).<br />
#Incubate at 37-55 °C for 5-10 minutes until the gel slice is completely dissolved2. For DNA fragments &gt;8 kb, following the incubation step, add one additional volume (equal to that of the gel slice) of water to the mixture for better DNA recovery (e.g. 100 μl agarose, 300 μl ADB and 100 μl water).<br />
#Transfer the melted agarose solution to a Zymo-SpinTM I Column in a Collection Tube.<br />
#Centrifuge at ≥10,000 x g for 30-60 seconds. Discard the flow-through.<br />
#Add 200 μl of Wash Buffer to the column and centrifuge at ≥10,000 x g for 30 seconds. Discard the flow-through. Repeat the wash step.<br />
#Add ≥6 μl of water3,4 directly to the column matrix. Place column into a 1.5 ml tube and centrifuge ≥10,000 x g for 30-60 seconds to elute DNA.<br>Ultra-pure DNA in water is now ready for use.<br><br />
<br />
<center>''' Miniprep '''</center><br />
<br />
'''Protocol:'''<br />
<br />
# Add 600 μl of bacterial culture grown in LB medium to a 1.5 ml microcentrifuge tube.<br />
# Add 100 μl of 7X Lysis Buffer (Blue)1 and mix by inverting the tube 4-6 times. Proceed to step 3 within 2 minutes. After addition of 7X Lysis Buffer the solution should change from opaque to clear blue, indicating complete lysis.<br />
# Add 350 μl of cold Neutralization Buffer (Yellow)2 and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.<br />
# Centrifuge at 11,000 – 16,000 x g for 2-4 minutes.<br />
# Transfer the supernatant (~900 μl) into the provided Zymo-Spin™ IIN column. Avoid disturbing the cell debris pellet.<br />
# Place the column into a Collection Tube and centrifuge for 15 seconds.<br />
# Discard the flow-through and place the column back into the same Collection Tube.<br />
# Add 200 μl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. It is not necessary to empty the collection tube.<br />
# Add 400 μl of Zyppy™ Wash Buffer2 to the column. Centrifuge for 30 seconds.<br />
# Transfer the column into a clean 1.5 ml microcentrifuge tube then add 30 μl of Zyppy™ Elution Buffer3 directly to the column matrix and let stand for one minute at room temperature.<br />
# Centrifuge for 15 seconds to elute the plasmid DNA.<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
*Digestion<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
<center>''' Restriction Digest '''</center><br />
<br />
{| width="60%" cellspacing="1" cellpadding="1" border="1"<br />
|-<br />
| Enzyme<br> <br />
| 10 units is sufficient, generally 1µl is used<br />
|-<br />
| DNA <br />
| 1 µg<br />
|-<br />
| 10X NEBuffer<br> <br />
| 5 µl (1X)<br />
|-<br />
| BSA <br />
| Add to a final concentration of 100 µg/ml (1X) if necessary<br />
|-<br />
| Total Reaction Volume <br />
| 50 µl<br />
|-<br />
| Incubation Time <br />
| 1 - 1.5 hour<br><br />
|-<br />
| Incubation Temperature Enzyme dependent <br />
| <br />
XbaI, SpeI, PstI, SpeI&nbsp;: 37 °C <br />
<br />
|}<br />
<br />
[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp activity of restriction enzymes in NEB buffers] <br> <br />
<br />
''' Biobrick standard <br> '''<br />
<br />
[http://openwetware.org/wiki/Restriction_digest Protocols for IGEM standard digestion] <br />
<br><br />
<center>'''Dephosphorylation'''</center><br />
using Antarctic Phosphatase<br />
#Add 1/10 volume of 10X Antarctic Phosphatase Reaction Buffer to 1-5 µg of DNA cut with any restriction endonuclease in any buffer.<br />
#Add 1 µl of Antarctic Phosphatase (5 units) and mix.<br />
#Incubate for 15 minutes at 37°C for 5´ extensions or blunt-ends, 60 minutes for 3´ extensions.<br />
#Heat inactivate (or as required to inactivate the restriction enzyme) for 5 minutes at 65°C.<br />
#Proceed with ligation.<br />
[http://www.neb.com/nebecomm/products/protocol76.asp from NEB]<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
*Ligation<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
''' Using T4 Ligase, New England Labs '''<br />
*1 µl T4 Ligase <span style="color: rgb(255, 102, 0);">(10.000 U)</span> <br />
*50 ng plasmid <br />
*3x mol(plasmid) insert <br />
*2 µl T4 Ligase 10x buffer <br />
*add H<sub>2</sub>O to reach final volume of 20 µl<br><br />
<br />
*incubation at 22°C for 1 h <br />
*storing at 16 °C for 40 min<br><br />
<br />
<br />
<u>'''Biobrick Standard'''</u><br> <br />
<br />
[http://parts2.mit.edu/wiki/index.php/Standard_Assembly Standard BioBrick assembly]<br> <br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
*Transformation<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
<br />
'''At Woehlke's S1-Lab !!!'''<br><br />
<br />
#Thaw competent cells on Ice<br />
#Add DNA, pipette gently to mix<br />
#Let sit for 30 minutes on ice<br />
#Incubate cells for 45 seconds at 42°C<br />
#Incubate cells on ice for 2 min<br />
#Add 1 ml LB0<br />
#Incubate for 1 hour at 37oC on shaker<br />
#Spread 100-300 μl onto a plate made with appropriate antibiotic.<br />
#Grow overnight at 37 °C.<br />
#Save the rest of the transformants in liquid culture at 4 °C<br />
<br />
modified from [http://openwetware.org/wiki/Transforming_chemically_competent_cells open wetware]<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
*Gel electrophoresis<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
<center>'''Agarose Gels '''</center><br />
usual volume needed: 80 ml<br />
[http://www.promega.com/enotes/faqspeak/fq0065.htm Optimum resolution according to NEB]<br><br />
[http://www.cwcboe.org/19992051412432550/lib/19992051412432550/Molecular%20Biology/Gel%20electrophoresis/Lab_manual_8_gel_elect.pdf further information on optimizing gel electrophoresis, e.g. recommanded voltage per cm2 gel]<br />
<br />
'''stain'''<br />
<br />
*SybrGold ([http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Nucleic-Acid-Detection-and-Genomics-Technology/Nucleic-Acid-Detection-and-Quantitation-in-Electrophoretic-Gels-and-Capillaries.html invitrogen])<br />
**Cover Gel with 1x TAE<br />
**Add SybrGold to a 1:10000 dilution<br />
**cover with aluminium foil (light sensitive)<br />
**shake&incubate 20 min (for 2% Agarose Gels at least 45 min!)<br />
<br><br />
*SybrSafe<br />
**used just like SybrGold<br />
<br />
''' standards<br> '''<br />
<br />
<br />
<br />
<br> <br />
<br />
{| width="600" height="386" cellspacing="1" cellpadding="1" border="1" align="center" style=""<br />
|-<br />
| [[Image:TUM2010_Dna lmw.gif]] <br> <br />
| [[Image:TUM2010_N3232_fig1_v1_000034.gif]]<br> <br />
| [[Image:TUM2010_N3200_fig1_v1_000036.gif]]<br><br />
|-<br />
| low molecular weight (NEB)<br> <br />
| 1 kb standard (NEB)<br> <br />
| 2-log standard (NEB)<br><br />
|}<br />
<br />
<br><br />
<center>'''Polyacrylamide Gels'''</center><br />
'''Preparation of Gels'''<br />
<br />
Recipe for denaturing gels: <br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| Gel type<br />
| 1 big gel <br />
| 2 big gels<br />
| 1 small gel<br />
| 2 small gels<br />
|-<br />
| Urea<br />
| 28.8 g <br />
| 57.6 g<br />
| x <br />
| x<br />
|-<br />
| Acrylamide 40%<br />
| 22.5 ml<br />
| 45 ml<br />
| x <br />
| x<br />
|-<br />
| Buffer 10x<br />
| 6 ml<br />
| 12 ml<br />
| x<br />
| x<br />
|-<br />
| End volume (reach by adding water)<br />
| 60 ml<br />
| 120 ml<br />
| x<br />
| x<br />
|-<br />
| APS<br />
| 600 µl<br />
| 1200 µl<br />
| x<br />
| x<br />
|-<br />
| TEMED<br />
| 60 µl<br />
| 120 µl<br />
| x<br />
| x<br />
|-<br />
|}<br />
<br><br />
*Dissolve Urea in Acrylamide-buffer mixture (use Ultrasound bath), this may take more than an hour!<br />
*Tighten the Gel chamber<br />
*add water to desired end volume <br />
*Add APS, then TEMED, mix<br />
*Pipette mixture into gel chamber<br />
*Add desired comb<br />
*let gel polymerize overnight; add buffer in the evening<br />
<br />
'''Running of Gels'''<br />
<br />
mix samples 1:1 with formamide loading dye (stored @ -20°C)<br />
carefully remove comb<br />
blow air into pockets with a 50 µl syringe<br />
fill samples into pockets<br />
run the gel <br />
(usually about 200 V)<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==''In vivo'' Measurement==<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
===Bacterial Cell Growth===<br />
Bacteria from over night cultures were diluted 1:50 into 20 ml culture in LBamp and incubated at 37 °C. Upon OD<sub>600</sub> of 0.7-0.8 the cultures were induced with 0.4% Arabinose and 0.4% Arabinose + 1mM IPTG, respectively. Subsequently Cultures were incubated at 25°C for at least 12 h.<br />
<br />
===Fluorescence Measurement===<br />
Cell samples for the fluorescence measurement were diluted to OD<sub>600</sub>=0.03 and analyzed in a JASCO fluorimeter. eGFP excitation wavelength was set to 501 nm and mCherry fluorescence was measured with an excitation at 587 nm. Standard parameters for the fluorimeter included scanning speed of 100 nm/ min and data points every 0.2 nm as well as medium detector sensivity. The cuvette holder was temperated to 25 °C.<br />
The resulting spectra were corrected for instrumental wavelength dependencies and quantum yield of the fluorescent proteins. A pure LBamp spectrum was subtracted and the corrected spectra were normalized using eGFP fluorescence as reference.<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==''In vitro'' Translation==<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
The [http://www.promega.com/catalog/catalogproducts.aspx?categoryname=productleaf_335&ckt=1 Promega Kit] is used according to the provided protocols. Further Information about this Kit can be found in the [http://partsregistry.org/Cell-free_chassis/Commercial_E._coli_S30 Parts Registry].<br />
<br />
Fluorescence kinetics are recorded for at least 3 hours, settings are applied as in the in vitro measurement. <br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==''In vitro'' Transcription==<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
===Buffers===<br />
Three different buffers were used for ''in vitro'' transcription experiments:<br />
* For Epicentre E. coli RNA Polymerase the recommended buffer was used<br />
* For T7 RNA Polymerase experiments two buffers were tested: <br />
**T7 RPO buffer as recommended and used by members of the Simmel group <br />
**T7 RPO "paper buffer", as used in the paper of XXX<br />
Of all buffers 2x concentrated stocks were prepared. Malachite green was usually added to the buffer stocks.<br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| <br />
| E. coli RPO buffer <br />
| T7 RPO buffer<br />
| T7 RPO buffer "paper"<br />
|-<br />
| Tris<br />
| 40 mM <br />
| 40 mM<br />
| 40 mM<br />
|-<br />
| pH<br />
| 7.5<br />
| 7.1<br />
| 7.9<br />
|-<br />
| MgCl2<br />
| 10 mM<br />
| 40 mM<br />
| 6 mM<br />
|-<br />
| KCl<br />
| 150 mM<br />
| /<br />
| 100 mM<br />
|-<br />
| Triton X-100<br />
| 0.01%<br />
| /<br />
| /<br />
|-<br />
|}<br />
===Sample Preparation===<br />
Different concentrations were tested for malachite green and DNA templates. Components of a standard experiment are listed in the table below. <br />
<br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| <br />
| E. coli RPO buffer <br />
| T7 RPO buffer<br />
| T7 RPO buffer "paper"<br />
|-<br />
| buffer<br />
| 1x<br />
| 1x<br />
| 1x<br />
|-<br />
| DTT <br />
| 10 mM<br />
| 10 mM<br />
| 10 mM<br />
|-<br />
| Malachite green<br />
| 5-10 µM<br />
| 5-10 µM<br />
| 5-10 µM<br />
|-<br />
| NTP's<br />
| 1 mM<br />
| 4 mM<br />
| 0.8 mM<br />
|-<br />
| RPO<br />
| 2 U<br />
| 125 U<br />
| 125 U<br />
|-<br />
|}<br />
In each run up to 4 samples are measured simultaneously. Components that are the same in each of the 4 samples (buffer, DTT, NTPs, RPO, Water) are prepared as a 4.1x MasterMix in a loBind tube and split to the 4 cuvettes. Final Volume of each sample is 100 µl.<br />
===Cary Eclipse===<br />
For fluorescence measurements, a Cary Eclipse Spectrofluorimeter with a Multicellholder (4 cells) is used. Kinetics are recorded at 37° C. Excitation wavelength is 630 nm, emission is followed at 650 nm and 655 nm. After each kinetics measurement, spectra are to be recorded. <br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
=References=<br />
<html><a name="ref1"></a></html>[1] http://www.promega.com/catalog/catalogproducts.aspx?categoryname=productleaf_335&ckt=1<br />
<html><a name="ref2"></a></html>[2] Zubay, G. (1980) Meth. Enzymol. 65, 856–77, Zubay, G. (1973) Ann. Rev. Genet. 7, 267–87.<br />
<br />
<!-- ############## WIKI-PAGE STOPS HERE ############## --><br />
{{:Team:TU_Munich/Templates/End}}<br />
<br />
<includeonly><br />
= Experiments =<br />
We designed several experiments to test our switches, all of them based on fluorescence measurements. We designed experiment setting for measurements ''in vivo'' as well as ''in vitro''. Our ''in vitro'' measurements relied on two different experiment set-ups. While the first was based on a commercial ''E. coli''-lysate, the latter was reporting on a transcriptional level only, eliminating most of the possible side-effects one could expect in the complex behaviour of a living cell or cell-lysate. [[Experiments_main|Read more]]<br />
<br />
== The Experiments ==<br />
===Fluorescent proteins as reporter===<br />
<div align="justify"><br />
Our initial idea to prove our concept of antitermination was to use flourescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks ''in vivo'' as well as ''in vitro'', the latter using a translation kit based on <i>E. coli</i> lysate. <br>We decided to use the flourescent proteins GFP and RFP, as their spectra do not overlap and we would not measure any FRET. We would use GFP fluorescence as internal control and RFP fluorescence as signal to detect termination/antitermination by our switch we cloned in between the coding sequences of the proteins. Both protein sequences are under the control of one (L-arabinose induced) promoter.<br />
[[Image:TUM2010_gfprfp_schalter_klein.gif|center|our idea]]<br />
<br><br />
When measuring the termination of our BioBricks and the antitermination by their corresponding signal-RNA, we should be able to observe an increasing RFP emission compared to the GFP emission upon induced signal-RNA production in the cells/in the kit:<br><br />
[[Image:TUM2010_Expected_emission_spextra.png|center|our idea]]<br />
Wiith these measurements, it should also be possible to observe differences in efficiency of termination as well as antitermination between our designed switches. <br><br><br />
<br />
===Measurements with the malachite green aptamer as reporter===<br />
A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramaticly (about 3000 times) upon binding of a specific RNA-aptamer.<br><br>#<br />
[[Image:TUM2010_Malachitgruen-2.png|500px|center|our idea]]<br />
---concept to be desribed, as well as literature---<br />
<ref>refs</ref><br />
<br><br><br />
To study the switches on the transcriptional level gives the advantage, that we would have less interferences and possible artefacts. Also, we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems.<br><br />
<br />
[[Image:TUM2010_Malachit_emission.png|200px|thumb|left|Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA]]We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.<br>Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
<br />
= Results =<br />
We ...blablabla<br />
[[Resultss_main|Read more]]<br />
==Flourescent proteins==<br />
Unfortunatly, we had to change the reporter construct two times during our experiments as several problems occured in our measurements:<br><br><br />
<br />
===First Try: based on the measurement plasmid pSB1A10===<br />
At the beginning, we decided to use the reporter plasmid [http://partsregistry.org/Part:pSB1A10 pSB1A10] from the registry. It consists of the fluorescent proteins eGFP and mRFP1. Each sequence includes a ribosome binding site and a stop-codon; the two genes are divided by a cloning side including the BioBrick cleavage sites.[[Image:ScreeningPlasmid1.0.PNG|300px|thumb|right|pSB1A10]]<br> In front of the eGFP sequence, the plasmid includes an arabinose-inducable promoter. The plasmid also contains an ampicilline resistence.<br><br />
We cloned our switches into the cloning site of the measurement plasmid and used an empty cloning site as control; our signal-RNAs we cloned into the [http://partsregistry.org/Part:pSB1K3 pSB1K3] vector, together with the BioBricks R0011 (Lac promoter) and B0014 (double terminator of transcription). Afterwards, we cut pSB1K3 with Aat2 and Pst1 and pSB1A10 with Nsi1 and Aat2 and ligated those fragments of each plasmid that contained our Bricks to get a Monsterplasmid.<br>We had to do so, as both plasmids contain the same ori mechanism. In addition, having both the switch and the signal RNA transcribed from the same plasmid gives us a high local concentration of the signal, once its transcription is induced.<br><br />
[[Image:TUM2010_Messplasmid_nr1.png|left|the measurement plasmid]]<br />
<br />
We transformed BL21(DE3) cells with the plasmid. We set up cultures, induced the arabinose promoter and measured the GFP and mRFP1 excitation/emission spectra within time.<br><br><br />
Unfortunatly, we were ot able to detect any RFP signal, not even in the positive control with no switch in between the GFP/RFP sequences.<br><br><br />
From these experiments, we concluded, that the mRNA of the RFP variant used was instable and rapidly degraded by RNases, so the RFP was not synthesized in the cells. This was also the conclusion from XXX...<br />
<br><br><br />
As solution to this, we decided to design a measurement plasmid ourselves:<br><br />
<hr width="300"><br />
<br><br />
<br />
===Second Try: A measurement plasmid of our own design===<br />
To design our own plasmid to overcome the problems that occurred in our first try gave us tghe possibility to overcome several other problems:<br><br />
#<br />
#<br />
#<br />
[[Image:TUM2010_Construct_no2.png|400px|our construct|center]]<br />
<br />
===Third Try: One promoter for each protein===<br />
We decided to use the measuremnt plasmid we developed in our second try but to clone another L-arabinose induced promoter into the plasmid, in front of our switch followed by the mCherry sequence.<br><br />
<br>[[Image:TUM2010_Construct_no3.png|left|400px|Construct #3]]In this way, we still can use GFP fluorescence as internal control, because both protein transcription is under the control of a promoter of identical design.<br />
Though we are still not able to tell exactly why our previous measurements did not work, but with this construct we measured the first time fluorescence of the mCherry protein in our positive control.<br />
<br><br><br><br><br />
<br />
On this page you can find our protocols for standard molecular biology procedure as well as the full notebook containing lab progress.<br />
<br />
<!-- ############## scheiß teil, wieso ist alles zentriert? ############## --><br />
=Protocols=<br />
==Gels==<br />
Agarose Gels<br />
*1-3 % agarose gels were used<br />
* TAE buffer<br />
** 0.4 M Tris<br />
** 0.01 M EDTA <br />
** 0.01 M acetic acid<br />
** pH=8.0<br />
;Stain<br />
*SybrGold stain<br />
**Cover Gel with 1x TAE<br />
**Add SybrGold to a 1:10000 dilution<br />
**cover with aluminium foil (light sensitive)<br />
**shake&incubate 20 min (for 2% Agarose Gels at least 45 min!)<br />
<br><br />
*SybrSafe<br />
**used just like SybrGold<br />
<br><br />
;Molecular weight marker<br />
*all molecular weight marker were purchased from NEB<br />
*in use: <br />
**low molecular weight<br />
**1 kb<br />
**2-log<br />
<br><br />
;Polyacrylamide Gels<br />
*Preparation of denaturing gels<br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| Gel type<br />
| 1 big gel <br />
| 2 big gels<br />
| 1 small gel<br />
| 2 small gels<br />
|-<br />
| Urea<br />
| 28.8 g <br />
| 57.6 g<br />
| x <br />
| x<br />
|-<br />
| Acrylamide 40%<br />
| 22.5 ml<br />
| 45 ml<br />
| x <br />
| x<br />
|-<br />
| Buffer 10x<br />
| 6 ml<br />
| 12 ml<br />
| x<br />
| x<br />
|-<br />
| End volume (reach by adding water)<br />
| 60 ml<br />
| 120 ml<br />
| x<br />
| x<br />
|-<br />
| APS<br />
| 600 µl<br />
| 1200 µl<br />
| x<br />
| x<br />
|-<br />
| TEMED<br />
| 60 µl<br />
| 120 µl<br />
| x<br />
| x<br />
|-<br />
|}<br />
<br><br />
*Dissolve Urea in Acrylamide-buffer mixture (use Ultrasound bath), this may take more than an hour!<br />
*Tighten the Gel chamber<br />
*add water to desired end volume <br />
*Add APS, then TEMED, mix<br />
*Pipette mixture into gel chamber<br />
*Add desired comb<br />
*let gel polymerize overnight; add buffer in the evening<br />
<br />
'''Running of Gels'''<br />
mix samples 1:1 with formamide loading dye (stored @ -20°C)<br />
carefully remove comb<br />
blow air into pockets with a 50 µl syringe<br />
fill samples into pockets<br />
run the gel <br />
(usually about 200 V)<br />
<br />
== PCR ==<br />
<br> <br />
<br />
=== used protocols<br> ===<br />
<br />
'''a) Taq Polymerase''' ''''Hot Start'''' <br />
<br />
'''PCR Pippeting plan: '''<br> <br />
<br />
1 µl template <br> <br />
<br />
1 µl dNTP 10 µM <br> <br />
<br />
1 µl G1004 (Primer) 10 µM<br> <br />
<br />
1 µl G1005 (Primer) 10 µM<br> <br />
<br />
5 µl 10x Taq-buffer&nbsp; (500 mM KCl, 100 mM Tris-HCl (pH 8.3), 15 mM&nbsp;MgCl<sub>2</sub>)&nbsp; <br />
<br />
0,2 µl Taq-Polymerase (add last) 5,000 U/ml<span style="background-color: rgb(255, 0, 0);"><br />
</span> <br />
<br />
<br> <br />
<br />
40.8 µl Water<br> <br />
<br />
Final volume 50µl <br />
<br />
'''<br>''' <br />
<br />
'''Processing: '''( program saved as '''IGEMPCR ''' )'''<br>''' <br />
<br />
*preheating of PCR chamber to 94 °C<br />
<br />
&nbsp;&nbsp; --&gt; insert sample <br />
<br />
*2 min at 94 °C <br />
*loop 35x:<br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp; - 30 sat 94°C (according to IGEM protocols) <br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp; - 30 s at 56 °C <br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp; - 45s at 72°C <br />
<br />
*7 min at 72°C <br />
*stay at 4°C <br><br />
<br />
<br> <br />
<br />
'''b) colony PCR'''<br />
*Colony PCR<br />
**pick colonies and resuspend them in 20 µl LB+Antibiotic (each)<br />
**PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified), store remaining 18 µl for overnight cultures<br />
**afterwards, mix 15 µl of each PCR product with 3 µl GLPn and load to Gel<br />
**make overnight cultures of positive clones by adding the remaining 18 µl to 5 ml LB+AB<br />
<br />
'''program:colonypcr '''' <br />
<br />
*preheating of PCR chamber to 94 °C<br />
<br />
&nbsp;&nbsp; --&gt; insert sample <br />
<br />
*5 min 30 sec at 94 °C <br />
*loop 35x:<br />
**30 sat 94°C (according to IGEM protocols) <br />
**30 s at 58 °C <br />
**60s at 72°C <br />
*7 min at 72°C <br />
*stay at 4°C <br><br />
<br />
<br><br />
<br />
== DNA Purification<br> ==<br />
<br />
=== PCR samples ===<br />
<br />
'''ZYMO RESEARCH DNA Clean&amp;Concentration Kit'''<br />
<br />
[http://www.zymoresearch.com/zrc/pdf/D4003i.pdf Protocol and Information]<br> <br />
<br />
#In a 1.5 ml microcentrifuge tube, add 2-7 volumes of DNA Binding Buffer to each volume of DNA sample (see table below). Mix briefly by vortexing.<br><br><br />
<br />
{| cellspacing="1" cellpadding="1" border="1" align="center" width="80%"<br />
|-<br />
| Application <br />
| DNA Binding Buffer&nbsp;: Sample <br />
| Example<br />
|-<br />
| Plasmid, genomic DNA (&gt;2 kb) <br />
| 2&nbsp;: 1 <br />
| 200 µl&nbsp;: 100 µl<br />
|-<br />
| PCR, cDNA, DNA fragment <br />
| 5&nbsp;: 1 <br />
| 500 µl&nbsp;: 100 µl<br />
|-<br />
| ssDNA (e.g., M13 phage) <br />
| 7&nbsp;: 1 <br />
| 700 µl&nbsp;: 100 µl<br />
|}<br />
<br />
#Transfer mixture to a provided Zymo-Spin™ Column1 in a Collection Tube.<br> <br />
#Centrifuge at =10,000 x g for 30 seconds. Discard the flow-through.<br> <br />
#Add 200 µl Wash Buffer to the column. Centrifuge at =10,000 x g for 30 seconds. Repeat wash step.<br> <br />
#Add =6 µl water2,3 directly to the column matrix. Transfer the column to a 1.5 ml microcentrifuge tube and centrifuge at =10,000 x g for 30 seconds to elute the DNA.<br>Ultra-pure DNA in water is now ready for use.<br><br />
<br />
<br> <br />
<br />
'''QIAquick purification Kit''' <br> <br />
<br />
[http://www1.qiagen.com/literature/render.aspx?id=103715 Handbook] <br />
<br />
Procedure<br> 1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene. For example, add 500 µl of Buffer PB to 100 µl PCR sample (not including oil).<br> 2. If pH indicator I has beein added to Buffer PB, check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.<br> 3. Place a QIAquick spin column in a provided 2 ml collection tube. <br>4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s. '''We changed it to 3 min @ 6000rpm&nbsp;! '''<br>5. Discard flow-through. Place the QIAquick column back into the same tube. Collection tubes are re-used to reduce plastic waste.<br> 6. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.<br> 7. Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 min.'''repeat!'''<br> IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.<br> 8. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.<br> 9. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.<br> IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 µl from 50 µl elution buffer volume, and 28 µl from 30 µl elution buffer. Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.<br> 10. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.<br><br />
<br />
=== Gel samples<br> ===<br />
<br />
'''ZYMO RESEARCH Gel DNA Recovery Kit'''<br />
<br />
[http://www.acgtinc.com/PDF_files/Sample%20Prepation_ACGT/Zymoclean%20Gel%20DNA%20Recovery%20Kit_Zymo%20Research.pdf Product informartion]<br />
<br />
'''Protocol'''<br><br />
<br />
#Excise the DNA fragment1 from the agarose gel using a razor blade or scalpel and transfer it to a 1.5 ml microcentrifuge tube.<br />
#Add 3 volumes of ADB to each volume of agarose excised from the gel (e.g. for 100 µl (mg) of agarose gel slice add 300 µl of ADB).<br />
#Incubate at 37-55 °C for 5-10 minutes until the gel slice is completely dissolved2. For DNA fragments &gt;8 kb, following the incubation step, add one additional volume (equal to that of the gel slice) of water to the mixture for better DNA recovery (e.g. 100 µl agarose, 300 µl ADB and 100 µl water).<br />
#Transfer the melted agarose solution to a Zymo-SpinTM I Column in a Collection Tube.<br />
#Centrifuge at =10,000 x g for 30-60 seconds. Discard the flow-through.<br />
#Add 200 µl of Wash Buffer to the column and centrifuge at =10,000 x g for 30 seconds. Discard the flow-through. Repeat the wash step.<br />
#Add =6 µl of water3,4 directly to the column matrix. Place column into a 1.5 ml tube and centrifuge =10,000 x g for 30-60 seconds to elute DNA.<br>Ultra-pure DNA in water is now ready for use.<br><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
*add other kits here...<br />
<br />
<br><br />
== Restriction <br> ==<br />
<br />
{| width="60%" cellspacing="1" cellpadding="1" border="1"<br />
|-<br />
| Enzyme<br> <br />
| 10 units is sufficient, generally 1µl is used<br />
|-<br />
| DNA <br />
| 1 µg<br />
|-<br />
| 10X NEBuffer<br> <br />
| 5 µl (1X)<br />
|-<br />
| BSA <br />
| Add to a final concentration of 100 µg/ml (1X) if necessary<br />
|-<br />
| Total Reaction Volume <br />
| 50 µl<br />
|-<br />
| Incubation Time <br />
| 1 - 1.5 hour<br><br />
|-<br />
| Incubation Temperature Enzyme dependent <br />
| <br />
XbaI, SpeI, PstI, SpeI&nbsp;: 37 °C <br />
<br />
|}<br />
<br />
[http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp activity of restriction enzymes in NEB buffers] <br> <br />
<br />
=== Biobrick standard <br> ===<br />
<br />
[http://openwetware.org/wiki/Restriction_digest Protocols for IGEM standard digestion] <br />
<br />
<br><br />
<br />
==Dephosphorylation==<br />
using Antarctic Phosphatase<br />
#Add 1/10 volume of 10X Antarctic Phosphatase Reaction Buffer to 1-5 µg of DNA cut with any restriction endonuclease in any buffer.<br />
#Add 1 µl of Antarctic Phosphatase (5 units) and mix.<br />
#Incubate for 15 minutes at 37°C for 5´ extensions or blunt-ends, 60 minutes for 3´ extensions.<br />
#Heat inactivate (or as required to inactivate the restriction enzyme) for 5 minutes at 65°C.<br />
#Proceed with ligation.<br />
[http://www.neb.com/nebecomm/products/protocol76.asp from NEB]<br />
<br />
== Ligation<br> ==<br />
<br />
'''Using T4 Ligase, New England Labs''' '''<u></u>'''<br />
<br />
*1 µl T4 Ligase <span style="color: rgb(255, 102, 0);">(10.000 U)</span> <br />
*50 ng plasmid <br />
*3x mol(plasmid) insert <br />
*2 µl T4 Ligase 10x buffer <br />
*add H<sub>2</sub>O to reach final volume of 20 µl<br><br />
<br />
<br> <br />
<br />
*incubation at 22°C for 1 h <br />
*storing at 16 °C for 40 min<br><br />
<br />
<br> <br />
<br />
<u>'''Biobrick Standard'''</u><br> <br />
<br />
[http://parts2.mit.edu/wiki/index.php/Standard_Assembly Standard BioBrick assembly]<br> <br />
<br />
<br> <br />
<br />
<br />
<br />
==Transformation==<br />
'''At Woehlke's S1-Lab !!!'''<br><br />
<br />
#Thaw competent cells on Ice<br />
#Add DNA, pipette gently to mix<br />
#Let sit for 30 minutes on ice<br />
#Incubate cells for 45 seconds at 42°C<br />
#Incubate cells on ice for 2 min<br />
#Add 1 ml LB0<br />
#Incubate for 1 hour at 37oC on shaker<br />
#Spread 100-300 µl onto a plate made with appropriate antibiotic.<br />
#Grow overnight at 37 °C.<br />
#Save the rest of the transformants in liquid culture at 4 °C<br />
<br />
modified from [http://openwetware.org/wiki/Transforming_chemically_competent_cells open wetware]<br />
<br />
==Miniprep==<br />
==Preparation of BioBricks from distribution 2008==<br />
<br />
==Sequencing==<br />
* Monsterplasmids contain GATC-Standard-Primer pBR1 (CGAAAAGTGCCACCTGAC ) directly in front of AATII cleavage site. <br />
* Monsterplasmids contain GATC-Standard-Primer pGFP-FP () approx. 100 bp upstream of Biobrick insert site.<br />
<br> !!! Please always fill in iGEM-Sequencing-YYMMDD (e.g. iGEM-Sequencing-100625 for today´s date) as internal billing number!<br />
<br />
=Notebook=<br />
<br />
Boxes: for example Cloning --> work done in this week<br />
<br />
'''Week 1 (BOX1) (BOX2)'''{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Tag 1 <br><br />
<br />
Tag2<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Week 2==<br />
<br />
<br />
<br />
</includeonly></div>Hartlmuellerhttp://2010.igem.org/Team:TU_Munich/ProjectTeam:TU Munich/Project2010-10-28T03:49:54Z<p>Hartlmueller: /* Network construction */</p>
<hr />
<div>{{:Team:TU_Munich/Templates/Beginn}}<br />
<!-- Title of this page here--><br />
Project<br />
{{:Team:TU_Munich/Templates/Middle}}<br />
<!-- ############## WIKI-PAGE STARTS HERE ############## --><br />
<center><font size="5pt" color="#000000">'''bioLOGICS'''</font><font size="4pt" color="#000000">: Logical RNA-Devices Enabling BioBrick-Network Formation</font></center><hr color="black"><br><br />
= Vision=<br />
<br />
Until today, 13.628 biobrick sequences<sup>[[Team:TU_Munich/Project#ref1|&#91;1&#93;]]</sup> have been submitted to partsregistry, thereof 102 reporter units and 12 signaling bricks.<br />
Since then, people are trying to arrange these single biological building blocks in such a manner that allows producing special biotechnological products (metabolic engineering), developing biological sensory circuits (biosensors) and even giving microorganisms the ability to react on multiple environmental factors and serve both as disease indicator and drug. These examples and further promising ideas were implemented on previous iGEM-competitions.<sup>[[Team:TU_Munich/Project#ref2|&#91;2&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref3|&#91;3&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref4|&#91;4&#93;]]</sup> <br><br><br />
The idea of combining the outcome of several iGEM competitions to construct complex synthetic biological systems falls at the last hurdle - the fact, that each team uses a different principle how to access and functionally connect the respectively used biobricks. For example, it is a major challenge to create a system that uses several sensoring BioBricks from different iGEM-teams which in turn regulates reportering BioBricks from various teams. In order to combine and fully take advantage of these promising projects, our vision is to develop an adapter that allows interconnecting arbitrary biobricks on a functional level. Such a system easily allows to setup sensor-reporter circuits and interconnect them to complete biological chips... A further step towards artificial cells.<br><br><br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, the above adapter has to meet the following requirements:<br />
*'''Universality'''<br />
:The adapter has to be compatible to as many BioBricks as possible. This objective will guarantee that a large number of BioBricks can be connected.<br />
*'''Scalability'''<br />
:Once the basic design of the system is established, the construction of the system is supposed to be automated in silico. This way it will be possible to create an adapter connecting a large amount of BioBricks.<br />
*'''Biological orthogonality'''<br />
:Interference with cellular components has to be as low as possible in order to avoid unwanted and perturbing side effects.<br />
*'''Logic'''<br />
:The adapter is supposed to not only associate different BioBricks, but to functionally connect BioBricks in a precisely determined manner (including operations such as AND/OR/NOT).<br />
<br><br />
Several biological logic units, devices and circuits have been developed so far<sup>[[Team:TU_Munich/Project#ref5|&#91;5&#93;]]</sup>, but to our knowledge, none was shown to meet all requirements listed above.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
=Implementation=<br />
To functionally connect BioBricks, there are several possibilities including genetic switches, riboswitches and direct protein-protein interactions. We investigated several hypothetically principles, and decided to focus our practical work on the development of a RNA-RNA interaction-based switch. These switches are capable of changing between two states, a state of antitermination and termination, and make use of highly-specific RNA-RNA interaction. In principle such a switch can fulfill all requirements mentioned previously. The following text clarifies how these switches work in detail.<br />
==How to connect BioBricks==<br />
Our adapter is a system, that activates or disables BioBricks (output BioBricks) in response to the presence of other Biobricks (input Biobricks). Our approach uses a molecular network to put this into practice and consists of four major elements:<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
<br><br />
{|<br />
|-<br />
|[[Image:Networks.png|center|thumb|730px|The general principle how different inputs can be connect to various outputs. For details see text.<br>Inputs (such as proteins or small molecules) are indicated on the left side. blue lines represent transmitter molecules whereas organe lines present logic gates. The type of logic gate is indicated. Green lines indicate transmitter RNA that can function as mRNA and consequently generate any output gene (indicated on the very right).]]<br />
|}<br />
In order to connect different BioBricks, our network requires four major types of components:<br />
*Input elements<br />
*Transmitter molecules<br />
*Logic gates<br />
*Output elements<br />
<br />
{{:Team:TU_Munich/Templates/InfoBoxStart}}'''Computer vs. molecular network - and our approach'''<br><br />
Logic gates in a molecular network are often compared to transistors used in a computer, where billions of transistors are incorporated<sup>[[Team:TU_Munich/Project#ref7|&#91;7&#93;]]</sup>. The main advantage on a computer chip is, all transistors share the same functional principle, and only the way connecting them in a special sequence allows specific addressing of only a subset of other transistors by an input. However, spatially fixed connections of molecular logic gates are not possible in a living cell. The "wiring" within a cell relies on the specific interaction between transmitter molecule and their corresponding logic gates, for example implemented by protein-protein/ligand-protein interactions or specific ligand-riboswitch interactions.<sup>[[Team:TU_Munich/Project#ref8|&#91;8&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref9|&#91;9&#93;]]</sup> As a result, in a cell, each occurring logic gate ("transistor") has to be different, at least in a special recognition site<sup>[[Team:TU_Munich/Project#ref10|&#91;10&#93;]]</sup> - for example like different transcription factors, recognizing different DNA-sites. Thanks to evolution, nature easily can invent a new transistor for each task - science achieves this only on a limited scale, and producing synthetic molecular logic gates artificially by either rational or evolutionary protein or riboswitch engineering, is limited to small circuits so far<sup>[[Team:TU_Munich/Project#ref11|&#91;11&#93;]]</sup>. Our project aims to establish a molecular switch as close as possible to a electronic transistor, thus sharing the same functional principle for all logic gates. At the same time, we want to design a easily exchangeable recognition site, which can individually be designed by everyone! {{:Team:TU_Munich/Templates/InfoBoxEnd}}<br />
<br />
These elements can be combined to build up a molecular network (see illustration). Each input molecule (such as a BioBrick) produces a unique transmitter molecule. All transmitters belong to the same type of molecule and share a common design. However, each transmitter molecule can only interact and activate a certain subset of logic gates. In other words, logic gates have to recognize as well as bind the corresponding transmitter molecules and are capable of producing a new output transmitter molecule. Depending on the type of the logic gate (AND, OR or NOT<sup>[[Team:TU_Munich/Project#ref6|&#91;6&#93;]]</sup>), an output transmitter is only created if both input transmitter molecules are present (AND), at least one of two input transmitters is present (OR) or if no input transmitter is present at all (NOT). Once a logic gate has produced a new output transmitter, these transmitters can in turn address another subset ("layer") of logic gates. In theory many layers of logic gates can be connected this way allowing the creation of large networks. Until this step, various transmitter molecules might have been produced. But in order to create a Biobrick output, the last layer of logic gates finally generates transmitter molecules that will not active logic gates, but will rather interact with the cell metabolism to produce a BioBrick response. In other words, the last layer of transmitter molecules is capable of regulating BioBrick formation.<br />
<br />
<br />
Summarizing, the network establishes a connection between input BioBricks and output BioBricks in a functional manner.<br />
Having addressed the basic layout of the molecular network, the next step is to determine what type of molecules can perform the required functions. We decided to use RNA, both as transmitter molecules and for constructing logic gates. Several advantages result from the utilization of RNA as the central element:<br />
*During the last years, many Biobricks were designed that are sensitive to various chemicals and substances. These BioBricks often function as a transcription factor that binds to a specific DNA sequence and consequently would be capable to produce a specific transmitter RNA molecule. Thus, in principle each BioBrick which involves transcription can be integrated in our network.<br />
*Since all logic gates are capable of producing transmitter RNA, they can also produce functional mRNA encoding any protein. This means, each BioBrick consisting of protein or RNA can be produced as an output of our network.<br />
*If RNA forms both, the transmitter molecule and the logic gates, they can specifically interact by RNA-RNA interaction, which is highly predictable compared to protein interactions. This allows to generate a library of transmitters and gates ''in silico''. Such a library is essential for the creation of large networks.<br />
*RNA production is fast and energy saving for a cell. Consequently, operating a network that only produces RNA rather than proteins will also be faster and more efficient for the host cell. Since our logic gates are based on transcription, translation and resource consuming protein production will only be required at the very last step. <br />
*As the half-time of RNA can be rather short, transmitter RNA will not accumulate within the cell and it is therefore less likely for the system to become saturated.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Design and functional principle of logic gates==<br />
The concept introduced above provides a framework that can potentially serve as an universal adapter between different BioBricks. However, the [[Team:TU_Munich/Glossary#logic gate | logic gates]] have not been specified more precisely so far. This will be done in the following section.<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, our logic gates are to possess the following characteristics:<br />
*Logic gates, such as AND, OR and NOT, have to be implemented by RNA-interaction based principles (see [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]).<br />
*All logic gates have to recognize their corresponding [[Team:TU_Munich/Glossary#Transmitter (bioLOGICS)| transmitter RNAs]] and, in response, produce an output transmitter molecule.<br />
*Logic gates should follow a basic design rule, in such a way, that their creation can be automated ''in silico''.<br />
*The response efficiency of a logic gate toward a transmitter molecule should be comparable for all logic gates to provide calculable robustness and sensitivity. This will ensure comparable molecular concentrations and functionality of large networks.<br />
*The system has to be designed for ''in vivo'' utilization at the first place. As a reference we always assumed a temperature of 37 °C and an ''E. coli'' environment.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}} <br />
In order to build logic gates for our bioLOGICS system we will first create a simple switch. A switch can be activated by one transmitter RNA and produce an output transmitter RNA. In contrast to a logic gate, a switch does not perform logic operations. However by combining switches, logic gates can be created. The following text will first describe how the developed switch works and secondly, how logic gates such as AND/OR/NOT can be created using these switches.<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Read more{{:Team:TU Munich/Templates/ToggleBoxStart2}}<br />
[[Image:toggle_switch.png|500px|thumb|center|id="hideOnReadMore"|'''A''' The basic structure of a bioLOGICS switch (left) and a transmitter molecule (right).<br>'''B'''The process of switching. See the text in the close-by "Read more" section for details.<br>Rectangles present the composition of our functional units on the level of DNA. Fringed lines represent RNA produced by RNA polymerase. The stem loop structure depicts the switchable terminator. Terminator and target site are illustrated in blue and turquoise, respectively. Recognition sites are indicated in different colors, in this case red for the input transmitter and green for the output transmitter.Each switch and or later logical unit has to be flanked by a promotor and another constitutive terminator, to allow RNA-production by RNA-polymerase in a proper way. ]]<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===Switch===<br />
[[Image:TUM2010_switch-and-transmitter.jpg|550px|right|thumb|The basic strcutrue of a switch (left) and a transmitter RNA (right). See text for details.]]<br />
Roughly speaking, a switch can be regarded as an enhanced switchable transcriptional terminator. The enhancement can be described easier by dividing a switch into its functional components: <br />
*'''Target site'''<br><br />
:The target site is the functional core element of our switches, allowing a shift between an "on" and "off" state. Since we work on the level of RNA-production (transcription), a "switchable" transcriptional terminator is suitable for this purpose. By allowing or preventing formation of a transcriptional terminator, that is by switching between termination and antitermination it is possible to represent an "off" and an "on" state, respectively. Therefore, the target site is the 5' ending of the terminator and is required for a stable terminator formation. It should be noted that this principle was also observed in nature.<br />
:To highlight and illustrate the functional principle of our switches, only the part of the terminator which is involved in interacting with a transmitter molecule and which is responsible for shifting between "on" and "off" state is called target site. The remaining terminator sequence is called terminator in the following, even if both, target site and terminator build up the terminator structure occurring in nature. <br />
:The important aspect of our switches is the fact that all switches will hold the same identical target site. Therefore having found one functional "switchable" terminator, will allow almost unlimited upscaling since this terminator can be used for a large library of switches. This is the main difference to previous works done on this field, which always required developing a new shifting principle for each switch.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup> Beside this scalability, this principle provides a comparable on/off shifting rate (responds function) for all switches, avoiding complex fine tuning of molecular networks.<br />
:To sum it up, the target site, allows to switch between an "on" and "off" state. But so far, the switch is not capable of performing specific interaction with transmitter molecules. This is where the recognition site comes into play.<br />
*'''Recognition site'''<br />
:The recognition site defines which transmitter molecule can actually interact with the switch. Therefore, a unique recognition site is generated for each switch and is positioned right upstream of the target site. In principle the recognition can be any random sequence as long as it remains unique within the molecular network.<br />
Summing up, the recognition site allows a specific interaction between switches and transmitter molecules. Once this interaction is formed, an interaction between the transmitter and the target will actually switch the state of the terminator. This allows the specific arrangement and interconnection of numerous of these switches by transmitter molecules, without changing the target site. Comparable to wires connecting many identical transistors, our target site remains the same.<br />
<br><br />
<br />
===Transmitter RNA´s===<br />
As desccribed above, transmitter RNAs are the input and output of bioLOGICS switches (compare [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]). These transmitters are short ssRNA molecules representing the "trigger" to shift switches between the "on" and "off" state. To fulfill this role, they need to posses the following properties:<br />
*A transmitter may only interact with certain switches. That is, a transmitter has to find the corresponding recognition site of a switch.<br />
*Once an interaction is established between a transmitter and a switch, a transmitter has to be capable of changing the secondary structure of a terminator and thus cause antitermination.<br />
Again, these two properties are fulfilled by two components of the transmitter:<br />
*'''Identity site'''<br />
:This site is capable of forcing an interaction between the transmitter and the switch. Therefore it is complementary to the recognition site of this switch. As the recognition site is unique within a network, so is the identity site. However, the single identity site is not capable of changing the state of the switch. That is were the trigger site comes into play.<br />
*'''Trigger site'''<br />
:Once an interaction is created by the identity site, the trigger site is capable of actually shifting the switch since it is complementary to the target site of the switch. To fulfill this role, it is placed upstream at the 5' end of the identity site. As the target site is the same for all switches, the trigger site is the same for all signals. Therefore it is important, that similar to the identity site, a trigger site cannot function on its own. That is, a single trigger site cannot shift the state of a switch without the help of an identity site.<br />
<br />
Summing up, we applied the principle introduced for the switches to the transmitter molecules. In contrast to previous approaches on this field <sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup>, we introduced the described synthetic trigger site in such a manner that it is not able to change the state of the terminator on its own, but only in combination with the identity site. So the challenge is to arrange and optimize these elementary building blocks thermodynamically, that a trigger site is only able to switch in combination with its respective identity site. This was done by ''in silico'' design using [[TU Munich/Glossary#NUPACK| NUPACK]], presented in section [[TU Munich/Modeling#in silico design based on thermodynamic calculations| in silico design]].<br />
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===Putting it all together: the switching process===<br />
[[Image:TUM2010_switching-process.jpg|550px|right|thumb|The basic structure of a switch (left) and a transmitter RNA (right). See text for details.]]The functional principle of the designed switches is illustrated in the figure. The switch is positioned on DNA upstream of a desired output transmitter. So in the absence of a triggering transmitter molecule, transcription will be canceled by the formation of a RNA stem loop in the nascent RNA-chain. This will cause the RNA polymerase to stop transcription and fall off the DNA and consequently no output RNA will be produced. This process only relies on [[Team:TU_Munich/Glossary#Termination| rho-independent termination]].<br />
On the other hand, in the presence of a [[Team:TU_Munich/Project#RNA_transmitters | input transmitter]], this small functional RNA inhibits the stem loop formation by complementary base-pairing and hence avoids termination of transcription. In detail, the identity site (red part on transmitter) binds the recognition site (red part on switch) and serves as [[Team:TU_Munich/Glossary#Toehold|toehold]], which will thermodynamically allow the trigger site (turquoise part on transmitter) to perform a [[Team:TU_Munich/Glossary#Strand Displacement| strand displacement]] and open up the stem loop structure. Consequently the polymerase can read all the way through and form the output RNA.<br>Summing up, we use this concept to create a switch that can be toggled by a transmitter RNA molecule and in response, is able to produce another transmitter RNA.<br />
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<br />
===From switches towards bioLOGICS logic gates===<br />
As described, each switch can be accessed by a specific RNA-transmitter molecule, representing the input. In turn, another RNA-transmitter molecule will be produced if the switch shifts its state. This output transmitter of one switch can serve as input transmitter for the next switch by meaningful selection and design of the respective recognition sites. This easily allows arranging several switches in specific sequences and faulty wiring - the corner stone of a logical network.<br><br />
To ease the building of logical networks we want to create a switch capable of Boolean logics, a common mathematical principle fundamental for computational science. Since AND/OR/NOT are basic logic operations which can be implemented with the presented switches, all remaining operations (such as XOR, NAND, ...) can be expressed by these three operators according to laws of boolean logics.<br />
Creating logic gates is achieved by combining two switches in two different ways, as illustrated below.<br />
*AND gate<br />
:An AND gate can be constrcuted by positioning two switches right next to each other. For the output transmitter to be created, both input transmitter have to be present.[[Image:AND2.png|500px|thumb|center|Combining two switches in series creates a logic AND gate.]]<br />
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*OR gate<br />
:An OR gate is created by utilizing two independent switches sharing the same output transmitter. If each one of both switches is activated, an output transmitter is generated. Therefore, one input transmitter is enough to produce an output transmitter.[[Image:OR2.png|500px|thumb|center|Combining two switches in parallel creates a logic OR gate.]]<br />
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*NOT gate<br />
:A NOT gate is supposed to function as an inverter. In contrast to the gates described above, a not gate requires only one sitch. However, to meet the design rule for transmitter molecules, this switch shows some differences compared to the switches used for AND and OR gates.<br><br />
:Since the transcriptional terminator may not form if no transmitter is present. Consequently, the switch needs an internal trigger site, capable of preventing terminator formation. To allow the binding of an input transmitter molecule, the switch contains a recognition site upstream of a second target site. The additional target site is mandatory since all transmitter molecules have to carry a trigger. In the case of the NOT switch this trigger site may not bind the actual target site within the transcriptional terminator. In other words, a second target site further upstream is required to catch the trigger site of the transmitter molecule. At the same time, the identity site of the transmitter may not bind right upstream of the terminator. This is accomplished by placing an other identity site right upstream of the terminator rather than an recognition site (compare switches used for AND or OR gate). Due to these two difference, the input transmitter is forced to bind further upstream to the recognition site, displacing the internal trigger site of the switch. This will allow the RNA polymerase to read through an create the output transmitter.<br />
[[Image:NOT2.png|500px|thumb|center|Structure and switching process of a NOT gate.<br>]]<br />
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<br />
==Network construction==<br />
Designing complex biological networks based on either traditional protein engineering or our new bioLOGICS is still a complex task. As described above our bioLOGICS design allows the creation and precise connection of logic gates. To illustrate how a bioLOGICS network is put together we developed a software allowing the fast construction of a custom-made network.<br><br />
To read more about this, take a look at our [https://2010.igem.org/Team:TU_Munich/Software software page]<br />
<br />
=Our Objective=<br />
Putting the implementation described above into practice, will be a major challenge. For this year's iGEM competition our goal is to do the first step: design and build a switch that can be toggled by a RNA molecule. To be precise, we want to apply the design rules of our switch to modify a transcription terminator in such a way that it interacts with a second RNA molecule and, as a result, is no longer capable of forming a stem loop. This objective will require intensive ''in silico'' designing and modeling of switches based on different terminators and their corresponding transmitters. In connection to this theoretical part, we also have to test and verify the switches. For this step, we establish custom-made assays, ''in vitro'' and ''in vivo''.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Once the objective mentioned above is accomplished, these basic RNA/RNA-interactions have to be modified in such a manner that the described identity/trigger site pattern for the transmitter and the complementary recognition/target site switch composition has to be established. The most important requirement is to is to optimize these modules that the transmitter is only able to switches specifically, meaning only in the presence of both, identity AND trigger site. <br />
<br><br />
Once the objective mentioned above is accomplished, the creation of an OR gate will be rather simple since it only requires two switches. However the creation of an AND or NOT gate and optimizing the logic gates to improve their responds function will remain the goal of future work. Also the creation of small networks and the correct integration of BioBricks as input and output molecules will be future challenges. Furthermore, we wanted to rather focus on the development and the testing of our structural design of the switches, rather than developing a variety of new BioBricks.<br />
<br />
==''In silico'' design==<br />
As described above, our switches are based on certain design rules. However, there still are different structural parameters that need to be tested and optimized (length of recognition site and target site, choice of terminator, etc.).<br />
We used [[Team:TU_Munich/Project#in silico design |''in silico'' design]] and [[Team:TU_Munich/Modeling| modeling]]) to test different parameters. Furthermore we tried to use the [[Team:TU_Munich/Glossary#Antitermination|antitermination principle]] observed in nature, such as [[Team:TU_Munich/Glossary#Attenuation| attenuation]] in ''E. coli'' or [[Team:TU_Munich/Glossary#Tiny Abortive RNA´s| tiny abortive RNA´s]] of T7-phage.<br />
==Evaluation and Measurements==<br />
To evaluate the functionality of our molecular switches, we first had to establish several assays. Therefore, we improved an existing [[Team:TU_Munich/Lab#In vivo Measurements |''in vivo'' assay]] and developed an [[Team:TU_Munich/Lab#In vitro Transcription | ''in vitro'' assay]] for this purpose. For more information please refer to the [[Team:TU_Munich/Lab | lab]] section.<br />
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Summarizing, the main challenges are <br />
* to find a suitable terminator construct and design a complementary trigger unit, which is only functional in combination with a specificity site - meaning an optimization of the '''thermodynamically parameters''' (see[[Team:TU_Munich/Project#in silico design| in silico design]])<br />
* to investigate whether the transmitter/switch interaction reaction is on a timescale to be competitive to terminator formation - meaning an comparison of '''kinetic parameters''' (see [[Team:TU_Munich/Modeling|Modeling page]])<br />
* to proof antitermination can be also be caused by synthetically RNA-interaction (see [[Team:TU_Munich/Glossary#Antitermination| Antitermination in nature]] and [[Team:TU_Munich/Project#Results| ''in vivo'' and ''in vitro'' measurements]] )<br />
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{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
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=Results=<br />
Every network starts with a basic unit. While our declared aim is to enable networks allowing fine-tuning of gene expression beyond the regular on/off, exploring such an on/off switch/signal pair is the first step towards a functional network. We constructed several units and tested their efficiency, robustness and reproducibility ''in vivo'', ''in vitro'' and ''in silico''. Furthermore we developed a software which allows easy constructions of networks based on our designed logic gates. Conclusive elaboration of a few first RNA-based logic units is the major contribution of our iGEM team.<br />
<br />
==in silico Design of Switching and Trigger Unit==<br />
As described on the [[Team:TU_Munich/Project | project]] page, one key aspect of our switches is the idea, that a [[Team:TU_Munich/Glossary#Transmitter_(bioLOGICS) | RNA transmitter molecule]] is capable to shift the state of a switch only if its [[Team:TU_Munich/Glossary#Trigger_Site_(bioLOGICS) | trigger site]] is present and its [[Team:TU_Munich/Glossary#Identity_Site_(bioLOGICS) | identity site]] corresponds to the [[Team:TU_Munich/Glossary#Recognition_Site_(bioLOGICS) | recognition site]] of the [[Team:TU_Munich/Glossary#Switch_(bioLOGICS) | switch]]. We successfully constructed several switches and their corresponding transmitter RNA ''in silico'' on a thermodynamical basis. We modified different transcriptional terminators in such a way, that the formation of the terminator was prevented by a transmitter molecule. As desired, this only occured if the transmitter molecule contained both, a trigger and an identity site. Analogously, we were able to design and verify a NOT gate using the same thermodynical approach.<br />
<br />
==Diffusion and RNA Folding Dynamics==<br />
We estimated the diffusion time for our constructs and modeled the folding dynamics of our bioLOGICS switches including the switching process with a stochastic RNA folding program. We were able to provide better insight in their folding dynamics and proved that they are able to interrupt termination. We also optimized the switches and the corresponding signals. Furthermore, we combined the switches what resulted in a logic gate. See our [[Team:TU Munich/Modeling|Modeling page]] for further details.<br />
<br />
==''in vivo'' Functionality Screening==<br />
Since our logic gates are intended to function in living cells, ''in vivo'' measurements were essential. In a set of experiments we concentrated on two different switches based on known [[Team:TU_Munich/Glossary#Attenuation|attenuators]] from nature: the [[Team:TU_Munich/Modeling#Switch|HisTerm]] and [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Focusing on fluorescent proteins for quantifiable input and output we designed a functional and robust screening system. For greater detail see [[Team:TU_Munich/Lab#Experiment_Design|Experimental Design]]. Unfortunately, setting up a working screening system failed twice. Only in redesigning and improving the screening plasmid pSB1A10 we succeeded, but lost precious time.<br />
<br />
Ultimately, the two switches displayed remarkable differences in their terminator efficiency, but neither of them responded to their corresponding signal. However, screening one transmitter signal does not disprove the basic working principle of our system. Limited by time, we hope for future teams to take up our work and to use our improved test system that we submitted to the parts registry, for performing successful in vivo measurement.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Considering the high complexity of ''in vivo'' measurements compared to other experimental challenges, a robust and easy to handle test system for [[Team:TU_Munich/Glossary#PoPS-based devices| PoPS-based devices]] is desirable. As described in [[Team:TU_Munich/Lab#Experiment_Design|Experimental design]], we used fluorescent proteins: RFP or mCherry to measure the amount of produced output and eGFP for normalization. Our first attempt, using the screening plasmid pSB1A10, yielded no interpretable results. Switching the fluorescent protein to mCherry did not work either, but after several experimental setups we determined a transcriptional problem causing no reporter protein expression regardless of the inserted part. Thereby we demonstrated the screening plasmid pSB1A10 to be [[Team:TU_Munich/Biobricks#Falsification| malfunctioning]]. <br />
Finally a new design based on pSB1A10 lead to a functional and robust screening system (compare [[Team:TU_Munich/Parts#Screening system: Backbone BBa_K494001| Screening system: Backbone BBa_K494001]]). A second promoter with identical induction properties inside the BioBrick cloning site enforces transcription of the PoPS-based device and the mCherry output.<br />
<br />
Exemplary, the graph below on the right shows the positive control, induced and uninduced at OD<sub>600</sub>=0.7 followed by 16 h incubation at 25 °C. Clearly visible are eGFP and mCherry fluorescence in the induced samples. The uninduced control showed no fluorescence at all, demonstrating the PBad promoter to be tight and providing very low basal transcription, what is a major advantage for the screening system. This newly designed screening approach renders the characterization of PoPS-based devices in general and switches in particular easy and robust. The low basal transcription furthermore fulfills one of the most important requirements for the designed switches, since output transmitters may only be produced in presence of an input transmitter. This helps to avoid strong "background" noise, which would extremely harden the successful interconnection of several switches. <br />
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[[Image:TUM2010_PosControlklein.JPG|200px||thumb|left|Bacteria containing positive control]]<br />
[[Image:TUM2010_graphPosControl1.png|355px|thumb|center|Emission spectra of induced (green/red) and uninduced(black) positive control BBa_K494002 ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
Due to the time limitations of the iGEM completion we had to focus our efforts on few switches after designing the screening system. Relying on the functionality of systems occurring in nature, we choose the [[Team:TU_Munich/Modeling#Switch|HisTerm]] as well as the [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Both switches are based on known natural [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|attenuators]]. Testing synthetic and none-naturally switchable terminators in vivo are goals for future work.<br />
Delorme et al. reported the His-Terminator to be a remarkable effective Terminator with more than 99% termination efficiency.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup> The exemplary measurement below on the right confirms the high terminator efficiency. In fact, we could not detect any mCherry fluorescence in any cells containing the [[Team:TU_Munich/Modeling#Switch|HisTerm]]. Even induction of the corresponding signal transmitter RNA via IPTG did not alter the Terminator efficiency. Again time was the limiting factor and prevented us from testing more than one corresponding transmitter, although the [[Team:TU_Munich/Modeling| Modeling]] highly suggested the necessarily of finding an optimized transmitter length. Thus, the results are insufficient either to prove or to disprove the functionality of the [[Team:TU_Munich/Modeling#Switch|HisTerm]] or our concept in general.<br />
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[[Image:TUM2010_HisSwitchklein.JPG|200px|thumb|left|Bacteria containing HisTerm]][[Image:TUM2010_HisSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing HisTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
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Attaining only 90% terminator efficiency, the natural Trp [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|Attenuator]] is known be less effective than the [[Team:TU_Munich/Modeling#Switch|HisTerm]].<sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup> The graph on the right depicts our designed [[Team:TU_Munich/Modeling#Switch|TrpTerm]] characteristic efficiency of about 40 %, notably below the natural standard. Allowing 60% transcription in the “off” state excludes the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] from possible candidates for a scalable network of logic gates, due to the mentioned required "yes or no" function (see [[Team:TU_Munich/Project#Implementation| Implementation and how to connect Biobricks]]). Thus the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] is inoperative as intended, but may still be useful in other contexts. Similar to the [[Team:TU_Munich/Modeling#Switch|HisTerm]], the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] also did not react to the induction of the corresponding signal. Under circumstances, termination efficiencies altered by the transmitter are on a low range and not resolvable within observed 40% basal transcription. <br />
<br><br />
[[Image:TUM2010_TrpSwitchklein.JPG|200px|thumb|left|Bacteria containing TrpTerm]][[Image:TUM2010_TrpSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing TrpTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
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Making use of our improved screening system we also carried out some ''in vivo'' kinetic measurements in addition to the end-point measurements above. In contrast to the ''in vitro'' experiments we did not obtain significant results for the characterization of our switches. As the switching process is many times faster than protein synthesis our ''in vivo'' kinetics include the synthesis of mCherry as well as its maturation. Therefore we centered our attention on end-point experiments. For more information browse the [[Team:TU_Munich/Lab#Lab_Book|lab book]]. <br><br />
Considering our ''in vivo'' measurements, neither of the tested switches showed any effect regarding the signal induction. But due to the small number of tested switches and signals this can hardly be regarded as disprove of concept. In particular in light of the recent findings by Sooncheol proving antitermination in principle using a T7 system.<sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup><br />
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<br />
==''in vitro'' Screening==<br />
To minimize the amount of disturbing factors we decided to countercheck our ''in vivo'' results with a set of ''in vitro'' measurements. While the ''in vitro'' systems are no doubt much less complex than living cells, the work with these set-ups proved to be quite as difficult.<br />
Just as with the ''in vivo'' measurements we could prove our switching system neither right nor wrong, leaving enough work for future iGEM teams.<br />
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===''in vitro translation''===<br />
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Beside optimization of the reporter proteins in use, the major problem occuring in the experiments was the low capacity of the kit. The signal intensity was very low, which made it difficult to observe any signal intensity alterations, so no conclusion could be drawn from these measurements.<br />
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===''in vitro'' transcription===<br />
We used two completely independent ''in vitro'' systems: Using ''E.coli'' RNA Polymerase we analyzed the His and Trp switches that had already been tested ''in vivo''. In a second set-up, we used the well-established T7 RNA Polymerase and switch based on the T7 terminator as well as several signal sequences.<br />
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====T7 System====<br />
In contradiction to the results of Kang and coworkers and other groups, in our ''in vitro'' set-up the T7 terminator did not seem to terminate at all. The negative control (Promoter_Terminator_malachite binding aptamer) showed a similar increase in fluorescence as the positive control (Promoter_random sequence_malachite binding aptamer). <br />
[[Image:TUM2010_T7Result1.png|350px||thumb|left|''in vitro'' transcription measurement of T7 terminator with no signal(upper left), nonsense signal (upper right) and two different designed signals (below)]]<br />
[[Image:TUM2010_T7Result3.png|350px||thumb|right|''in vitro'' transcription measurement of positive control(upper left and T7 terminator with three different designed signals (remaining traces)]]<br><br />
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Furthermore denaturing Polyacrylamide Gel Electrophoresis (PAGE) confirmed that there was no observeable termination of transcription. The addition of a signaling sequence led to a significantly lower increase in fluorescence, which can be attributed to the fact that both DNA sequences, switch and signal, compete for RNA Polymerases.<br />
However, there is almost no difference between the designed signals and random sequences, which is not a big surprise since there can be no antitermination if the terminator itself does not work.<br><br />
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Possible explanations for the contradiction between our results and those of Kang and coworkers might be the experimental set-up and the RNA Polymerases we used. Different variants of T7 RNA Polymerase might respond in different ways to terminator structures, and the termination might be influenced by the presence or absence of cofactors, depending on the purification methods used in producing the Polymerase.<br><br><br />
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This set-up offers a lot of possible experiments for the future, which we would have loved to conduct with a just a bit more time...<br />
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====''E.coli'' System====<br />
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Compared to the T7 System, the ''E. coli'' RPO system produced poor increases in fluorescence, indicating little RNA synthesis. It was shown that the presence of a terminator decreases, as expected, the production of downstream RNA.<br><br />
<br><br />
[[Image:TUM2010_101023kinetik.PNG|350px||thumb|left|''in vitro'' transcription measurement of Switch TrpTerm (upper traces) and positive control (lower traces). Left side: with Trp-signal, right side: no signal]]<br />
[[Image:TUM2010_101022_Kinetik.png|350px||thumb|right|''in vitro'' transcription measurement of positive control (left), Switch TrpTerm (center) and switch HisTerm(right)<br />
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]]<br><br />
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This result was also confirmed by denaturing PAGE. However, due to the poor changes in fluorescence we were not able to actually characterize the behaviour of our switches ''in vitro'', and the small RNA concentrations did not allow a quantitative interpretation of our gels.<br />
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[[Image:TUM2010_coliGel.png|500px||thumb|center|denaturing polyacrylamide gel electrophoresis of DNaseI digested samples from ''in vitro'' transcription of positive control (16z), Switch TrpTerm (W) and switch HisTerm(H). c marks the lanes in which the DNA was injected, the last three lanes show the undigested samples]]<br />
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A major problem with this method was the low concentration of the ordered Polymerase resulting in a much weaker overall signal as comparable measurements using the T7 Polymerase. <br><br><br />
In future experiments we might try to work with smaller volumes in order to reach higher concentration of RPO and of the synthesized RNA molecules, so measuring in 96 well plate readers might be a good choice. <br />
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==Software==<br />
Although we could not show the full functionality of bioLOGICS in the lab we still want to demonstrate the potential of our approach. Hence we implemented the idea behind our logic gates in a program which illustrates how bioLOGCIS theoretically would allow the construction of complex information processing networks interconnecting BioBricks. For further details take a look at our [[Team:TU Munich/Software|Software page]].<br />
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<br />
=References=<br />
<html><a name="ref1"></a></html>[1] http://partsregistry.org/cgi/partsdb/Statistics.cgi<br />
<html><a name="ref2"></a></html>[2] https://2009.igem.org/Team:Imperial_College_London/M1 encapsulation<br />
<html><a name="ref3"></a></html>[3] https://2009.igem.org/Team:TUDelft<br />
<html><a name="ref4"></a></html>[4] https://2008.igem.org/Team:Heidelberg<br />
<html><a name="ref5"></a></html>[5] Maung Nyan Win and Christina D. Smolke, Science Oct. 2008 Vol. 322. no. 5900, pp. 456 - 460<br />
<html><a name="ref6"></a></html>[6] Lu, T.K., A.S. Khalil, and J.J. Collins, Next-generation synthetic gene networks. Nature biotechnology, 2009. 27(12): p. 1139-1150. <br />
<html><a name="ref7"></a></html>[7] Schaller, R.R., Moore's law: past, present and future. Spectrum, IEEE, 2002. 34(6): p. 52-59.<br />
<html><a name="ref8"></a></html>[8] von Mering, C., et al., Comparative assessment of large-scale data sets of protein–protein interactions. Nature, 2002. 417(6887): p. 399-403.<br />
<html><a name="ref9"></a></html>[9] Mandal, M. and R.R. Breaker, Gene regulation by riboswitches. Nature Reviews Molecular Cell Biology, 2004. 5(6): p. 451-463. <br />
<html><a name="ref10"></a></html>[10] Benner, S.A. and A.M. Sismour, Synthetic biology. Nature Reviews Genetics, 2005. 6(7): p. 533-543.<br />
<html><a name="ref11"></a></html>[11] Beaudry, A. and G. Joyce, Directed evolution of an RNA enzyme. Science, 1992. 257(5070): p. 635-641.<br />
<html><a name="ref12"></a></html>[12] Delorme, Ehrlich and Renault, Regulation of Expression of the Lactococcus lactis Histidine Operon. Journal of Bacteriology, Apr. 1999, p. 2026–2037<br />
<html><a name="ref13"></a></html>[13] Trun and Trempy(2003): Fundamental Bacterial Genetics, Wiley-Blackwell, Chapter 12 <br />
<html><a name="ref14"></a></html>[14]Sooncheol Lee, Huong Minh Nguyen and Changwon Kang, Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator. Nucleic Acids Research, 2010, 1–9<br />
<br />
<!-- The idea behind our project is to change the way BioBricks have been used up to now. Over the years, many receptors and signals have been constructed as BioBricks during the annual iGEM competition, but still it is not possible to interconnect these Bricks in a complex biological network resuting in a cell, that is able to respond to its environment giving differenciated responses depending on the input signals. (Beispiel: cambridge hat das gemacht, xx dies, aber eine zelle kann nicht beides...<br><br />
We plan to create biological switches, that can function as locial gates inside a cell. Our switches rely on RNA/RNA-interactions, regulating transcriptional termination. This is a major advance of our concept, as regular switches rely on complex regulation including proteins and/or metabolites. Thus, our switches shall offer a greater robustness and their behaviour should be easier to predict. [[switch|Read more]] (hier sollte noch das hochskalieren erwähnt werden...<br><br />
These switches can further be used to build up a logical network inside a bacterial cell, enabling every scientist to connect as many functionalities (in form of BioBricks) as designated. We plan to offer simulation on each specifically designed network.<br />
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<br><br>Over the years, many teams participating in the iGEM competition spent their time on constructing receptors and systems to detect a certain input that a variety of gorgeous oppurtunities is available so far.[[Image:TUM2010 network.png|thumb|300 px|right|Our visioon: A logic network inside the cell]] Nevertheless, until now it is not possible to link all those functionalities and build up a network giving differenciated responses to several of those input signals, where the molecular response depends on the complex composition of the environment a cell faces. We would like to offer this possibility to everyone.<br />
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The logic network we want to apply will be based on devices, that can be easily upscaled and therefor offer the chance to build networks of any wanted complexicity. Our devices rely on pure RNA/RNA interactions and thus their behaviour is well predictable.<br />
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The concept we rely on for our design of RNA-switches is based on the principle of [https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation/ '''attenuation'''].<br />
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= Experiments =<br />
We designed several experiments to test our switches, all of them based on fluorescence measurements. We designed experiment setting for measurements ''in vivo'' as well as ''in vitro''. Our ''in vitro'' measurements relied on two different experiment set-ups. While the first was based on a commercial ''E. coli''-lysate, the latter was reporting on a transcriptional level only, eliminating most of the possible side-effects one could expect in the complex behaviour of a living cell or cell-lysate. [[Experiments_main|Read more]]<br />
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= Results =<br />
We ...blablabla<br />
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'''bioLOGICS: Logical RNA-Devices Enabling BioBrick-Network Formation'''<br />
'''Abstract'''<br />
Among the goals of iGEM is the creation of synthetic biological parts and their utilization to achieve novel features and behavior in biological systems. The emphasis of our project is put on this latter, "systems" aspect of iGEM. More precisely, we aim at the development and experimental demonstration of a scalable approach for the realization of logical functions in vivo.<br />
<br />
By developing a computational biological network based on RNA logical devices we will offer everyone the opportunity to 'program' their own cells with individual AND/OR/NOT connections between BioBricks of their choice. Thereby, BioBricks can finally fulfill their original assignment as biological parts that can be connected in many different ways. We will achieve this by engineering simple and easy-to-handle switches based on predictable RNA/RNA-interactions regulating transcriptional termination. These switches represent a complete set of logical functions and are capable of forming arbitrarily complex networks.<br />
<br />
== The Experiments ==<br />
===Fluorescent proteins as reporter===<br />
<div align="justify"><br />
Our initial idea to prove our concept of antitermination was to use flourescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks ''in vivo'' as well as ''in vitro'', the latter using a translation kit based on e.coli lysate. <br>We decided to use the flourescent proteins GFP and RFP, as their spectra do not overlap and we would not measure any FRET. We would use GFP fluorescence as internal control and RFP fluorescence as signal to detect termination/antitermination by our switch we cloned in between the coding sequences of the proteins. Both protein sequences are under the control of one (L-arabinose induced) promoter.<br />
[[Image:TUM2010_gfprfp_schalter_klein.gif|center|our idea]]<br />
<br><br />
When measuring the termination of our BioBricks and the antitermination by their corresponding signal-RNA, we should be able to observe an increasing RFP emission compared to the GFP emission upon induced signal-RNA production in the cells/in the kit:<br><br />
[[Image:TUM2010_Expected_emission_spextra.png|center|our idea]]<br />
Wiith these measurements, it should also be possible to observe differences in efficiency of termination as well as antitermination between our designed switches. <br><br><br />
<br />
===Measurements with the malachite green aptamer as reporter===<br />
A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramaticly (about 3000 times) upon binding of a specific RNA-aptamer.<br><br>#<br />
[[Image:TUM2010_Malachitgruen-2.png|500px|center|our idea]]<br />
---concept to be desribed, as well as literature---<br />
<ref>refs</ref><br />
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To study the switches on the transcriptional level gives the advantage, that we would have less interferences and possible artefacts. Also, we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems.<br><br />
<br />
[[Image:TUM2010_Malachit_emission.png|200px|thumb|left|Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA]]We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.<br>Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level.<br />
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Results <br />
==Flourescent proteins==<br />
Unfortunatly, we had to change the reporter construct two times during our experiments as several problems occured in our measurements:<br><br><br />
<br />
===First Try: based on the measurement plasmid pSB1A10===<br />
At the beginning, we decided to use the reporter plasmid [http://partsregistry.org/Part:pSB1A10 pSB1A10] from the registry. It consists of the fluorescent proteins eGFP and mRFP1. Each sequence includes a ribosome binding site and a stop-codon; the two genes are divided by a cloning side including the BioBrick cleavage sites.[[Image:ScreeningPlasmid1.0.PNG|300px|thumb|right|pSB1A10]]<br> In front of the eGFP sequence, the plasmid includes an arabinose-inducable promoter. The plasmid also contains an ampicilline resistence.<br><br />
We cloned our switches into the cloning site of the measurement plasmid and used an empty cloning site as control; our signal-RNAs we cloned into the [http://partsregistry.org/Part:pSB1K3 pSB1K3] vector, together with the BioBricks R0011 (Lac promoter) and B0014 (double terminator of transcription). Afterwards, we cut pSB1K3 with Aat2 and Pst1 and pSB1A10 with Nsi1 and Aat2 and ligated those fragments of each plasmid that contained our Bricks to get a Monsterplasmid.<br>We had to do so, as both plasmids contain the same ori mechanism. In addition, having both the switch and the signal RNA transcribed from the same plasmid gives us a high local concentration of the signal, once its transcription is induced.<br><br />
[[Image:TUM2010_Messplasmid_nr1.png|left|the measurement plasmid]]<br />
<br />
We transformed BL21(DE3) cells with the plasmid. We set up cultures, induced the arabinose promoter and measured the GFP and mRFP1 excitation/emission spectra within time.<br><br><br />
Unfortunatly, we were ot able to detect any RFP signal, not even in the positive control with no switch in between the GFP/RFP sequences.<br><br><br />
From these experiments, we concluded, that the mRNA of the RFP variant used was instable and rapidly degraded by RNases, so the RFP was not synthesized in the cells. This was also the conclusion from XXX...<br />
<br><br><br />
As solution to this, we decided to design a measurement plasmid ourselves:<br><br />
<hr width="300"><br />
<br><br />
<br />
===Second Try: A measurement plasmid of our own design===<br />
To design our own plasmid to overcome the problems that occurred in our first try gave us tghe possibility to overcome several other problems:<br><br />
#<br />
#<br />
#<br />
[[Image:TUM2010_Construct_no2.png|400px|our construct|center]]<br />
<br />
===Third Try: One promoter for each protein===<br />
We decided to use the measuremnt plasmid we developed in our second try but to clone another L-arabinose induced promoter into the plasmid, in front of our switch followed by the mCherry sequence.<br><br />
<br>[[Image:TUM2010_Construct_no3.png|left|400px|Construct #3]]In this way, we still can use GFP fluorescence as internal control, because both protein transcription is under the control of a promoter of identical design.<br />
Though we are still not able to tell exactly why our previous measurements did not work, but with this construct we measured the first time fluorescence of the mCherry protein in our positive control.<br />
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{{:Team:TU_Munich/Templates/End}}</div>Hartlmuellerhttp://2010.igem.org/Team:TU_Munich/ProjectTeam:TU Munich/Project2010-10-28T03:44:40Z<p>Hartlmueller: /* From switches towards bioLOGICS logic gates */</p>
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<center><font size="5pt" color="#000000">'''bioLOGICS'''</font><font size="4pt" color="#000000">: Logical RNA-Devices Enabling BioBrick-Network Formation</font></center><hr color="black"><br><br />
= Vision=<br />
<br />
Until today, 13.628 biobrick sequences<sup>[[Team:TU_Munich/Project#ref1|&#91;1&#93;]]</sup> have been submitted to partsregistry, thereof 102 reporter units and 12 signaling bricks.<br />
Since then, people are trying to arrange these single biological building blocks in such a manner that allows producing special biotechnological products (metabolic engineering), developing biological sensory circuits (biosensors) and even giving microorganisms the ability to react on multiple environmental factors and serve both as disease indicator and drug. These examples and further promising ideas were implemented on previous iGEM-competitions.<sup>[[Team:TU_Munich/Project#ref2|&#91;2&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref3|&#91;3&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref4|&#91;4&#93;]]</sup> <br><br><br />
The idea of combining the outcome of several iGEM competitions to construct complex synthetic biological systems falls at the last hurdle - the fact, that each team uses a different principle how to access and functionally connect the respectively used biobricks. For example, it is a major challenge to create a system that uses several sensoring BioBricks from different iGEM-teams which in turn regulates reportering BioBricks from various teams. In order to combine and fully take advantage of these promising projects, our vision is to develop an adapter that allows interconnecting arbitrary biobricks on a functional level. Such a system easily allows to setup sensor-reporter circuits and interconnect them to complete biological chips... A further step towards artificial cells.<br><br><br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, the above adapter has to meet the following requirements:<br />
*'''Universality'''<br />
:The adapter has to be compatible to as many BioBricks as possible. This objective will guarantee that a large number of BioBricks can be connected.<br />
*'''Scalability'''<br />
:Once the basic design of the system is established, the construction of the system is supposed to be automated in silico. This way it will be possible to create an adapter connecting a large amount of BioBricks.<br />
*'''Biological orthogonality'''<br />
:Interference with cellular components has to be as low as possible in order to avoid unwanted and perturbing side effects.<br />
*'''Logic'''<br />
:The adapter is supposed to not only associate different BioBricks, but to functionally connect BioBricks in a precisely determined manner (including operations such as AND/OR/NOT).<br />
<br><br />
Several biological logic units, devices and circuits have been developed so far<sup>[[Team:TU_Munich/Project#ref5|&#91;5&#93;]]</sup>, but to our knowledge, none was shown to meet all requirements listed above.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
=Implementation=<br />
To functionally connect BioBricks, there are several possibilities including genetic switches, riboswitches and direct protein-protein interactions. We investigated several hypothetically principles, and decided to focus our practical work on the development of a RNA-RNA interaction-based switch. These switches are capable of changing between two states, a state of antitermination and termination, and make use of highly-specific RNA-RNA interaction. In principle such a switch can fulfill all requirements mentioned previously. The following text clarifies how these switches work in detail.<br />
==How to connect BioBricks==<br />
Our adapter is a system, that activates or disables BioBricks (output BioBricks) in response to the presence of other Biobricks (input Biobricks). Our approach uses a molecular network to put this into practice and consists of four major elements:<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
<br><br />
{|<br />
|-<br />
|[[Image:Networks.png|center|thumb|730px|The general principle how different inputs can be connect to various outputs. For details see text.<br>Inputs (such as proteins or small molecules) are indicated on the left side. blue lines represent transmitter molecules whereas organe lines present logic gates. The type of logic gate is indicated. Green lines indicate transmitter RNA that can function as mRNA and consequently generate any output gene (indicated on the very right).]]<br />
|}<br />
In order to connect different BioBricks, our network requires four major types of components:<br />
*Input elements<br />
*Transmitter molecules<br />
*Logic gates<br />
*Output elements<br />
<br />
{{:Team:TU_Munich/Templates/InfoBoxStart}}'''Computer vs. molecular network - and our approach'''<br><br />
Logic gates in a molecular network are often compared to transistors used in a computer, where billions of transistors are incorporated<sup>[[Team:TU_Munich/Project#ref7|&#91;7&#93;]]</sup>. The main advantage on a computer chip is, all transistors share the same functional principle, and only the way connecting them in a special sequence allows specific addressing of only a subset of other transistors by an input. However, spatially fixed connections of molecular logic gates are not possible in a living cell. The "wiring" within a cell relies on the specific interaction between transmitter molecule and their corresponding logic gates, for example implemented by protein-protein/ligand-protein interactions or specific ligand-riboswitch interactions.<sup>[[Team:TU_Munich/Project#ref8|&#91;8&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref9|&#91;9&#93;]]</sup> As a result, in a cell, each occurring logic gate ("transistor") has to be different, at least in a special recognition site<sup>[[Team:TU_Munich/Project#ref10|&#91;10&#93;]]</sup> - for example like different transcription factors, recognizing different DNA-sites. Thanks to evolution, nature easily can invent a new transistor for each task - science achieves this only on a limited scale, and producing synthetic molecular logic gates artificially by either rational or evolutionary protein or riboswitch engineering, is limited to small circuits so far<sup>[[Team:TU_Munich/Project#ref11|&#91;11&#93;]]</sup>. Our project aims to establish a molecular switch as close as possible to a electronic transistor, thus sharing the same functional principle for all logic gates. At the same time, we want to design a easily exchangeable recognition site, which can individually be designed by everyone! {{:Team:TU_Munich/Templates/InfoBoxEnd}}<br />
<br />
These elements can be combined to build up a molecular network (see illustration). Each input molecule (such as a BioBrick) produces a unique transmitter molecule. All transmitters belong to the same type of molecule and share a common design. However, each transmitter molecule can only interact and activate a certain subset of logic gates. In other words, logic gates have to recognize as well as bind the corresponding transmitter molecules and are capable of producing a new output transmitter molecule. Depending on the type of the logic gate (AND, OR or NOT<sup>[[Team:TU_Munich/Project#ref6|&#91;6&#93;]]</sup>), an output transmitter is only created if both input transmitter molecules are present (AND), at least one of two input transmitters is present (OR) or if no input transmitter is present at all (NOT). Once a logic gate has produced a new output transmitter, these transmitters can in turn address another subset ("layer") of logic gates. In theory many layers of logic gates can be connected this way allowing the creation of large networks. Until this step, various transmitter molecules might have been produced. But in order to create a Biobrick output, the last layer of logic gates finally generates transmitter molecules that will not active logic gates, but will rather interact with the cell metabolism to produce a BioBrick response. In other words, the last layer of transmitter molecules is capable of regulating BioBrick formation.<br />
<br />
<br />
Summarizing, the network establishes a connection between input BioBricks and output BioBricks in a functional manner.<br />
Having addressed the basic layout of the molecular network, the next step is to determine what type of molecules can perform the required functions. We decided to use RNA, both as transmitter molecules and for constructing logic gates. Several advantages result from the utilization of RNA as the central element:<br />
*During the last years, many Biobricks were designed that are sensitive to various chemicals and substances. These BioBricks often function as a transcription factor that binds to a specific DNA sequence and consequently would be capable to produce a specific transmitter RNA molecule. Thus, in principle each BioBrick which involves transcription can be integrated in our network.<br />
*Since all logic gates are capable of producing transmitter RNA, they can also produce functional mRNA encoding any protein. This means, each BioBrick consisting of protein or RNA can be produced as an output of our network.<br />
*If RNA forms both, the transmitter molecule and the logic gates, they can specifically interact by RNA-RNA interaction, which is highly predictable compared to protein interactions. This allows to generate a library of transmitters and gates ''in silico''. Such a library is essential for the creation of large networks.<br />
*RNA production is fast and energy saving for a cell. Consequently, operating a network that only produces RNA rather than proteins will also be faster and more efficient for the host cell. Since our logic gates are based on transcription, translation and resource consuming protein production will only be required at the very last step. <br />
*As the half-time of RNA can be rather short, transmitter RNA will not accumulate within the cell and it is therefore less likely for the system to become saturated.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Design and functional principle of logic gates==<br />
The concept introduced above provides a framework that can potentially serve as an universal adapter between different BioBricks. However, the [[Team:TU_Munich/Glossary#logic gate | logic gates]] have not been specified more precisely so far. This will be done in the following section.<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, our logic gates are to possess the following characteristics:<br />
*Logic gates, such as AND, OR and NOT, have to be implemented by RNA-interaction based principles (see [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]).<br />
*All logic gates have to recognize their corresponding [[Team:TU_Munich/Glossary#Transmitter (bioLOGICS)| transmitter RNAs]] and, in response, produce an output transmitter molecule.<br />
*Logic gates should follow a basic design rule, in such a way, that their creation can be automated ''in silico''.<br />
*The response efficiency of a logic gate toward a transmitter molecule should be comparable for all logic gates to provide calculable robustness and sensitivity. This will ensure comparable molecular concentrations and functionality of large networks.<br />
*The system has to be designed for ''in vivo'' utilization at the first place. As a reference we always assumed a temperature of 37 °C and an ''E. coli'' environment.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}} <br />
In order to build logic gates for our bioLOGICS system we will first create a simple switch. A switch can be activated by one transmitter RNA and produce an output transmitter RNA. In contrast to a logic gate, a switch does not perform logic operations. However by combining switches, logic gates can be created. The following text will first describe how the developed switch works and secondly, how logic gates such as AND/OR/NOT can be created using these switches.<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Read more{{:Team:TU Munich/Templates/ToggleBoxStart2}}<br />
[[Image:toggle_switch.png|500px|thumb|center|id="hideOnReadMore"|'''A''' The basic structure of a bioLOGICS switch (left) and a transmitter molecule (right).<br>'''B'''The process of switching. See the text in the close-by "Read more" section for details.<br>Rectangles present the composition of our functional units on the level of DNA. Fringed lines represent RNA produced by RNA polymerase. The stem loop structure depicts the switchable terminator. Terminator and target site are illustrated in blue and turquoise, respectively. Recognition sites are indicated in different colors, in this case red for the input transmitter and green for the output transmitter.Each switch and or later logical unit has to be flanked by a promotor and another constitutive terminator, to allow RNA-production by RNA-polymerase in a proper way. ]]<br />
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===Switch===<br />
[[Image:TUM2010_switch-and-transmitter.jpg|550px|right|thumb|The basic strcutrue of a switch (left) and a transmitter RNA (right). See text for details.]]<br />
Roughly speaking, a switch can be regarded as an enhanced switchable transcriptional terminator. The enhancement can be described easier by dividing a switch into its functional components: <br />
*'''Target site'''<br><br />
:The target site is the functional core element of our switches, allowing a shift between an "on" and "off" state. Since we work on the level of RNA-production (transcription), a "switchable" transcriptional terminator is suitable for this purpose. By allowing or preventing formation of a transcriptional terminator, that is by switching between termination and antitermination it is possible to represent an "off" and an "on" state, respectively. Therefore, the target site is the 5' ending of the terminator and is required for a stable terminator formation. It should be noted that this principle was also observed in nature.<br />
:To highlight and illustrate the functional principle of our switches, only the part of the terminator which is involved in interacting with a transmitter molecule and which is responsible for shifting between "on" and "off" state is called target site. The remaining terminator sequence is called terminator in the following, even if both, target site and terminator build up the terminator structure occurring in nature. <br />
:The important aspect of our switches is the fact that all switches will hold the same identical target site. Therefore having found one functional "switchable" terminator, will allow almost unlimited upscaling since this terminator can be used for a large library of switches. This is the main difference to previous works done on this field, which always required developing a new shifting principle for each switch.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup> Beside this scalability, this principle provides a comparable on/off shifting rate (responds function) for all switches, avoiding complex fine tuning of molecular networks.<br />
:To sum it up, the target site, allows to switch between an "on" and "off" state. But so far, the switch is not capable of performing specific interaction with transmitter molecules. This is where the recognition site comes into play.<br />
*'''Recognition site'''<br />
:The recognition site defines which transmitter molecule can actually interact with the switch. Therefore, a unique recognition site is generated for each switch and is positioned right upstream of the target site. In principle the recognition can be any random sequence as long as it remains unique within the molecular network.<br />
Summing up, the recognition site allows a specific interaction between switches and transmitter molecules. Once this interaction is formed, an interaction between the transmitter and the target will actually switch the state of the terminator. This allows the specific arrangement and interconnection of numerous of these switches by transmitter molecules, without changing the target site. Comparable to wires connecting many identical transistors, our target site remains the same.<br />
<br><br />
<br />
===Transmitter RNA´s===<br />
As desccribed above, transmitter RNAs are the input and output of bioLOGICS switches (compare [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]). These transmitters are short ssRNA molecules representing the "trigger" to shift switches between the "on" and "off" state. To fulfill this role, they need to posses the following properties:<br />
*A transmitter may only interact with certain switches. That is, a transmitter has to find the corresponding recognition site of a switch.<br />
*Once an interaction is established between a transmitter and a switch, a transmitter has to be capable of changing the secondary structure of a terminator and thus cause antitermination.<br />
Again, these two properties are fulfilled by two components of the transmitter:<br />
*'''Identity site'''<br />
:This site is capable of forcing an interaction between the transmitter and the switch. Therefore it is complementary to the recognition site of this switch. As the recognition site is unique within a network, so is the identity site. However, the single identity site is not capable of changing the state of the switch. That is were the trigger site comes into play.<br />
*'''Trigger site'''<br />
:Once an interaction is created by the identity site, the trigger site is capable of actually shifting the switch since it is complementary to the target site of the switch. To fulfill this role, it is placed upstream at the 5' end of the identity site. As the target site is the same for all switches, the trigger site is the same for all signals. Therefore it is important, that similar to the identity site, a trigger site cannot function on its own. That is, a single trigger site cannot shift the state of a switch without the help of an identity site.<br />
<br />
Summing up, we applied the principle introduced for the switches to the transmitter molecules. In contrast to previous approaches on this field <sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup>, we introduced the described synthetic trigger site in such a manner that it is not able to change the state of the terminator on its own, but only in combination with the identity site. So the challenge is to arrange and optimize these elementary building blocks thermodynamically, that a trigger site is only able to switch in combination with its respective identity site. This was done by ''in silico'' design using [[TU Munich/Glossary#NUPACK| NUPACK]], presented in section [[TU Munich/Modeling#in silico design based on thermodynamic calculations| in silico design]].<br />
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===Putting it all together: the switching process===<br />
[[Image:TUM2010_switching-process.jpg|550px|right|thumb|The basic structure of a switch (left) and a transmitter RNA (right). See text for details.]]The functional principle of the designed switches is illustrated in the figure. The switch is positioned on DNA upstream of a desired output transmitter. So in the absence of a triggering transmitter molecule, transcription will be canceled by the formation of a RNA stem loop in the nascent RNA-chain. This will cause the RNA polymerase to stop transcription and fall off the DNA and consequently no output RNA will be produced. This process only relies on [[Team:TU_Munich/Glossary#Termination| rho-independent termination]].<br />
On the other hand, in the presence of a [[Team:TU_Munich/Project#RNA_transmitters | input transmitter]], this small functional RNA inhibits the stem loop formation by complementary base-pairing and hence avoids termination of transcription. In detail, the identity site (red part on transmitter) binds the recognition site (red part on switch) and serves as [[Team:TU_Munich/Glossary#Toehold|toehold]], which will thermodynamically allow the trigger site (turquoise part on transmitter) to perform a [[Team:TU_Munich/Glossary#Strand Displacement| strand displacement]] and open up the stem loop structure. Consequently the polymerase can read all the way through and form the output RNA.<br>Summing up, we use this concept to create a switch that can be toggled by a transmitter RNA molecule and in response, is able to produce another transmitter RNA.<br />
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<br />
===From switches towards bioLOGICS logic gates===<br />
As described, each switch can be accessed by a specific RNA-transmitter molecule, representing the input. In turn, another RNA-transmitter molecule will be produced if the switch shifts its state. This output transmitter of one switch can serve as input transmitter for the next switch by meaningful selection and design of the respective recognition sites. This easily allows arranging several switches in specific sequences and faulty wiring - the corner stone of a logical network.<br><br />
To ease the building of logical networks we want to create a switch capable of Boolean logics, a common mathematical principle fundamental for computational science. Since AND/OR/NOT are basic logic operations which can be implemented with the presented switches, all remaining operations (such as XOR, NAND, ...) can be expressed by these three operators according to laws of boolean logics.<br />
Creating logic gates is achieved by combining two switches in two different ways, as illustrated below.<br />
*AND gate<br />
:An AND gate can be constrcuted by positioning two switches right next to each other. For the output transmitter to be created, both input transmitter have to be present.[[Image:AND2.png|500px|thumb|center|Combining two switches in series creates a logic AND gate.]]<br />
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*OR gate<br />
:An OR gate is created by utilizing two independent switches sharing the same output transmitter. If each one of both switches is activated, an output transmitter is generated. Therefore, one input transmitter is enough to produce an output transmitter.[[Image:OR2.png|500px|thumb|center|Combining two switches in parallel creates a logic OR gate.]]<br />
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*NOT gate<br />
:A NOT gate is supposed to function as an inverter. In contrast to the gates described above, a not gate requires only one sitch. However, to meet the design rule for transmitter molecules, this switch shows some differences compared to the switches used for AND and OR gates.<br><br />
:Since the transcriptional terminator may not form if no transmitter is present. Consequently, the switch needs an internal trigger site, capable of preventing terminator formation. To allow the binding of an input transmitter molecule, the switch contains a recognition site upstream of a second target site. The additional target site is mandatory since all transmitter molecules have to carry a trigger. In the case of the NOT switch this trigger site may not bind the actual target site within the transcriptional terminator. In other words, a second target site further upstream is required to catch the trigger site of the transmitter molecule. At the same time, the identity site of the transmitter may not bind right upstream of the terminator. This is accomplished by placing an other identity site right upstream of the terminator rather than an recognition site (compare switches used for AND or OR gate). Due to these two difference, the input transmitter is forced to bind further upstream to the recognition site, displacing the internal trigger site of the switch. This will allow the RNA polymerase to read through an create the output transmitter.<br />
[[Image:NOT2.png|500px|thumb|center|Structure and switching process of a NOT gate.<br>]]<br />
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<br />
==Network construction==<br />
Designing complex biological networks based on either traditional protein engineering or our new bioLOGICS is still a complex task. We developed a software which allows the fast construction of a bioLOGICS based networks. <br><br />
To read more about this, look at our [https://2010.igem.org/Team:TU_Munich/Software Software page]<br />
<br />
=Our Objective=<br />
Putting the implementation described above into practice, will be a major challenge. For this year's iGEM competition our goal is to do the first step: design and build a switch that can be toggled by a RNA molecule. To be precise, we want to apply the design rules of our switch to modify a transcription terminator in such a way that it interacts with a second RNA molecule and, as a result, is no longer capable of forming a stem loop. This objective will require intensive ''in silico'' designing and modeling of switches based on different terminators and their corresponding transmitters. In connection to this theoretical part, we also have to test and verify the switches. For this step, we establish custom-made assays, ''in vitro'' and ''in vivo''.<br />
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Once the objective mentioned above is accomplished, these basic RNA/RNA-interactions have to be modified in such a manner that the described identity/trigger site pattern for the transmitter and the complementary recognition/target site switch composition has to be established. The most important requirement is to is to optimize these modules that the transmitter is only able to switches specifically, meaning only in the presence of both, identity AND trigger site. <br />
<br><br />
Once the objective mentioned above is accomplished, the creation of an OR gate will be rather simple since it only requires two switches. However the creation of an AND or NOT gate and optimizing the logic gates to improve their responds function will remain the goal of future work. Also the creation of small networks and the correct integration of BioBricks as input and output molecules will be future challenges. Furthermore, we wanted to rather focus on the development and the testing of our structural design of the switches, rather than developing a variety of new BioBricks.<br />
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==''In silico'' design==<br />
As described above, our switches are based on certain design rules. However, there still are different structural parameters that need to be tested and optimized (length of recognition site and target site, choice of terminator, etc.).<br />
We used [[Team:TU_Munich/Project#in silico design |''in silico'' design]] and [[Team:TU_Munich/Modeling| modeling]]) to test different parameters. Furthermore we tried to use the [[Team:TU_Munich/Glossary#Antitermination|antitermination principle]] observed in nature, such as [[Team:TU_Munich/Glossary#Attenuation| attenuation]] in ''E. coli'' or [[Team:TU_Munich/Glossary#Tiny Abortive RNA´s| tiny abortive RNA´s]] of T7-phage.<br />
==Evaluation and Measurements==<br />
To evaluate the functionality of our molecular switches, we first had to establish several assays. Therefore, we improved an existing [[Team:TU_Munich/Lab#In vivo Measurements |''in vivo'' assay]] and developed an [[Team:TU_Munich/Lab#In vitro Transcription | ''in vitro'' assay]] for this purpose. For more information please refer to the [[Team:TU_Munich/Lab | lab]] section.<br />
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<br><br />
Summarizing, the main challenges are <br />
* to find a suitable terminator construct and design a complementary trigger unit, which is only functional in combination with a specificity site - meaning an optimization of the '''thermodynamically parameters''' (see[[Team:TU_Munich/Project#in silico design| in silico design]])<br />
* to investigate whether the transmitter/switch interaction reaction is on a timescale to be competitive to terminator formation - meaning an comparison of '''kinetic parameters''' (see [[Team:TU_Munich/Modeling|Modeling page]])<br />
* to proof antitermination can be also be caused by synthetically RNA-interaction (see [[Team:TU_Munich/Glossary#Antitermination| Antitermination in nature]] and [[Team:TU_Munich/Project#Results| ''in vivo'' and ''in vitro'' measurements]] )<br />
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=Results=<br />
Every network starts with a basic unit. While our declared aim is to enable networks allowing fine-tuning of gene expression beyond the regular on/off, exploring such an on/off switch/signal pair is the first step towards a functional network. We constructed several units and tested their efficiency, robustness and reproducibility ''in vivo'', ''in vitro'' and ''in silico''. Furthermore we developed a software which allows easy constructions of networks based on our designed logic gates. Conclusive elaboration of a few first RNA-based logic units is the major contribution of our iGEM team.<br />
<br />
==in silico Design of Switching and Trigger Unit==<br />
As described on the [[Team:TU_Munich/Project | project]] page, one key aspect of our switches is the idea, that a [[Team:TU_Munich/Glossary#Transmitter_(bioLOGICS) | RNA transmitter molecule]] is capable to shift the state of a switch only if its [[Team:TU_Munich/Glossary#Trigger_Site_(bioLOGICS) | trigger site]] is present and its [[Team:TU_Munich/Glossary#Identity_Site_(bioLOGICS) | identity site]] corresponds to the [[Team:TU_Munich/Glossary#Recognition_Site_(bioLOGICS) | recognition site]] of the [[Team:TU_Munich/Glossary#Switch_(bioLOGICS) | switch]]. We successfully constructed several switches and their corresponding transmitter RNA ''in silico'' on a thermodynamical basis. We modified different transcriptional terminators in such a way, that the formation of the terminator was prevented by a transmitter molecule. As desired, this only occured if the transmitter molecule contained both, a trigger and an identity site. Analogously, we were able to design and verify a NOT gate using the same thermodynical approach.<br />
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==Diffusion and RNA Folding Dynamics==<br />
We estimated the diffusion time for our constructs and modeled the folding dynamics of our bioLOGICS switches including the switching process with a stochastic RNA folding program. We were able to provide better insight in their folding dynamics and proved that they are able to interrupt termination. We also optimized the switches and the corresponding signals. Furthermore, we combined the switches what resulted in a logic gate. See our [[Team:TU Munich/Modeling|Modeling page]] for further details.<br />
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==''in vivo'' Functionality Screening==<br />
Since our logic gates are intended to function in living cells, ''in vivo'' measurements were essential. In a set of experiments we concentrated on two different switches based on known [[Team:TU_Munich/Glossary#Attenuation|attenuators]] from nature: the [[Team:TU_Munich/Modeling#Switch|HisTerm]] and [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Focusing on fluorescent proteins for quantifiable input and output we designed a functional and robust screening system. For greater detail see [[Team:TU_Munich/Lab#Experiment_Design|Experimental Design]]. Unfortunately, setting up a working screening system failed twice. Only in redesigning and improving the screening plasmid pSB1A10 we succeeded, but lost precious time.<br />
<br />
Ultimately, the two switches displayed remarkable differences in their terminator efficiency, but neither of them responded to their corresponding signal. However, screening one transmitter signal does not disprove the basic working principle of our system. Limited by time, we hope for future teams to take up our work and to use our improved test system that we submitted to the parts registry, for performing successful in vivo measurement.<br />
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Considering the high complexity of ''in vivo'' measurements compared to other experimental challenges, a robust and easy to handle test system for [[Team:TU_Munich/Glossary#PoPS-based devices| PoPS-based devices]] is desirable. As described in [[Team:TU_Munich/Lab#Experiment_Design|Experimental design]], we used fluorescent proteins: RFP or mCherry to measure the amount of produced output and eGFP for normalization. Our first attempt, using the screening plasmid pSB1A10, yielded no interpretable results. Switching the fluorescent protein to mCherry did not work either, but after several experimental setups we determined a transcriptional problem causing no reporter protein expression regardless of the inserted part. Thereby we demonstrated the screening plasmid pSB1A10 to be [[Team:TU_Munich/Biobricks#Falsification| malfunctioning]]. <br />
Finally a new design based on pSB1A10 lead to a functional and robust screening system (compare [[Team:TU_Munich/Parts#Screening system: Backbone BBa_K494001| Screening system: Backbone BBa_K494001]]). A second promoter with identical induction properties inside the BioBrick cloning site enforces transcription of the PoPS-based device and the mCherry output.<br />
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Exemplary, the graph below on the right shows the positive control, induced and uninduced at OD<sub>600</sub>=0.7 followed by 16 h incubation at 25 °C. Clearly visible are eGFP and mCherry fluorescence in the induced samples. The uninduced control showed no fluorescence at all, demonstrating the PBad promoter to be tight and providing very low basal transcription, what is a major advantage for the screening system. This newly designed screening approach renders the characterization of PoPS-based devices in general and switches in particular easy and robust. The low basal transcription furthermore fulfills one of the most important requirements for the designed switches, since output transmitters may only be produced in presence of an input transmitter. This helps to avoid strong "background" noise, which would extremely harden the successful interconnection of several switches. <br />
<br><br />
[[Image:TUM2010_PosControlklein.JPG|200px||thumb|left|Bacteria containing positive control]]<br />
[[Image:TUM2010_graphPosControl1.png|355px|thumb|center|Emission spectra of induced (green/red) and uninduced(black) positive control BBa_K494002 ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
Due to the time limitations of the iGEM completion we had to focus our efforts on few switches after designing the screening system. Relying on the functionality of systems occurring in nature, we choose the [[Team:TU_Munich/Modeling#Switch|HisTerm]] as well as the [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Both switches are based on known natural [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|attenuators]]. Testing synthetic and none-naturally switchable terminators in vivo are goals for future work.<br />
Delorme et al. reported the His-Terminator to be a remarkable effective Terminator with more than 99% termination efficiency.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup> The exemplary measurement below on the right confirms the high terminator efficiency. In fact, we could not detect any mCherry fluorescence in any cells containing the [[Team:TU_Munich/Modeling#Switch|HisTerm]]. Even induction of the corresponding signal transmitter RNA via IPTG did not alter the Terminator efficiency. Again time was the limiting factor and prevented us from testing more than one corresponding transmitter, although the [[Team:TU_Munich/Modeling| Modeling]] highly suggested the necessarily of finding an optimized transmitter length. Thus, the results are insufficient either to prove or to disprove the functionality of the [[Team:TU_Munich/Modeling#Switch|HisTerm]] or our concept in general.<br />
<br><br />
[[Image:TUM2010_HisSwitchklein.JPG|200px|thumb|left|Bacteria containing HisTerm]][[Image:TUM2010_HisSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing HisTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
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Attaining only 90% terminator efficiency, the natural Trp [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|Attenuator]] is known be less effective than the [[Team:TU_Munich/Modeling#Switch|HisTerm]].<sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup> The graph on the right depicts our designed [[Team:TU_Munich/Modeling#Switch|TrpTerm]] characteristic efficiency of about 40 %, notably below the natural standard. Allowing 60% transcription in the “off” state excludes the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] from possible candidates for a scalable network of logic gates, due to the mentioned required "yes or no" function (see [[Team:TU_Munich/Project#Implementation| Implementation and how to connect Biobricks]]). Thus the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] is inoperative as intended, but may still be useful in other contexts. Similar to the [[Team:TU_Munich/Modeling#Switch|HisTerm]], the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] also did not react to the induction of the corresponding signal. Under circumstances, termination efficiencies altered by the transmitter are on a low range and not resolvable within observed 40% basal transcription. <br />
<br><br />
[[Image:TUM2010_TrpSwitchklein.JPG|200px|thumb|left|Bacteria containing TrpTerm]][[Image:TUM2010_TrpSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing TrpTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
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<br />
Making use of our improved screening system we also carried out some ''in vivo'' kinetic measurements in addition to the end-point measurements above. In contrast to the ''in vitro'' experiments we did not obtain significant results for the characterization of our switches. As the switching process is many times faster than protein synthesis our ''in vivo'' kinetics include the synthesis of mCherry as well as its maturation. Therefore we centered our attention on end-point experiments. For more information browse the [[Team:TU_Munich/Lab#Lab_Book|lab book]]. <br><br />
Considering our ''in vivo'' measurements, neither of the tested switches showed any effect regarding the signal induction. But due to the small number of tested switches and signals this can hardly be regarded as disprove of concept. In particular in light of the recent findings by Sooncheol proving antitermination in principle using a T7 system.<sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup><br />
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==''in vitro'' Screening==<br />
To minimize the amount of disturbing factors we decided to countercheck our ''in vivo'' results with a set of ''in vitro'' measurements. While the ''in vitro'' systems are no doubt much less complex than living cells, the work with these set-ups proved to be quite as difficult.<br />
Just as with the ''in vivo'' measurements we could prove our switching system neither right nor wrong, leaving enough work for future iGEM teams.<br />
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===''in vitro translation''===<br />
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Beside optimization of the reporter proteins in use, the major problem occuring in the experiments was the low capacity of the kit. The signal intensity was very low, which made it difficult to observe any signal intensity alterations, so no conclusion could be drawn from these measurements.<br />
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===''in vitro'' transcription===<br />
We used two completely independent ''in vitro'' systems: Using ''E.coli'' RNA Polymerase we analyzed the His and Trp switches that had already been tested ''in vivo''. In a second set-up, we used the well-established T7 RNA Polymerase and switch based on the T7 terminator as well as several signal sequences.<br />
<br />
====T7 System====<br />
In contradiction to the results of Kang and coworkers and other groups, in our ''in vitro'' set-up the T7 terminator did not seem to terminate at all. The negative control (Promoter_Terminator_malachite binding aptamer) showed a similar increase in fluorescence as the positive control (Promoter_random sequence_malachite binding aptamer). <br />
[[Image:TUM2010_T7Result1.png|350px||thumb|left|''in vitro'' transcription measurement of T7 terminator with no signal(upper left), nonsense signal (upper right) and two different designed signals (below)]]<br />
[[Image:TUM2010_T7Result3.png|350px||thumb|right|''in vitro'' transcription measurement of positive control(upper left and T7 terminator with three different designed signals (remaining traces)]]<br><br />
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Furthermore denaturing Polyacrylamide Gel Electrophoresis (PAGE) confirmed that there was no observeable termination of transcription. The addition of a signaling sequence led to a significantly lower increase in fluorescence, which can be attributed to the fact that both DNA sequences, switch and signal, compete for RNA Polymerases.<br />
However, there is almost no difference between the designed signals and random sequences, which is not a big surprise since there can be no antitermination if the terminator itself does not work.<br><br />
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Possible explanations for the contradiction between our results and those of Kang and coworkers might be the experimental set-up and the RNA Polymerases we used. Different variants of T7 RNA Polymerase might respond in different ways to terminator structures, and the termination might be influenced by the presence or absence of cofactors, depending on the purification methods used in producing the Polymerase.<br><br><br />
<br />
This set-up offers a lot of possible experiments for the future, which we would have loved to conduct with a just a bit more time...<br />
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====''E.coli'' System====<br />
<br />
Compared to the T7 System, the ''E. coli'' RPO system produced poor increases in fluorescence, indicating little RNA synthesis. It was shown that the presence of a terminator decreases, as expected, the production of downstream RNA.<br><br />
<br><br />
[[Image:TUM2010_101023kinetik.PNG|350px||thumb|left|''in vitro'' transcription measurement of Switch TrpTerm (upper traces) and positive control (lower traces). Left side: with Trp-signal, right side: no signal]]<br />
[[Image:TUM2010_101022_Kinetik.png|350px||thumb|right|''in vitro'' transcription measurement of positive control (left), Switch TrpTerm (center) and switch HisTerm(right)<br />
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]]<br><br />
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This result was also confirmed by denaturing PAGE. However, due to the poor changes in fluorescence we were not able to actually characterize the behaviour of our switches ''in vitro'', and the small RNA concentrations did not allow a quantitative interpretation of our gels.<br />
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[[Image:TUM2010_coliGel.png|500px||thumb|center|denaturing polyacrylamide gel electrophoresis of DNaseI digested samples from ''in vitro'' transcription of positive control (16z), Switch TrpTerm (W) and switch HisTerm(H). c marks the lanes in which the DNA was injected, the last three lanes show the undigested samples]]<br />
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A major problem with this method was the low concentration of the ordered Polymerase resulting in a much weaker overall signal as comparable measurements using the T7 Polymerase. <br><br><br />
In future experiments we might try to work with smaller volumes in order to reach higher concentration of RPO and of the synthesized RNA molecules, so measuring in 96 well plate readers might be a good choice. <br />
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==Software==<br />
Although we could not show the full functionality of bioLOGICS in the lab we still want to demonstrate the potential of our approach. Hence we implemented the idea behind our logic gates in a program which illustrates how bioLOGCIS theoretically would allow the construction of complex information processing networks interconnecting BioBricks. For further details take a look at our [[Team:TU Munich/Software|Software page]].<br />
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=References=<br />
<html><a name="ref1"></a></html>[1] http://partsregistry.org/cgi/partsdb/Statistics.cgi<br />
<html><a name="ref2"></a></html>[2] https://2009.igem.org/Team:Imperial_College_London/M1 encapsulation<br />
<html><a name="ref3"></a></html>[3] https://2009.igem.org/Team:TUDelft<br />
<html><a name="ref4"></a></html>[4] https://2008.igem.org/Team:Heidelberg<br />
<html><a name="ref5"></a></html>[5] Maung Nyan Win and Christina D. Smolke, Science Oct. 2008 Vol. 322. no. 5900, pp. 456 - 460<br />
<html><a name="ref6"></a></html>[6] Lu, T.K., A.S. Khalil, and J.J. Collins, Next-generation synthetic gene networks. Nature biotechnology, 2009. 27(12): p. 1139-1150. <br />
<html><a name="ref7"></a></html>[7] Schaller, R.R., Moore's law: past, present and future. Spectrum, IEEE, 2002. 34(6): p. 52-59.<br />
<html><a name="ref8"></a></html>[8] von Mering, C., et al., Comparative assessment of large-scale data sets of protein–protein interactions. Nature, 2002. 417(6887): p. 399-403.<br />
<html><a name="ref9"></a></html>[9] Mandal, M. and R.R. Breaker, Gene regulation by riboswitches. Nature Reviews Molecular Cell Biology, 2004. 5(6): p. 451-463. <br />
<html><a name="ref10"></a></html>[10] Benner, S.A. and A.M. Sismour, Synthetic biology. Nature Reviews Genetics, 2005. 6(7): p. 533-543.<br />
<html><a name="ref11"></a></html>[11] Beaudry, A. and G. Joyce, Directed evolution of an RNA enzyme. Science, 1992. 257(5070): p. 635-641.<br />
<html><a name="ref12"></a></html>[12] Delorme, Ehrlich and Renault, Regulation of Expression of the Lactococcus lactis Histidine Operon. Journal of Bacteriology, Apr. 1999, p. 2026–2037<br />
<html><a name="ref13"></a></html>[13] Trun and Trempy(2003): Fundamental Bacterial Genetics, Wiley-Blackwell, Chapter 12 <br />
<html><a name="ref14"></a></html>[14]Sooncheol Lee, Huong Minh Nguyen and Changwon Kang, Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator. Nucleic Acids Research, 2010, 1–9<br />
<br />
<!-- The idea behind our project is to change the way BioBricks have been used up to now. Over the years, many receptors and signals have been constructed as BioBricks during the annual iGEM competition, but still it is not possible to interconnect these Bricks in a complex biological network resuting in a cell, that is able to respond to its environment giving differenciated responses depending on the input signals. (Beispiel: cambridge hat das gemacht, xx dies, aber eine zelle kann nicht beides...<br><br />
We plan to create biological switches, that can function as locial gates inside a cell. Our switches rely on RNA/RNA-interactions, regulating transcriptional termination. This is a major advance of our concept, as regular switches rely on complex regulation including proteins and/or metabolites. Thus, our switches shall offer a greater robustness and their behaviour should be easier to predict. [[switch|Read more]] (hier sollte noch das hochskalieren erwähnt werden...<br><br />
These switches can further be used to build up a logical network inside a bacterial cell, enabling every scientist to connect as many functionalities (in form of BioBricks) as designated. We plan to offer simulation on each specifically designed network.<br />
<br />
<br><br>Over the years, many teams participating in the iGEM competition spent their time on constructing receptors and systems to detect a certain input that a variety of gorgeous oppurtunities is available so far.[[Image:TUM2010 network.png|thumb|300 px|right|Our visioon: A logic network inside the cell]] Nevertheless, until now it is not possible to link all those functionalities and build up a network giving differenciated responses to several of those input signals, where the molecular response depends on the complex composition of the environment a cell faces. We would like to offer this possibility to everyone.<br />
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The logic network we want to apply will be based on devices, that can be easily upscaled and therefor offer the chance to build networks of any wanted complexicity. Our devices rely on pure RNA/RNA interactions and thus their behaviour is well predictable.<br />
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The concept we rely on for our design of RNA-switches is based on the principle of [https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation/ '''attenuation'''].<br />
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= Experiments =<br />
We designed several experiments to test our switches, all of them based on fluorescence measurements. We designed experiment setting for measurements ''in vivo'' as well as ''in vitro''. Our ''in vitro'' measurements relied on two different experiment set-ups. While the first was based on a commercial ''E. coli''-lysate, the latter was reporting on a transcriptional level only, eliminating most of the possible side-effects one could expect in the complex behaviour of a living cell or cell-lysate. [[Experiments_main|Read more]]<br />
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= Results =<br />
We ...blablabla<br />
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Text that will present our results...<br />
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= thing to move =<br />
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'''bioLOGICS: Logical RNA-Devices Enabling BioBrick-Network Formation'''<br />
'''Abstract'''<br />
Among the goals of iGEM is the creation of synthetic biological parts and their utilization to achieve novel features and behavior in biological systems. The emphasis of our project is put on this latter, "systems" aspect of iGEM. More precisely, we aim at the development and experimental demonstration of a scalable approach for the realization of logical functions in vivo.<br />
<br />
By developing a computational biological network based on RNA logical devices we will offer everyone the opportunity to 'program' their own cells with individual AND/OR/NOT connections between BioBricks of their choice. Thereby, BioBricks can finally fulfill their original assignment as biological parts that can be connected in many different ways. We will achieve this by engineering simple and easy-to-handle switches based on predictable RNA/RNA-interactions regulating transcriptional termination. These switches represent a complete set of logical functions and are capable of forming arbitrarily complex networks.<br />
<br />
== The Experiments ==<br />
===Fluorescent proteins as reporter===<br />
<div align="justify"><br />
Our initial idea to prove our concept of antitermination was to use flourescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks ''in vivo'' as well as ''in vitro'', the latter using a translation kit based on e.coli lysate. <br>We decided to use the flourescent proteins GFP and RFP, as their spectra do not overlap and we would not measure any FRET. We would use GFP fluorescence as internal control and RFP fluorescence as signal to detect termination/antitermination by our switch we cloned in between the coding sequences of the proteins. Both protein sequences are under the control of one (L-arabinose induced) promoter.<br />
[[Image:TUM2010_gfprfp_schalter_klein.gif|center|our idea]]<br />
<br><br />
When measuring the termination of our BioBricks and the antitermination by their corresponding signal-RNA, we should be able to observe an increasing RFP emission compared to the GFP emission upon induced signal-RNA production in the cells/in the kit:<br><br />
[[Image:TUM2010_Expected_emission_spextra.png|center|our idea]]<br />
Wiith these measurements, it should also be possible to observe differences in efficiency of termination as well as antitermination between our designed switches. <br><br><br />
<br />
===Measurements with the malachite green aptamer as reporter===<br />
A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramaticly (about 3000 times) upon binding of a specific RNA-aptamer.<br><br>#<br />
[[Image:TUM2010_Malachitgruen-2.png|500px|center|our idea]]<br />
---concept to be desribed, as well as literature---<br />
<ref>refs</ref><br />
<br><br><br />
To study the switches on the transcriptional level gives the advantage, that we would have less interferences and possible artefacts. Also, we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems.<br><br />
<br />
[[Image:TUM2010_Malachit_emission.png|200px|thumb|left|Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA]]We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.<br>Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level.<br />
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<br />
Results <br />
==Flourescent proteins==<br />
Unfortunatly, we had to change the reporter construct two times during our experiments as several problems occured in our measurements:<br><br><br />
<br />
===First Try: based on the measurement plasmid pSB1A10===<br />
At the beginning, we decided to use the reporter plasmid [http://partsregistry.org/Part:pSB1A10 pSB1A10] from the registry. It consists of the fluorescent proteins eGFP and mRFP1. Each sequence includes a ribosome binding site and a stop-codon; the two genes are divided by a cloning side including the BioBrick cleavage sites.[[Image:ScreeningPlasmid1.0.PNG|300px|thumb|right|pSB1A10]]<br> In front of the eGFP sequence, the plasmid includes an arabinose-inducable promoter. The plasmid also contains an ampicilline resistence.<br><br />
We cloned our switches into the cloning site of the measurement plasmid and used an empty cloning site as control; our signal-RNAs we cloned into the [http://partsregistry.org/Part:pSB1K3 pSB1K3] vector, together with the BioBricks R0011 (Lac promoter) and B0014 (double terminator of transcription). Afterwards, we cut pSB1K3 with Aat2 and Pst1 and pSB1A10 with Nsi1 and Aat2 and ligated those fragments of each plasmid that contained our Bricks to get a Monsterplasmid.<br>We had to do so, as both plasmids contain the same ori mechanism. In addition, having both the switch and the signal RNA transcribed from the same plasmid gives us a high local concentration of the signal, once its transcription is induced.<br><br />
[[Image:TUM2010_Messplasmid_nr1.png|left|the measurement plasmid]]<br />
<br />
We transformed BL21(DE3) cells with the plasmid. We set up cultures, induced the arabinose promoter and measured the GFP and mRFP1 excitation/emission spectra within time.<br><br><br />
Unfortunatly, we were ot able to detect any RFP signal, not even in the positive control with no switch in between the GFP/RFP sequences.<br><br><br />
From these experiments, we concluded, that the mRNA of the RFP variant used was instable and rapidly degraded by RNases, so the RFP was not synthesized in the cells. This was also the conclusion from XXX...<br />
<br><br><br />
As solution to this, we decided to design a measurement plasmid ourselves:<br><br />
<hr width="300"><br />
<br><br />
<br />
===Second Try: A measurement plasmid of our own design===<br />
To design our own plasmid to overcome the problems that occurred in our first try gave us tghe possibility to overcome several other problems:<br><br />
#<br />
#<br />
#<br />
[[Image:TUM2010_Construct_no2.png|400px|our construct|center]]<br />
<br />
===Third Try: One promoter for each protein===<br />
We decided to use the measuremnt plasmid we developed in our second try but to clone another L-arabinose induced promoter into the plasmid, in front of our switch followed by the mCherry sequence.<br><br />
<br>[[Image:TUM2010_Construct_no3.png|left|400px|Construct #3]]In this way, we still can use GFP fluorescence as internal control, because both protein transcription is under the control of a promoter of identical design.<br />
Though we are still not able to tell exactly why our previous measurements did not work, but with this construct we measured the first time fluorescence of the mCherry protein in our positive control.<br />
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{{:Team:TU_Munich/Templates/End}}</div>Hartlmuellerhttp://2010.igem.org/Team:TU_Munich/ProjectTeam:TU Munich/Project2010-10-28T03:28:13Z<p>Hartlmueller: /* From switches towards bioLOGICS logic gates */</p>
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<center><font size="5pt" color="#000000">'''bioLOGICS'''</font><font size="4pt" color="#000000">: Logical RNA-Devices Enabling BioBrick-Network Formation</font></center><hr color="black"><br><br />
= Vision=<br />
<br />
Until today, 13.628 biobrick sequences<sup>[[Team:TU_Munich/Project#ref1|&#91;1&#93;]]</sup> have been submitted to partsregistry, thereof 102 reporter units and 12 signaling bricks.<br />
Since then, people are trying to arrange these single biological building blocks in such a manner that allows producing special biotechnological products (metabolic engineering), developing biological sensory circuits (biosensors) and even giving microorganisms the ability to react on multiple environmental factors and serve both as disease indicator and drug. These examples and further promising ideas were implemented on previous iGEM-competitions.<sup>[[Team:TU_Munich/Project#ref2|&#91;2&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref3|&#91;3&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref4|&#91;4&#93;]]</sup> <br><br><br />
The idea of combining the outcome of several iGEM competitions to construct complex synthetic biological systems falls at the last hurdle - the fact, that each team uses a different principle how to access and functionally connect the respectively used biobricks. For example, it is a major challenge to create a system that uses several sensoring BioBricks from different iGEM-teams which in turn regulates reportering BioBricks from various teams. In order to combine and fully take advantage of these promising projects, our vision is to develop an adapter that allows interconnecting arbitrary biobricks on a functional level. Such a system easily allows to setup sensor-reporter circuits and interconnect them to complete biological chips... A further step towards artificial cells.<br><br><br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, the above adapter has to meet the following requirements:<br />
*'''Universality'''<br />
:The adapter has to be compatible to as many BioBricks as possible. This objective will guarantee that a large number of BioBricks can be connected.<br />
*'''Scalability'''<br />
:Once the basic design of the system is established, the construction of the system is supposed to be automated in silico. This way it will be possible to create an adapter connecting a large amount of BioBricks.<br />
*'''Biological orthogonality'''<br />
:Interference with cellular components has to be as low as possible in order to avoid unwanted and perturbing side effects.<br />
*'''Logic'''<br />
:The adapter is supposed to not only associate different BioBricks, but to functionally connect BioBricks in a precisely determined manner (including operations such as AND/OR/NOT).<br />
<br><br />
Several biological logic units, devices and circuits have been developed so far<sup>[[Team:TU_Munich/Project#ref5|&#91;5&#93;]]</sup>, but to our knowledge, none was shown to meet all requirements listed above.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
=Implementation=<br />
To functionally connect BioBricks, there are several possibilities including genetic switches, riboswitches and direct protein-protein interactions. We investigated several hypothetically principles, and decided to focus our practical work on the development of a RNA-RNA interaction-based switch. These switches are capable of changing between two states, a state of antitermination and termination, and make use of highly-specific RNA-RNA interaction. In principle such a switch can fulfill all requirements mentioned previously. The following text clarifies how these switches work in detail.<br />
==How to connect BioBricks==<br />
Our adapter is a system, that activates or disables BioBricks (output BioBricks) in response to the presence of other Biobricks (input Biobricks). Our approach uses a molecular network to put this into practice and consists of four major elements:<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
<br><br />
{|<br />
|-<br />
|[[Image:Networks.png|center|thumb|730px|The general principle how different inputs can be connect to various outputs. For details see text.<br>Inputs (such as proteins or small molecules) are indicated on the left side. blue lines represent transmitter molecules whereas organe lines present logic gates. The type of logic gate is indicated. Green lines indicate transmitter RNA that can function as mRNA and consequently generate any output gene (indicated on the very right).]]<br />
|}<br />
In order to connect different BioBricks, our network requires four major types of components:<br />
*Input elements<br />
*Transmitter molecules<br />
*Logic gates<br />
*Output elements<br />
<br />
{{:Team:TU_Munich/Templates/InfoBoxStart}}'''Computer vs. molecular network - and our approach'''<br><br />
Logic gates in a molecular network are often compared to transistors used in a computer, where billions of transistors are incorporated<sup>[[Team:TU_Munich/Project#ref7|&#91;7&#93;]]</sup>. The main advantage on a computer chip is, all transistors share the same functional principle, and only the way connecting them in a special sequence allows specific addressing of only a subset of other transistors by an input. However, spatially fixed connections of molecular logic gates are not possible in a living cell. The "wiring" within a cell relies on the specific interaction between transmitter molecule and their corresponding logic gates, for example implemented by protein-protein/ligand-protein interactions or specific ligand-riboswitch interactions.<sup>[[Team:TU_Munich/Project#ref8|&#91;8&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref9|&#91;9&#93;]]</sup> As a result, in a cell, each occurring logic gate ("transistor") has to be different, at least in a special recognition site<sup>[[Team:TU_Munich/Project#ref10|&#91;10&#93;]]</sup> - for example like different transcription factors, recognizing different DNA-sites. Thanks to evolution, nature easily can invent a new transistor for each task - science achieves this only on a limited scale, and producing synthetic molecular logic gates artificially by either rational or evolutionary protein or riboswitch engineering, is limited to small circuits so far<sup>[[Team:TU_Munich/Project#ref11|&#91;11&#93;]]</sup>. Our project aims to establish a molecular switch as close as possible to a electronic transistor, thus sharing the same functional principle for all logic gates. At the same time, we want to design a easily exchangeable recognition site, which can individually be designed by everyone! {{:Team:TU_Munich/Templates/InfoBoxEnd}}<br />
<br />
These elements can be combined to build up a molecular network (see illustration). Each input molecule (such as a BioBrick) produces a unique transmitter molecule. All transmitters belong to the same type of molecule and share a common design. However, each transmitter molecule can only interact and activate a certain subset of logic gates. In other words, logic gates have to recognize as well as bind the corresponding transmitter molecules and are capable of producing a new output transmitter molecule. Depending on the type of the logic gate (AND, OR or NOT<sup>[[Team:TU_Munich/Project#ref6|&#91;6&#93;]]</sup>), an output transmitter is only created if both input transmitter molecules are present (AND), at least one of two input transmitters is present (OR) or if no input transmitter is present at all (NOT). Once a logic gate has produced a new output transmitter, these transmitters can in turn address another subset ("layer") of logic gates. In theory many layers of logic gates can be connected this way allowing the creation of large networks. Until this step, various transmitter molecules might have been produced. But in order to create a Biobrick output, the last layer of logic gates finally generates transmitter molecules that will not active logic gates, but will rather interact with the cell metabolism to produce a BioBrick response. In other words, the last layer of transmitter molecules is capable of regulating BioBrick formation.<br />
<br />
<br />
Summarizing, the network establishes a connection between input BioBricks and output BioBricks in a functional manner.<br />
Having addressed the basic layout of the molecular network, the next step is to determine what type of molecules can perform the required functions. We decided to use RNA, both as transmitter molecules and for constructing logic gates. Several advantages result from the utilization of RNA as the central element:<br />
*During the last years, many Biobricks were designed that are sensitive to various chemicals and substances. These BioBricks often function as a transcription factor that binds to a specific DNA sequence and consequently would be capable to produce a specific transmitter RNA molecule. Thus, in principle each BioBrick which involves transcription can be integrated in our network.<br />
*Since all logic gates are capable of producing transmitter RNA, they can also produce functional mRNA encoding any protein. This means, each BioBrick consisting of protein or RNA can be produced as an output of our network.<br />
*If RNA forms both, the transmitter molecule and the logic gates, they can specifically interact by RNA-RNA interaction, which is highly predictable compared to protein interactions. This allows to generate a library of transmitters and gates ''in silico''. Such a library is essential for the creation of large networks.<br />
*RNA production is fast and energy saving for a cell. Consequently, operating a network that only produces RNA rather than proteins will also be faster and more efficient for the host cell. Since our logic gates are based on transcription, translation and resource consuming protein production will only be required at the very last step. <br />
*As the half-time of RNA can be rather short, transmitter RNA will not accumulate within the cell and it is therefore less likely for the system to become saturated.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Design and functional principle of logic gates==<br />
The concept introduced above provides a framework that can potentially serve as an universal adapter between different BioBricks. However, the [[Team:TU_Munich/Glossary#logic gate | logic gates]] have not been specified more precisely so far. This will be done in the following section.<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, our logic gates are to possess the following characteristics:<br />
*Logic gates, such as AND, OR and NOT, have to be implemented by RNA-interaction based principles (see [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]).<br />
*All logic gates have to recognize their corresponding [[Team:TU_Munich/Glossary#Transmitter (bioLOGICS)| transmitter RNAs]] and, in response, produce an output transmitter molecule.<br />
*Logic gates should follow a basic design rule, in such a way, that their creation can be automated ''in silico''.<br />
*The response efficiency of a logic gate toward a transmitter molecule should be comparable for all logic gates to provide calculable robustness and sensitivity. This will ensure comparable molecular concentrations and functionality of large networks.<br />
*The system has to be designed for ''in vivo'' utilization at the first place. As a reference we always assumed a temperature of 37 °C and an ''E. coli'' environment.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}} <br />
In order to build logic gates for our bioLOGICS system we will first create a simple switch. A switch can be activated by one transmitter RNA and produce an output transmitter RNA. In contrast to a logic gate, a switch does not perform logic operations. However by combining switches, logic gates can be created. The following text will first describe how the developed switch works and secondly, how logic gates such as AND/OR/NOT can be created using these switches.<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Read more{{:Team:TU Munich/Templates/ToggleBoxStart2}}<br />
[[Image:toggle_switch.png|500px|thumb|center|id="hideOnReadMore"|'''A''' The basic structure of a bioLOGICS switch (left) and a transmitter molecule (right).<br>'''B'''The process of switching. See the text in the close-by "Read more" section for details.<br>Rectangles present the composition of our functional units on the level of DNA. Fringed lines represent RNA produced by RNA polymerase. The stem loop structure depicts the switchable terminator. Terminator and target site are illustrated in blue and turquoise, respectively. Recognition sites are indicated in different colors, in this case red for the input transmitter and green for the output transmitter.Each switch and or later logical unit has to be flanked by a promotor and another constitutive terminator, to allow RNA-production by RNA-polymerase in a proper way. ]]<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===Switch===<br />
[[Image:TUM2010_switch-and-transmitter.jpg|550px|right|thumb|The basic strcutrue of a switch (left) and a transmitter RNA (right). See text for details.]]<br />
Roughly speaking, a switch can be regarded as an enhanced switchable transcriptional terminator. The enhancement can be described easier by dividing a switch into its functional components: <br />
*'''Target site'''<br><br />
:The target site is the functional core element of our switches, allowing a shift between an "on" and "off" state. Since we work on the level of RNA-production (transcription), a "switchable" transcriptional terminator is suitable for this purpose. By allowing or preventing formation of a transcriptional terminator, that is by switching between termination and antitermination it is possible to represent an "off" and an "on" state, respectively. Therefore, the target site is the 5' ending of the terminator and is required for a stable terminator formation. It should be noted that this principle was also observed in nature.<br />
:To highlight and illustrate the functional principle of our switches, only the part of the terminator which is involved in interacting with a transmitter molecule and which is responsible for shifting between "on" and "off" state is called target site. The remaining terminator sequence is called terminator in the following, even if both, target site and terminator build up the terminator structure occurring in nature. <br />
:The important aspect of our switches is the fact that all switches will hold the same identical target site. Therefore having found one functional "switchable" terminator, will allow almost unlimited upscaling since this terminator can be used for a large library of switches. This is the main difference to previous works done on this field, which always required developing a new shifting principle for each switch.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup> Beside this scalability, this principle provides a comparable on/off shifting rate (responds function) for all switches, avoiding complex fine tuning of molecular networks.<br />
:To sum it up, the target site, allows to switch between an "on" and "off" state. But so far, the switch is not capable of performing specific interaction with transmitter molecules. This is where the recognition site comes into play.<br />
*'''Recognition site'''<br />
:The recognition site defines which transmitter molecule can actually interact with the switch. Therefore, a unique recognition site is generated for each switch and is positioned right upstream of the target site. In principle the recognition can be any random sequence as long as it remains unique within the molecular network.<br />
Summing up, the recognition site allows a specific interaction between switches and transmitter molecules. Once this interaction is formed, an interaction between the transmitter and the target will actually switch the state of the terminator. This allows the specific arrangement and interconnection of numerous of these switches by transmitter molecules, without changing the target site. Comparable to wires connecting many identical transistors, our target site remains the same.<br />
<br><br />
<br />
===Transmitter RNA´s===<br />
As desccribed above, transmitter RNAs are the input and output of bioLOGICS switches (compare [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]). These transmitters are short ssRNA molecules representing the "trigger" to shift switches between the "on" and "off" state. To fulfill this role, they need to posses the following properties:<br />
*A transmitter may only interact with certain switches. That is, a transmitter has to find the corresponding recognition site of a switch.<br />
*Once an interaction is established between a transmitter and a switch, a transmitter has to be capable of changing the secondary structure of a terminator and thus cause antitermination.<br />
Again, these two properties are fulfilled by two components of the transmitter:<br />
*'''Identity site'''<br />
:This site is capable of forcing an interaction between the transmitter and the switch. Therefore it is complementary to the recognition site of this switch. As the recognition site is unique within a network, so is the identity site. However, the single identity site is not capable of changing the state of the switch. That is were the trigger site comes into play.<br />
*'''Trigger site'''<br />
:Once an interaction is created by the identity site, the trigger site is capable of actually shifting the switch since it is complementary to the target site of the switch. To fulfill this role, it is placed upstream at the 5' end of the identity site. As the target site is the same for all switches, the trigger site is the same for all signals. Therefore it is important, that similar to the identity site, a trigger site cannot function on its own. That is, a single trigger site cannot shift the state of a switch without the help of an identity site.<br />
<br />
Summing up, we applied the principle introduced for the switches to the transmitter molecules. In contrast to previous approaches on this field <sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup>, we introduced the described synthetic trigger site in such a manner that it is not able to change the state of the terminator on its own, but only in combination with the identity site. So the challenge is to arrange and optimize these elementary building blocks thermodynamically, that a trigger site is only able to switch in combination with its respective identity site. This was done by ''in silico'' design using [[TU Munich/Glossary#NUPACK| NUPACK]], presented in section [[TU Munich/Modeling#in silico design based on thermodynamic calculations| in silico design]].<br />
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===Putting it all together: the switching process===<br />
[[Image:TUM2010_switching-process.jpg|550px|right|thumb|The basic structure of a switch (left) and a transmitter RNA (right). See text for details.]]The functional principle of the designed switches is illustrated in the figure. The switch is positioned on DNA upstream of a desired output transmitter. So in the absence of a triggering transmitter molecule, transcription will be canceled by the formation of a RNA stem loop in the nascent RNA-chain. This will cause the RNA polymerase to stop transcription and fall off the DNA and consequently no output RNA will be produced. This process only relies on [[Team:TU_Munich/Glossary#Termination| rho-independent termination]].<br />
On the other hand, in the presence of a [[Team:TU_Munich/Project#RNA_transmitters | input transmitter]], this small functional RNA inhibits the stem loop formation by complementary base-pairing and hence avoids termination of transcription. In detail, the identity site (red part on transmitter) binds the recognition site (red part on switch) and serves as [[Team:TU_Munich/Glossary#Toehold|toehold]], which will thermodynamically allow the trigger site (turquoise part on transmitter) to perform a [[Team:TU_Munich/Glossary#Strand Displacement| strand displacement]] and open up the stem loop structure. Consequently the polymerase can read all the way through and form the output RNA.<br>Summing up, we use this concept to create a switch that can be toggled by a transmitter RNA molecule and in response, is able to produce another transmitter RNA.<br />
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===From switches towards bioLOGICS logic gates===<br />
As described, each switch can be accessed by a specific RNA-transmitter molecule, representing the input. In turn, another RNA-transmitter molecule will be produced if the switch shifts its state. This output transmitter of one switch can serve as input transmitter for the next switch by meaningful selection and design of the respective recognition sites. This easily allows arranging several switches in specific sequences and faulty wiring - the corner stone of a logical network.<br><br />
To ease the building of logical networks we want to create a switch capable of Boolean logics, a common mathematical principle fundamental for computational science. Since AND/OR/NOT are basic logic operations which can be implemented with the presented switches, all remaining operations (such as XOR, NAND, ...) can be expressed by these three operators according to laws of boolean logics.<br />
Creating logic gates is achieved by combining two switches in two different ways, as illustrated below.<br />
*AND gate<br />
:An AND gate can be constrcuted by positioning two switches right next to each other. For the output transmitter to be created, both input transmitter have to be present.[[Image:AND2.png|500px|thumb|center|Combining two switches in series creates a logic AND gate.]]<br />
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*OR gate<br />
:An OR gate is created by utilizing two independent switches sharing the same output transmitter. If each one of both switches is activated, an output transmitter is generated. Therefore, one input transmitter is enough to produce an output transmitter.[[Image:OR2.png|500px|thumb|center|Combining two switches in parallel creates a logic OR gate.]]<br />
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*NOT gate<br />
:A NOT gate is supposed to function as an inverter. In contrast to the gates described above, a not gate requires only one sitch. However, to meet the design rule for transmitter molecules, this switch shows some differences compared to the switches used for AND and OR gates.<br><br />
In principle, it consists only of one switch which contains its respective signal molecule intrinsic, so via intramolecular interaction, antitermination is the initial state. The signal is intrinsically of the same components as usual to allow interconnection with other logic gates.<br />
[[Image:NOT2.png|500px|thumb|center]]<br />
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{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
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==Network construction==<br />
Designing complex biological networks based on either traditional protein engineering or our new bioLOGICS is still a complex task. We developed a software which allows the fast construction of a bioLOGICS based networks. <br><br />
To read more about this, look at our [https://2010.igem.org/Team:TU_Munich/Software Software page]<br />
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=Our Objective=<br />
Putting the implementation described above into practice, will be a major challenge. For this year's iGEM competition our goal is to do the first step: design and build a switch that can be toggled by a RNA molecule. To be precise, we want to apply the design rules of our switch to modify a transcription terminator in such a way that it interacts with a second RNA molecule and, as a result, is no longer capable of forming a stem loop. This objective will require intensive ''in silico'' designing and modeling of switches based on different terminators and their corresponding transmitters. In connection to this theoretical part, we also have to test and verify the switches. For this step, we establish custom-made assays, ''in vitro'' and ''in vivo''.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Once the objective mentioned above is accomplished, these basic RNA/RNA-interactions have to be modified in such a manner that the described identity/trigger site pattern for the transmitter and the complementary recognition/target site switch composition has to be established. The most important requirement is to is to optimize these modules that the transmitter is only able to switches specifically, meaning only in the presence of both, identity AND trigger site. <br />
<br><br />
Once the objective mentioned above is accomplished, the creation of an OR gate will be rather simple since it only requires two switches. However the creation of an AND or NOT gate and optimizing the logic gates to improve their responds function will remain the goal of future work. Also the creation of small networks and the correct integration of BioBricks as input and output molecules will be future challenges. Furthermore, we wanted to rather focus on the development and the testing of our structural design of the switches, rather than developing a variety of new BioBricks.<br />
<br />
==''In silico'' design==<br />
As described above, our switches are based on certain design rules. However, there still are different structural parameters that need to be tested and optimized (length of recognition site and target site, choice of terminator, etc.).<br />
We used [[Team:TU_Munich/Project#in silico design |''in silico'' design]] and [[Team:TU_Munich/Modeling| modeling]]) to test different parameters. Furthermore we tried to use the [[Team:TU_Munich/Glossary#Antitermination|antitermination principle]] observed in nature, such as [[Team:TU_Munich/Glossary#Attenuation| attenuation]] in ''E. coli'' or [[Team:TU_Munich/Glossary#Tiny Abortive RNA´s| tiny abortive RNA´s]] of T7-phage.<br />
==Evaluation and Measurements==<br />
To evaluate the functionality of our molecular switches, we first had to establish several assays. Therefore, we improved an existing [[Team:TU_Munich/Lab#In vivo Measurements |''in vivo'' assay]] and developed an [[Team:TU_Munich/Lab#In vitro Transcription | ''in vitro'' assay]] for this purpose. For more information please refer to the [[Team:TU_Munich/Lab | lab]] section.<br />
<br><br />
<br><br />
Summarizing, the main challenges are <br />
* to find a suitable terminator construct and design a complementary trigger unit, which is only functional in combination with a specificity site - meaning an optimization of the '''thermodynamically parameters''' (see[[Team:TU_Munich/Project#in silico design| in silico design]])<br />
* to investigate whether the transmitter/switch interaction reaction is on a timescale to be competitive to terminator formation - meaning an comparison of '''kinetic parameters''' (see [[Team:TU_Munich/Modeling|Modeling page]])<br />
* to proof antitermination can be also be caused by synthetically RNA-interaction (see [[Team:TU_Munich/Glossary#Antitermination| Antitermination in nature]] and [[Team:TU_Munich/Project#Results| ''in vivo'' and ''in vitro'' measurements]] )<br />
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=Results=<br />
Every network starts with a basic unit. While our declared aim is to enable networks allowing fine-tuning of gene expression beyond the regular on/off, exploring such an on/off switch/signal pair is the first step towards a functional network. We constructed several units and tested their efficiency, robustness and reproducibility ''in vivo'', ''in vitro'' and ''in silico''. Furthermore we developed a software which allows easy constructions of networks based on our designed logic gates. Conclusive elaboration of a few first RNA-based logic units is the major contribution of our iGEM team.<br />
<br />
==in silico Design of Switching and Trigger Unit==<br />
As described on the [[Team:TU_Munich/Project | project]] page, one key aspect of our switches is the idea, that a [[Team:TU_Munich/Glossary#Transmitter_(bioLOGICS) | RNA transmitter molecule]] is capable to shift the state of a switch only if its [[Team:TU_Munich/Glossary#Trigger_Site_(bioLOGICS) | trigger site]] is present and its [[Team:TU_Munich/Glossary#Identity_Site_(bioLOGICS) | identity site]] corresponds to the [[Team:TU_Munich/Glossary#Recognition_Site_(bioLOGICS) | recognition site]] of the [[Team:TU_Munich/Glossary#Switch_(bioLOGICS) | switch]]. We successfully constructed several switches and their corresponding transmitter RNA ''in silico'' on a thermodynamical basis. We modified different transcriptional terminators in such a way, that the formation of the terminator was prevented by a transmitter molecule. As desired, this only occured if the transmitter molecule contained both, a trigger and an identity site. Analogously, we were able to design and verify a NOT gate using the same thermodynical approach.<br />
<br />
==Diffusion and RNA Folding Dynamics==<br />
We estimated the diffusion time for our constructs and modeled the folding dynamics of our bioLOGICS switches including the switching process with a stochastic RNA folding program. We were able to provide better insight in their folding dynamics and proved that they are able to interrupt termination. We also optimized the switches and the corresponding signals. Furthermore, we combined the switches what resulted in a logic gate. See our [[Team:TU Munich/Modeling|Modeling page]] for further details.<br />
<br />
==''in vivo'' Functionality Screening==<br />
Since our logic gates are intended to function in living cells, ''in vivo'' measurements were essential. In a set of experiments we concentrated on two different switches based on known [[Team:TU_Munich/Glossary#Attenuation|attenuators]] from nature: the [[Team:TU_Munich/Modeling#Switch|HisTerm]] and [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Focusing on fluorescent proteins for quantifiable input and output we designed a functional and robust screening system. For greater detail see [[Team:TU_Munich/Lab#Experiment_Design|Experimental Design]]. Unfortunately, setting up a working screening system failed twice. Only in redesigning and improving the screening plasmid pSB1A10 we succeeded, but lost precious time.<br />
<br />
Ultimately, the two switches displayed remarkable differences in their terminator efficiency, but neither of them responded to their corresponding signal. However, screening one transmitter signal does not disprove the basic working principle of our system. Limited by time, we hope for future teams to take up our work and to use our improved test system that we submitted to the parts registry, for performing successful in vivo measurement.<br />
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Considering the high complexity of ''in vivo'' measurements compared to other experimental challenges, a robust and easy to handle test system for [[Team:TU_Munich/Glossary#PoPS-based devices| PoPS-based devices]] is desirable. As described in [[Team:TU_Munich/Lab#Experiment_Design|Experimental design]], we used fluorescent proteins: RFP or mCherry to measure the amount of produced output and eGFP for normalization. Our first attempt, using the screening plasmid pSB1A10, yielded no interpretable results. Switching the fluorescent protein to mCherry did not work either, but after several experimental setups we determined a transcriptional problem causing no reporter protein expression regardless of the inserted part. Thereby we demonstrated the screening plasmid pSB1A10 to be [[Team:TU_Munich/Biobricks#Falsification| malfunctioning]]. <br />
Finally a new design based on pSB1A10 lead to a functional and robust screening system (compare [[Team:TU_Munich/Parts#Screening system: Backbone BBa_K494001| Screening system: Backbone BBa_K494001]]). A second promoter with identical induction properties inside the BioBrick cloning site enforces transcription of the PoPS-based device and the mCherry output.<br />
<br />
Exemplary, the graph below on the right shows the positive control, induced and uninduced at OD<sub>600</sub>=0.7 followed by 16 h incubation at 25 °C. Clearly visible are eGFP and mCherry fluorescence in the induced samples. The uninduced control showed no fluorescence at all, demonstrating the PBad promoter to be tight and providing very low basal transcription, what is a major advantage for the screening system. This newly designed screening approach renders the characterization of PoPS-based devices in general and switches in particular easy and robust. The low basal transcription furthermore fulfills one of the most important requirements for the designed switches, since output transmitters may only be produced in presence of an input transmitter. This helps to avoid strong "background" noise, which would extremely harden the successful interconnection of several switches. <br />
<br><br />
[[Image:TUM2010_PosControlklein.JPG|200px||thumb|left|Bacteria containing positive control]]<br />
[[Image:TUM2010_graphPosControl1.png|355px|thumb|center|Emission spectra of induced (green/red) and uninduced(black) positive control BBa_K494002 ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
Due to the time limitations of the iGEM completion we had to focus our efforts on few switches after designing the screening system. Relying on the functionality of systems occurring in nature, we choose the [[Team:TU_Munich/Modeling#Switch|HisTerm]] as well as the [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Both switches are based on known natural [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|attenuators]]. Testing synthetic and none-naturally switchable terminators in vivo are goals for future work.<br />
Delorme et al. reported the His-Terminator to be a remarkable effective Terminator with more than 99% termination efficiency.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup> The exemplary measurement below on the right confirms the high terminator efficiency. In fact, we could not detect any mCherry fluorescence in any cells containing the [[Team:TU_Munich/Modeling#Switch|HisTerm]]. Even induction of the corresponding signal transmitter RNA via IPTG did not alter the Terminator efficiency. Again time was the limiting factor and prevented us from testing more than one corresponding transmitter, although the [[Team:TU_Munich/Modeling| Modeling]] highly suggested the necessarily of finding an optimized transmitter length. Thus, the results are insufficient either to prove or to disprove the functionality of the [[Team:TU_Munich/Modeling#Switch|HisTerm]] or our concept in general.<br />
<br><br />
[[Image:TUM2010_HisSwitchklein.JPG|200px|thumb|left|Bacteria containing HisTerm]][[Image:TUM2010_HisSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing HisTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
<br />
Attaining only 90% terminator efficiency, the natural Trp [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|Attenuator]] is known be less effective than the [[Team:TU_Munich/Modeling#Switch|HisTerm]].<sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup> The graph on the right depicts our designed [[Team:TU_Munich/Modeling#Switch|TrpTerm]] characteristic efficiency of about 40 %, notably below the natural standard. Allowing 60% transcription in the “off” state excludes the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] from possible candidates for a scalable network of logic gates, due to the mentioned required "yes or no" function (see [[Team:TU_Munich/Project#Implementation| Implementation and how to connect Biobricks]]). Thus the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] is inoperative as intended, but may still be useful in other contexts. Similar to the [[Team:TU_Munich/Modeling#Switch|HisTerm]], the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] also did not react to the induction of the corresponding signal. Under circumstances, termination efficiencies altered by the transmitter are on a low range and not resolvable within observed 40% basal transcription. <br />
<br><br />
[[Image:TUM2010_TrpSwitchklein.JPG|200px|thumb|left|Bacteria containing TrpTerm]][[Image:TUM2010_TrpSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing TrpTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
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<br />
Making use of our improved screening system we also carried out some ''in vivo'' kinetic measurements in addition to the end-point measurements above. In contrast to the ''in vitro'' experiments we did not obtain significant results for the characterization of our switches. As the switching process is many times faster than protein synthesis our ''in vivo'' kinetics include the synthesis of mCherry as well as its maturation. Therefore we centered our attention on end-point experiments. For more information browse the [[Team:TU_Munich/Lab#Lab_Book|lab book]]. <br><br />
Considering our ''in vivo'' measurements, neither of the tested switches showed any effect regarding the signal induction. But due to the small number of tested switches and signals this can hardly be regarded as disprove of concept. In particular in light of the recent findings by Sooncheol proving antitermination in principle using a T7 system.<sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup><br />
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<br />
==''in vitro'' Screening==<br />
To minimize the amount of disturbing factors we decided to countercheck our ''in vivo'' results with a set of ''in vitro'' measurements. While the ''in vitro'' systems are no doubt much less complex than living cells, the work with these set-ups proved to be quite as difficult.<br />
Just as with the ''in vivo'' measurements we could prove our switching system neither right nor wrong, leaving enough work for future iGEM teams.<br />
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===''in vitro translation''===<br />
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Beside optimization of the reporter proteins in use, the major problem occuring in the experiments was the low capacity of the kit. The signal intensity was very low, which made it difficult to observe any signal intensity alterations, so no conclusion could be drawn from these measurements.<br />
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===''in vitro'' transcription===<br />
We used two completely independent ''in vitro'' systems: Using ''E.coli'' RNA Polymerase we analyzed the His and Trp switches that had already been tested ''in vivo''. In a second set-up, we used the well-established T7 RNA Polymerase and switch based on the T7 terminator as well as several signal sequences.<br />
<br />
====T7 System====<br />
In contradiction to the results of Kang and coworkers and other groups, in our ''in vitro'' set-up the T7 terminator did not seem to terminate at all. The negative control (Promoter_Terminator_malachite binding aptamer) showed a similar increase in fluorescence as the positive control (Promoter_random sequence_malachite binding aptamer). <br />
[[Image:TUM2010_T7Result1.png|360px||thumb|left|''in vitro'' transcription measurement of T7 terminator with no signal(upper left), nonsense signal (upper right) and two different designed signals (below)]]<br />
[[Image:TUM2010_T7Result3.png|360px||thumb|right|''in vitro'' transcription measurement of positive control(upper left and T7 terminator with three different designed signals (remaining traces)]]<br />
Furthermore denaturing Polyacrylamide Gel Electrophoresis (PAGE) confirmed that there was no observeable termination of transcription. The addition of a signaling sequence led to a significantly lower increase in fluorescence, which can be attributed to the fact that both DNA sequences, switch and signal, compete for RNA Polymerases.<br />
However, there is almost no difference between the designed signals and random sequences, which is not a big surprise since there can be no antitermination if the terminator itself does not work.<br><br />
<br />
Possible explanations for the contradiction between our results and those of Kang and coworkers might be the experimental set-up and the RNA Polymerases we used. Different variants of T7 RNA Polymerase might respond in different ways to terminator structures, and the termination might be influenced by the presence or absence of cofactors, depending on the purification methods used in producing the Polymerase.<br><br><br />
<br />
This set-up offers a lot of possible experiments for the future, which we would have loved to conduct with a just a bit more time...<br />
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====''E.coli'' System====<br />
<br />
Compared to the T7 System, the ''E. coli'' RPO system produced poor increases in fluorescence, indicating little RNA synthesis. It was shown that the presence of a terminator decreases, as expected, the production of downstream RNA.<br />
[Image:TUM2010_101023kinetik.PNG|360px||thumb|left|''in vitro'' transcription measurement of Switch TrpTerm (upper traces) and positive control (lower traces). Left side: with Trp-signal, right side: no signal]]<br />
[Image:TUM2010_101022_kinetik.PNG|360px||thumb|right|''in vitro'' transcription measurement of positive control (left), Switch TrpTerm (center) and switch HisTerm(right)]]<br />
<br />
This result was also confirmed by denaturing PAGE. However, due to the poor changes in fluorescence we were not able to actually characterize the behaviour of our switches ''in vitro'', and the small RNA concentrations did not allow a quantitative interpretation of our gels.<br />
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[Image:TUM2010_coliGel.PNG|500px||thumb|center|denaturing polyacrylamide gel electrophoresis of DNaseI digested samples from ''in vitro'' transcription of positive control (16z), Switch TrpTerm (W) and switch HisTerm(H). c marks the lanes in which the DNA was injected, the last three lanes show the undigested samples]]<br />
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A major problem with this method was the low concentration of the ordered Polymerase resulting in a much weaker overall signal as comparable measurements using the T7 Polymerase. <br><br><br />
In future experiments we might try to work with smaller volumes in order to reach higher concentration of RPO and of the synthesized RNA molecules, so measuring in 96 well plate readers might be a good choice. <br />
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{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
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==Software==<br />
Although we could not show the full functionality of bioLOGICS in the lab we still want to demonstrate the potential of our approach. Hence we implemented the idea behind our logic gates in a program which illustrates how bioLOGCIS theoretically would allow the construction of complex information processing networks interconnecting BioBricks. For further details take a look at our [[Team:TU Munich/Software|Software page]].<br />
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<br />
=References=<br />
<html><a name="ref1"></a></html>[1] http://partsregistry.org/cgi/partsdb/Statistics.cgi<br />
<html><a name="ref2"></a></html>[2] https://2009.igem.org/Team:Imperial_College_London/M1 encapsulation<br />
<html><a name="ref3"></a></html>[3] https://2009.igem.org/Team:TUDelft<br />
<html><a name="ref4"></a></html>[4] https://2008.igem.org/Team:Heidelberg<br />
<html><a name="ref5"></a></html>[5] Maung Nyan Win and Christina D. Smolke, Science Oct. 2008 Vol. 322. no. 5900, pp. 456 - 460<br />
<html><a name="ref6"></a></html>[6] Lu, T.K., A.S. Khalil, and J.J. Collins, Next-generation synthetic gene networks. Nature biotechnology, 2009. 27(12): p. 1139-1150. <br />
<html><a name="ref7"></a></html>[7] Schaller, R.R., Moore's law: past, present and future. Spectrum, IEEE, 2002. 34(6): p. 52-59.<br />
<html><a name="ref8"></a></html>[8] von Mering, C., et al., Comparative assessment of large-scale data sets of protein–protein interactions. Nature, 2002. 417(6887): p. 399-403.<br />
<html><a name="ref9"></a></html>[9] Mandal, M. and R.R. Breaker, Gene regulation by riboswitches. Nature Reviews Molecular Cell Biology, 2004. 5(6): p. 451-463. <br />
<html><a name="ref10"></a></html>[10] Benner, S.A. and A.M. Sismour, Synthetic biology. Nature Reviews Genetics, 2005. 6(7): p. 533-543.<br />
<html><a name="ref11"></a></html>[11] Beaudry, A. and G. Joyce, Directed evolution of an RNA enzyme. Science, 1992. 257(5070): p. 635-641.<br />
<html><a name="ref12"></a></html>[12] Delorme, Ehrlich and Renault, Regulation of Expression of the Lactococcus lactis Histidine Operon. Journal of Bacteriology, Apr. 1999, p. 2026–2037<br />
<html><a name="ref13"></a></html>[13] Trun and Trempy(2003): Fundamental Bacterial Genetics, Wiley-Blackwell, Chapter 12 <br />
<html><a name="ref14"></a></html>[14]Sooncheol Lee, Huong Minh Nguyen and Changwon Kang, Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator. Nucleic Acids Research, 2010, 1–9<br />
<br />
<!-- The idea behind our project is to change the way BioBricks have been used up to now. Over the years, many receptors and signals have been constructed as BioBricks during the annual iGEM competition, but still it is not possible to interconnect these Bricks in a complex biological network resuting in a cell, that is able to respond to its environment giving differenciated responses depending on the input signals. (Beispiel: cambridge hat das gemacht, xx dies, aber eine zelle kann nicht beides...<br><br />
We plan to create biological switches, that can function as locial gates inside a cell. Our switches rely on RNA/RNA-interactions, regulating transcriptional termination. This is a major advance of our concept, as regular switches rely on complex regulation including proteins and/or metabolites. Thus, our switches shall offer a greater robustness and their behaviour should be easier to predict. [[switch|Read more]] (hier sollte noch das hochskalieren erwähnt werden...<br><br />
These switches can further be used to build up a logical network inside a bacterial cell, enabling every scientist to connect as many functionalities (in form of BioBricks) as designated. We plan to offer simulation on each specifically designed network.<br />
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<br><br>Over the years, many teams participating in the iGEM competition spent their time on constructing receptors and systems to detect a certain input that a variety of gorgeous oppurtunities is available so far.[[Image:TUM2010 network.png|thumb|300 px|right|Our visioon: A logic network inside the cell]] Nevertheless, until now it is not possible to link all those functionalities and build up a network giving differenciated responses to several of those input signals, where the molecular response depends on the complex composition of the environment a cell faces. We would like to offer this possibility to everyone.<br />
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The logic network we want to apply will be based on devices, that can be easily upscaled and therefor offer the chance to build networks of any wanted complexicity. Our devices rely on pure RNA/RNA interactions and thus their behaviour is well predictable.<br />
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The concept we rely on for our design of RNA-switches is based on the principle of [https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation/ '''attenuation'''].<br />
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= Experiments =<br />
We designed several experiments to test our switches, all of them based on fluorescence measurements. We designed experiment setting for measurements ''in vivo'' as well as ''in vitro''. Our ''in vitro'' measurements relied on two different experiment set-ups. While the first was based on a commercial ''E. coli''-lysate, the latter was reporting on a transcriptional level only, eliminating most of the possible side-effects one could expect in the complex behaviour of a living cell or cell-lysate. [[Experiments_main|Read more]]<br />
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= Results =<br />
We ...blablabla<br />
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Text that will present our results...<br />
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= thing to move =<br />
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'''bioLOGICS: Logical RNA-Devices Enabling BioBrick-Network Formation'''<br />
'''Abstract'''<br />
Among the goals of iGEM is the creation of synthetic biological parts and their utilization to achieve novel features and behavior in biological systems. The emphasis of our project is put on this latter, "systems" aspect of iGEM. More precisely, we aim at the development and experimental demonstration of a scalable approach for the realization of logical functions in vivo.<br />
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By developing a computational biological network based on RNA logical devices we will offer everyone the opportunity to 'program' their own cells with individual AND/OR/NOT connections between BioBricks of their choice. Thereby, BioBricks can finally fulfill their original assignment as biological parts that can be connected in many different ways. We will achieve this by engineering simple and easy-to-handle switches based on predictable RNA/RNA-interactions regulating transcriptional termination. These switches represent a complete set of logical functions and are capable of forming arbitrarily complex networks.<br />
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== The Experiments ==<br />
===Fluorescent proteins as reporter===<br />
<div align="justify"><br />
Our initial idea to prove our concept of antitermination was to use flourescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks ''in vivo'' as well as ''in vitro'', the latter using a translation kit based on e.coli lysate. <br>We decided to use the flourescent proteins GFP and RFP, as their spectra do not overlap and we would not measure any FRET. We would use GFP fluorescence as internal control and RFP fluorescence as signal to detect termination/antitermination by our switch we cloned in between the coding sequences of the proteins. Both protein sequences are under the control of one (L-arabinose induced) promoter.<br />
[[Image:TUM2010_gfprfp_schalter_klein.gif|center|our idea]]<br />
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When measuring the termination of our BioBricks and the antitermination by their corresponding signal-RNA, we should be able to observe an increasing RFP emission compared to the GFP emission upon induced signal-RNA production in the cells/in the kit:<br><br />
[[Image:TUM2010_Expected_emission_spextra.png|center|our idea]]<br />
Wiith these measurements, it should also be possible to observe differences in efficiency of termination as well as antitermination between our designed switches. <br><br><br />
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===Measurements with the malachite green aptamer as reporter===<br />
A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramaticly (about 3000 times) upon binding of a specific RNA-aptamer.<br><br>#<br />
[[Image:TUM2010_Malachitgruen-2.png|500px|center|our idea]]<br />
---concept to be desribed, as well as literature---<br />
<ref>refs</ref><br />
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To study the switches on the transcriptional level gives the advantage, that we would have less interferences and possible artefacts. Also, we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems.<br><br />
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[[Image:TUM2010_Malachit_emission.png|200px|thumb|left|Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA]]We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.<br>Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level.<br />
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Results <br />
==Flourescent proteins==<br />
Unfortunatly, we had to change the reporter construct two times during our experiments as several problems occured in our measurements:<br><br><br />
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===First Try: based on the measurement plasmid pSB1A10===<br />
At the beginning, we decided to use the reporter plasmid [http://partsregistry.org/Part:pSB1A10 pSB1A10] from the registry. It consists of the fluorescent proteins eGFP and mRFP1. Each sequence includes a ribosome binding site and a stop-codon; the two genes are divided by a cloning side including the BioBrick cleavage sites.[[Image:ScreeningPlasmid1.0.PNG|300px|thumb|right|pSB1A10]]<br> In front of the eGFP sequence, the plasmid includes an arabinose-inducable promoter. The plasmid also contains an ampicilline resistence.<br><br />
We cloned our switches into the cloning site of the measurement plasmid and used an empty cloning site as control; our signal-RNAs we cloned into the [http://partsregistry.org/Part:pSB1K3 pSB1K3] vector, together with the BioBricks R0011 (Lac promoter) and B0014 (double terminator of transcription). Afterwards, we cut pSB1K3 with Aat2 and Pst1 and pSB1A10 with Nsi1 and Aat2 and ligated those fragments of each plasmid that contained our Bricks to get a Monsterplasmid.<br>We had to do so, as both plasmids contain the same ori mechanism. In addition, having both the switch and the signal RNA transcribed from the same plasmid gives us a high local concentration of the signal, once its transcription is induced.<br><br />
[[Image:TUM2010_Messplasmid_nr1.png|left|the measurement plasmid]]<br />
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We transformed BL21(DE3) cells with the plasmid. We set up cultures, induced the arabinose promoter and measured the GFP and mRFP1 excitation/emission spectra within time.<br><br><br />
Unfortunatly, we were ot able to detect any RFP signal, not even in the positive control with no switch in between the GFP/RFP sequences.<br><br><br />
From these experiments, we concluded, that the mRNA of the RFP variant used was instable and rapidly degraded by RNases, so the RFP was not synthesized in the cells. This was also the conclusion from XXX...<br />
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As solution to this, we decided to design a measurement plasmid ourselves:<br><br />
<hr width="300"><br />
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===Second Try: A measurement plasmid of our own design===<br />
To design our own plasmid to overcome the problems that occurred in our first try gave us tghe possibility to overcome several other problems:<br><br />
#<br />
#<br />
#<br />
[[Image:TUM2010_Construct_no2.png|400px|our construct|center]]<br />
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===Third Try: One promoter for each protein===<br />
We decided to use the measuremnt plasmid we developed in our second try but to clone another L-arabinose induced promoter into the plasmid, in front of our switch followed by the mCherry sequence.<br><br />
<br>[[Image:TUM2010_Construct_no3.png|left|400px|Construct #3]]In this way, we still can use GFP fluorescence as internal control, because both protein transcription is under the control of a promoter of identical design.<br />
Though we are still not able to tell exactly why our previous measurements did not work, but with this construct we measured the first time fluorescence of the mCherry protein in our positive control.<br />
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{{:Team:TU_Munich/Templates/End}}</div>Hartlmuellerhttp://2010.igem.org/Team:TU_Munich/ProjectTeam:TU Munich/Project2010-10-28T03:23:18Z<p>Hartlmueller: /* From switches towards bioLOGICS logic gates */</p>
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<center><font size="5pt" color="#000000">'''bioLOGICS'''</font><font size="4pt" color="#000000">: Logical RNA-Devices Enabling BioBrick-Network Formation</font></center><hr color="black"><br><br />
= Vision=<br />
<br />
Until today, 13.628 biobrick sequences<sup>[[Team:TU_Munich/Project#ref1|&#91;1&#93;]]</sup> have been submitted to partsregistry, thereof 102 reporter units and 12 signaling bricks.<br />
Since then, people are trying to arrange these single biological building blocks in such a manner that allows producing special biotechnological products (metabolic engineering), developing biological sensory circuits (biosensors) and even giving microorganisms the ability to react on multiple environmental factors and serve both as disease indicator and drug. These examples and further promising ideas were implemented on previous iGEM-competitions.<sup>[[Team:TU_Munich/Project#ref2|&#91;2&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref3|&#91;3&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref4|&#91;4&#93;]]</sup> <br><br><br />
The idea of combining the outcome of several iGEM competitions to construct complex synthetic biological systems falls at the last hurdle - the fact, that each team uses a different principle how to access and functionally connect the respectively used biobricks. For example, it is a major challenge to create a system that uses several sensoring BioBricks from different iGEM-teams which in turn regulates reportering BioBricks from various teams. In order to combine and fully take advantage of these promising projects, our vision is to develop an adapter that allows interconnecting arbitrary biobricks on a functional level. Such a system easily allows to setup sensor-reporter circuits and interconnect them to complete biological chips... A further step towards artificial cells.<br><br><br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, the above adapter has to meet the following requirements:<br />
*'''Universality'''<br />
:The adapter has to be compatible to as many BioBricks as possible. This objective will guarantee that a large number of BioBricks can be connected.<br />
*'''Scalability'''<br />
:Once the basic design of the system is established, the construction of the system is supposed to be automated in silico. This way it will be possible to create an adapter connecting a large amount of BioBricks.<br />
*'''Biological orthogonality'''<br />
:Interference with cellular components has to be as low as possible in order to avoid unwanted and perturbing side effects.<br />
*'''Logic'''<br />
:The adapter is supposed to not only associate different BioBricks, but to functionally connect BioBricks in a precisely determined manner (including operations such as AND/OR/NOT).<br />
<br><br />
Several biological logic units, devices and circuits have been developed so far<sup>[[Team:TU_Munich/Project#ref5|&#91;5&#93;]]</sup>, but to our knowledge, none was shown to meet all requirements listed above.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
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=Implementation=<br />
To functionally connect BioBricks, there are several possibilities including genetic switches, riboswitches and direct protein-protein interactions. We investigated several hypothetically principles, and decided to focus our practical work on the development of a RNA-RNA interaction-based switch. These switches are capable of changing between two states, a state of antitermination and termination, and make use of highly-specific RNA-RNA interaction. In principle such a switch can fulfill all requirements mentioned previously. The following text clarifies how these switches work in detail.<br />
==How to connect BioBricks==<br />
Our adapter is a system, that activates or disables BioBricks (output BioBricks) in response to the presence of other Biobricks (input Biobricks). Our approach uses a molecular network to put this into practice and consists of four major elements:<br />
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<br><br />
{|<br />
|-<br />
|[[Image:Networks.png|center|thumb|730px|The general principle how different inputs can be connect to various outputs. For details see text.<br>Inputs (such as proteins or small molecules) are indicated on the left side. blue lines represent transmitter molecules whereas organe lines present logic gates. The type of logic gate is indicated. Green lines indicate transmitter RNA that can function as mRNA and consequently generate any output gene (indicated on the very right).]]<br />
|}<br />
In order to connect different BioBricks, our network requires four major types of components:<br />
*Input elements<br />
*Transmitter molecules<br />
*Logic gates<br />
*Output elements<br />
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{{:Team:TU_Munich/Templates/InfoBoxStart}}'''Computer vs. molecular network - and our approach'''<br><br />
Logic gates in a molecular network are often compared to transistors used in a computer, where billions of transistors are incorporated<sup>[[Team:TU_Munich/Project#ref7|&#91;7&#93;]]</sup>. The main advantage on a computer chip is, all transistors share the same functional principle, and only the way connecting them in a special sequence allows specific addressing of only a subset of other transistors by an input. However, spatially fixed connections of molecular logic gates are not possible in a living cell. The "wiring" within a cell relies on the specific interaction between transmitter molecule and their corresponding logic gates, for example implemented by protein-protein/ligand-protein interactions or specific ligand-riboswitch interactions.<sup>[[Team:TU_Munich/Project#ref8|&#91;8&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref9|&#91;9&#93;]]</sup> As a result, in a cell, each occurring logic gate ("transistor") has to be different, at least in a special recognition site<sup>[[Team:TU_Munich/Project#ref10|&#91;10&#93;]]</sup> - for example like different transcription factors, recognizing different DNA-sites. Thanks to evolution, nature easily can invent a new transistor for each task - science achieves this only on a limited scale, and producing synthetic molecular logic gates artificially by either rational or evolutionary protein or riboswitch engineering, is limited to small circuits so far<sup>[[Team:TU_Munich/Project#ref11|&#91;11&#93;]]</sup>. Our project aims to establish a molecular switch as close as possible to a electronic transistor, thus sharing the same functional principle for all logic gates. At the same time, we want to design a easily exchangeable recognition site, which can individually be designed by everyone! {{:Team:TU_Munich/Templates/InfoBoxEnd}}<br />
<br />
These elements can be combined to build up a molecular network (see illustration). Each input molecule (such as a BioBrick) produces a unique transmitter molecule. All transmitters belong to the same type of molecule and share a common design. However, each transmitter molecule can only interact and activate a certain subset of logic gates. In other words, logic gates have to recognize as well as bind the corresponding transmitter molecules and are capable of producing a new output transmitter molecule. Depending on the type of the logic gate (AND, OR or NOT<sup>[[Team:TU_Munich/Project#ref6|&#91;6&#93;]]</sup>), an output transmitter is only created if both input transmitter molecules are present (AND), at least one of two input transmitters is present (OR) or if no input transmitter is present at all (NOT). Once a logic gate has produced a new output transmitter, these transmitters can in turn address another subset ("layer") of logic gates. In theory many layers of logic gates can be connected this way allowing the creation of large networks. Until this step, various transmitter molecules might have been produced. But in order to create a Biobrick output, the last layer of logic gates finally generates transmitter molecules that will not active logic gates, but will rather interact with the cell metabolism to produce a BioBrick response. In other words, the last layer of transmitter molecules is capable of regulating BioBrick formation.<br />
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<br />
Summarizing, the network establishes a connection between input BioBricks and output BioBricks in a functional manner.<br />
Having addressed the basic layout of the molecular network, the next step is to determine what type of molecules can perform the required functions. We decided to use RNA, both as transmitter molecules and for constructing logic gates. Several advantages result from the utilization of RNA as the central element:<br />
*During the last years, many Biobricks were designed that are sensitive to various chemicals and substances. These BioBricks often function as a transcription factor that binds to a specific DNA sequence and consequently would be capable to produce a specific transmitter RNA molecule. Thus, in principle each BioBrick which involves transcription can be integrated in our network.<br />
*Since all logic gates are capable of producing transmitter RNA, they can also produce functional mRNA encoding any protein. This means, each BioBrick consisting of protein or RNA can be produced as an output of our network.<br />
*If RNA forms both, the transmitter molecule and the logic gates, they can specifically interact by RNA-RNA interaction, which is highly predictable compared to protein interactions. This allows to generate a library of transmitters and gates ''in silico''. Such a library is essential for the creation of large networks.<br />
*RNA production is fast and energy saving for a cell. Consequently, operating a network that only produces RNA rather than proteins will also be faster and more efficient for the host cell. Since our logic gates are based on transcription, translation and resource consuming protein production will only be required at the very last step. <br />
*As the half-time of RNA can be rather short, transmitter RNA will not accumulate within the cell and it is therefore less likely for the system to become saturated.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Design and functional principle of logic gates==<br />
The concept introduced above provides a framework that can potentially serve as an universal adapter between different BioBricks. However, the [[Team:TU_Munich/Glossary#logic gate | logic gates]] have not been specified more precisely so far. This will be done in the following section.<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, our logic gates are to possess the following characteristics:<br />
*Logic gates, such as AND, OR and NOT, have to be implemented by RNA-interaction based principles (see [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]).<br />
*All logic gates have to recognize their corresponding [[Team:TU_Munich/Glossary#Transmitter (bioLOGICS)| transmitter RNAs]] and, in response, produce an output transmitter molecule.<br />
*Logic gates should follow a basic design rule, in such a way, that their creation can be automated ''in silico''.<br />
*The response efficiency of a logic gate toward a transmitter molecule should be comparable for all logic gates to provide calculable robustness and sensitivity. This will ensure comparable molecular concentrations and functionality of large networks.<br />
*The system has to be designed for ''in vivo'' utilization at the first place. As a reference we always assumed a temperature of 37 °C and an ''E. coli'' environment.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}} <br />
In order to build logic gates for our bioLOGICS system we will first create a simple switch. A switch can be activated by one transmitter RNA and produce an output transmitter RNA. In contrast to a logic gate, a switch does not perform logic operations. However by combining switches, logic gates can be created. The following text will first describe how the developed switch works and secondly, how logic gates such as AND/OR/NOT can be created using these switches.<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Read more{{:Team:TU Munich/Templates/ToggleBoxStart2}}<br />
[[Image:toggle_switch.png|500px|thumb|center|id="hideOnReadMore"|'''A''' The basic structure of a bioLOGICS switch (left) and a transmitter molecule (right).<br>'''B'''The process of switching. See the text in the close-by "Read more" section for details.<br>Rectangles present the composition of our functional units on the level of DNA. Fringed lines represent RNA produced by RNA polymerase. The stem loop structure depicts the switchable terminator. Terminator and target site are illustrated in blue and turquoise, respectively. Recognition sites are indicated in different colors, in this case red for the input transmitter and green for the output transmitter.Each switch and or later logical unit has to be flanked by a promotor and another constitutive terminator, to allow RNA-production by RNA-polymerase in a proper way. ]]<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===Switch===<br />
[[Image:TUM2010_switch-and-transmitter.jpg|550px|right|thumb|The basic strcutrue of a switch (left) and a transmitter RNA (right). See text for details.]]<br />
Roughly speaking, a switch can be regarded as an enhanced switchable transcriptional terminator. The enhancement can be described easier by dividing a switch into its functional components: <br />
*'''Target site'''<br><br />
:The target site is the functional core element of our switches, allowing a shift between an "on" and "off" state. Since we work on the level of RNA-production (transcription), a "switchable" transcriptional terminator is suitable for this purpose. By allowing or preventing formation of a transcriptional terminator, that is by switching between termination and antitermination it is possible to represent an "off" and an "on" state, respectively. Therefore, the target site is the 5' ending of the terminator and is required for a stable terminator formation. It should be noted that this principle was also observed in nature.<br />
:To highlight and illustrate the functional principle of our switches, only the part of the terminator which is involved in interacting with a transmitter molecule and which is responsible for shifting between "on" and "off" state is called target site. The remaining terminator sequence is called terminator in the following, even if both, target site and terminator build up the terminator structure occurring in nature. <br />
:The important aspect of our switches is the fact that all switches will hold the same identical target site. Therefore having found one functional "switchable" terminator, will allow almost unlimited upscaling since this terminator can be used for a large library of switches. This is the main difference to previous works done on this field, which always required developing a new shifting principle for each switch.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup> Beside this scalability, this principle provides a comparable on/off shifting rate (responds function) for all switches, avoiding complex fine tuning of molecular networks.<br />
:To sum it up, the target site, allows to switch between an "on" and "off" state. But so far, the switch is not capable of performing specific interaction with transmitter molecules. This is where the recognition site comes into play.<br />
*'''Recognition site'''<br />
:The recognition site defines which transmitter molecule can actually interact with the switch. Therefore, a unique recognition site is generated for each switch and is positioned right upstream of the target site. In principle the recognition can be any random sequence as long as it remains unique within the molecular network.<br />
Summing up, the recognition site allows a specific interaction between switches and transmitter molecules. Once this interaction is formed, an interaction between the transmitter and the target will actually switch the state of the terminator. This allows the specific arrangement and interconnection of numerous of these switches by transmitter molecules, without changing the target site. Comparable to wires connecting many identical transistors, our target site remains the same.<br />
<br><br />
<br />
===Transmitter RNA´s===<br />
As desccribed above, transmitter RNAs are the input and output of bioLOGICS switches (compare [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]). These transmitters are short ssRNA molecules representing the "trigger" to shift switches between the "on" and "off" state. To fulfill this role, they need to posses the following properties:<br />
*A transmitter may only interact with certain switches. That is, a transmitter has to find the corresponding recognition site of a switch.<br />
*Once an interaction is established between a transmitter and a switch, a transmitter has to be capable of changing the secondary structure of a terminator and thus cause antitermination.<br />
Again, these two properties are fulfilled by two components of the transmitter:<br />
*'''Identity site'''<br />
:This site is capable of forcing an interaction between the transmitter and the switch. Therefore it is complementary to the recognition site of this switch. As the recognition site is unique within a network, so is the identity site. However, the single identity site is not capable of changing the state of the switch. That is were the trigger site comes into play.<br />
*'''Trigger site'''<br />
:Once an interaction is created by the identity site, the trigger site is capable of actually shifting the switch since it is complementary to the target site of the switch. To fulfill this role, it is placed upstream at the 5' end of the identity site. As the target site is the same for all switches, the trigger site is the same for all signals. Therefore it is important, that similar to the identity site, a trigger site cannot function on its own. That is, a single trigger site cannot shift the state of a switch without the help of an identity site.<br />
<br />
Summing up, we applied the principle introduced for the switches to the transmitter molecules. In contrast to previous approaches on this field <sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup>, we introduced the described synthetic trigger site in such a manner that it is not able to change the state of the terminator on its own, but only in combination with the identity site. So the challenge is to arrange and optimize these elementary building blocks thermodynamically, that a trigger site is only able to switch in combination with its respective identity site. This was done by ''in silico'' design using [[TU Munich/Glossary#NUPACK| NUPACK]], presented in section [[TU Munich/Modeling#in silico design based on thermodynamic calculations| in silico design]].<br />
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===Putting it all together: the switching process===<br />
[[Image:TUM2010_switching-process.jpg|550px|right|thumb|The basic structure of a switch (left) and a transmitter RNA (right). See text for details.]]The functional principle of the designed switches is illustrated in the figure. The switch is positioned on DNA upstream of a desired output transmitter. So in the absence of a triggering transmitter molecule, transcription will be canceled by the formation of a RNA stem loop in the nascent RNA-chain. This will cause the RNA polymerase to stop transcription and fall off the DNA and consequently no output RNA will be produced. This process only relies on [[Team:TU_Munich/Glossary#Termination| rho-independent termination]].<br />
On the other hand, in the presence of a [[Team:TU_Munich/Project#RNA_transmitters | input transmitter]], this small functional RNA inhibits the stem loop formation by complementary base-pairing and hence avoids termination of transcription. In detail, the identity site (red part on transmitter) binds the recognition site (red part on switch) and serves as [[Team:TU_Munich/Glossary#Toehold|toehold]], which will thermodynamically allow the trigger site (turquoise part on transmitter) to perform a [[Team:TU_Munich/Glossary#Strand Displacement| strand displacement]] and open up the stem loop structure. Consequently the polymerase can read all the way through and form the output RNA.<br>Summing up, we use this concept to create a switch that can be toggled by a transmitter RNA molecule and in response, is able to produce another transmitter RNA.<br />
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===From switches towards bioLOGICS logic gates===<br />
As described, each switch can be accessed by a specific RNA-transmitter molecule, representing the input. In turn, another RNA-transmitter molecule will be produced if the switch shifts its state. This output transmitter of one switch can serve as input transmitter for the next switch by meaningful selection and design of the respective recognition sites. This easily allows arranging several switches in specific sequences and faulty wiring - the corner stone of a logical network.<br><br />
To ease the building of logical networks we want to create a switch capable of Boolean logics, a common mathematical principle fundamental for computational science. Since AND/OR/NOT are basic logic operations which can be implemented with the presented switches, all remaining operations (such as XOR, NAND, ...) can be expressed by these three operators according to laws of boolean logics.<br />
Creating logic gates is achieved by combining two switches in two different ways, as illustrated below.<br />
*AND gate<br />
:[[Image:AND2.png|500px|thumb|center|Combining two switches in series creates a logic AND gate.]]An AND gate can be constrcuted by positioning two switches right next to each other. For the output transmitter to be created, both input transmitter have to be present.<br />
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*OR gate<br />
:[[Image:OR2.png|500px|thumb|center|Combining two switches in parallel creates a logic OR gate.]]An OR gate is created by utilizing two independent switches sharing the same output transmitter. If each one of both switches is activated, an output transmitter is generated. Therefore, one input transmitter is enough to produce an output transmitter.<br />
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*NOT is more complex to explain. In principle, it consists only of one switch which contains its respective signal molecule intrinsic, so via intramolecular interaction, antitermination is the initial state. The signal is intrinsically of the same components as usual to allow interconnection with other logic gates.<br />
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[[Image:NOT2.png|500px|thumb|center]]<br />
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==Network construction==<br />
Designing complex biological networks based on either traditional protein engineering or our new bioLOGICS is still a complex task. We developed a software which allows the fast construction of a bioLOGICS based networks. <br><br />
To read more about this, look at our [https://2010.igem.org/Team:TU_Munich/Software Software page]<br />
<br />
=Our Objective=<br />
Putting the implementation described above into practice, will be a major challenge. For this year's iGEM competition our goal is to do the first step: design and build a switch that can be toggled by a RNA molecule. To be precise, we want to apply the design rules of our switch to modify a transcription terminator in such a way that it interacts with a second RNA molecule and, as a result, is no longer capable of forming a stem loop. This objective will require intensive ''in silico'' designing and modeling of switches based on different terminators and their corresponding transmitters. In connection to this theoretical part, we also have to test and verify the switches. For this step, we establish custom-made assays, ''in vitro'' and ''in vivo''.<br />
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Once the objective mentioned above is accomplished, these basic RNA/RNA-interactions have to be modified in such a manner that the described identity/trigger site pattern for the transmitter and the complementary recognition/target site switch composition has to be established. The most important requirement is to is to optimize these modules that the transmitter is only able to switches specifically, meaning only in the presence of both, identity AND trigger site. <br />
<br><br />
Once the objective mentioned above is accomplished, the creation of an OR gate will be rather simple since it only requires two switches. However the creation of an AND or NOT gate and optimizing the logic gates to improve their responds function will remain the goal of future work. Also the creation of small networks and the correct integration of BioBricks as input and output molecules will be future challenges. Furthermore, we wanted to rather focus on the development and the testing of our structural design of the switches, rather than developing a variety of new BioBricks.<br />
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==''In silico'' design==<br />
As described above, our switches are based on certain design rules. However, there still are different structural parameters that need to be tested and optimized (length of recognition site and target site, choice of terminator, etc.).<br />
We used [[Team:TU_Munich/Project#in silico design |''in silico'' design]] and [[Team:TU_Munich/Modeling| modeling]]) to test different parameters. Furthermore we tried to use the [[Team:TU_Munich/Glossary#Antitermination|antitermination principle]] observed in nature, such as [[Team:TU_Munich/Glossary#Attenuation| attenuation]] in ''E. coli'' or [[Team:TU_Munich/Glossary#Tiny Abortive RNA´s| tiny abortive RNA´s]] of T7-phage.<br />
==Evaluation and Measurements==<br />
To evaluate the functionality of our molecular switches, we first had to establish several assays. Therefore, we improved an existing [[Team:TU_Munich/Lab#In vivo Measurements |''in vivo'' assay]] and developed an [[Team:TU_Munich/Lab#In vitro Transcription | ''in vitro'' assay]] for this purpose. For more information please refer to the [[Team:TU_Munich/Lab | lab]] section.<br />
<br><br />
<br><br />
Summarizing, the main challenges are <br />
* to find a suitable terminator construct and design a complementary trigger unit, which is only functional in combination with a specificity site - meaning an optimization of the '''thermodynamically parameters''' (see[[Team:TU_Munich/Project#in silico design| in silico design]])<br />
* to investigate whether the transmitter/switch interaction reaction is on a timescale to be competitive to terminator formation - meaning an comparison of '''kinetic parameters''' (see [[Team:TU_Munich/Modeling|Modeling page]])<br />
* to proof antitermination can be also be caused by synthetically RNA-interaction (see [[Team:TU_Munich/Glossary#Antitermination| Antitermination in nature]] and [[Team:TU_Munich/Project#Results| ''in vivo'' and ''in vitro'' measurements]] )<br />
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=Results=<br />
Every network starts with a basic unit. While our declared aim is to enable networks allowing fine-tuning of gene expression beyond the regular on/off, exploring such an on/off switch/signal pair is the first step towards a functional network. We constructed several units and tested their efficiency, robustness and reproducibility ''in vivo'', ''in vitro'' and ''in silico''. Furthermore we developed a software which allows easy constructions of networks based on our designed logic gates. Conclusive elaboration of a few first RNA-based logic units is the major contribution of our iGEM team.<br />
<br />
==in silico Design of Switching and Trigger Unit==<br />
As described on the [[Team:TU_Munich/Project | project]] page, one key aspect of our switches is the idea, that a [[Team:TU_Munich/Glossary#Transmitter_(bioLOGICS) | RNA transmitter molecule]] is capable to shift the state of a switch only if its [[Team:TU_Munich/Glossary#Trigger_Site_(bioLOGICS) | trigger site]] is present and its [[Team:TU_Munich/Glossary#Identity_Site_(bioLOGICS) | identity site]] corresponds to the [[Team:TU_Munich/Glossary#Recognition_Site_(bioLOGICS) | recognition site]] of the [[Team:TU_Munich/Glossary#Switch_(bioLOGICS) | switch]]. We successfully constructed several switches and their corresponding transmitter RNA ''in silico'' on a thermodynamical basis. We modified different transcriptional terminators in such a way, that the formation of the terminator was prevented by a transmitter molecule. As desired, this only occured if the transmitter molecule contained both, a trigger and an identity site. Analogously, we were able to design and verify a NOT gate using the same thermodynical approach.<br />
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==Diffusion and RNA Folding Dynamics==<br />
We estimated the diffusion time for our constructs and modeled the folding dynamics of our bioLOGICS switches including the switching process with a stochastic RNA folding program. We were able to provide better insight in their folding dynamics and proved that they are able to interrupt termination. We also optimized the switches and the corresponding signals. Furthermore, we combined the switches what resulted in a logic gate. See our [[Team:TU Munich/Modeling|Modeling page]] for further details.<br />
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==''in vivo'' Functionality Screening==<br />
Since our logic gates are intended to function in living cells, ''in vivo'' measurements were essential. In a set of experiments we concentrated on two different switches based on known [[Team:TU_Munich/Glossary#Attenuation|attenuators]] from nature: the [[Team:TU_Munich/Modeling#Switch|HisTerm]] and [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Focusing on fluorescent proteins for quantifiable input and output we designed a functional and robust screening system. For greater detail see [[Team:TU_Munich/Lab#Experiment_Design|Experimental Design]]. Unfortunately, setting up a working screening system failed twice. Only in redesigning and improving the screening plasmid pSB1A10 we succeeded, but lost precious time.<br />
<br />
Ultimately, the two switches displayed remarkable differences in their terminator efficiency, but neither of them responded to their corresponding signal. However, screening one transmitter signal does not disprove the basic working principle of our system. Limited by time, we hope for future teams to take up our work and to use our improved test system that we submitted to the parts registry, for performing successful in vivo measurement.<br />
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Considering the high complexity of ''in vivo'' measurements compared to other experimental challenges, a robust and easy to handle test system for [[Team:TU_Munich/Glossary#PoPS-based devices| PoPS-based devices]] is desirable. As described in [[Team:TU_Munich/Lab#Experiment_Design|Experimental design]], we used fluorescent proteins: RFP or mCherry to measure the amount of produced output and eGFP for normalization. Our first attempt, using the screening plasmid pSB1A10, yielded no interpretable results. Switching the fluorescent protein to mCherry did not work either, but after several experimental setups we determined a transcriptional problem causing no reporter protein expression regardless of the inserted part. Thereby we demonstrated the screening plasmid pSB1A10 to be [[Team:TU_Munich/Biobricks#Falsification| malfunctioning]]. <br />
Finally a new design based on pSB1A10 lead to a functional and robust screening system (compare [[Team:TU_Munich/Parts#Screening system: Backbone BBa_K494001| Screening system: Backbone BBa_K494001]]). A second promoter with identical induction properties inside the BioBrick cloning site enforces transcription of the PoPS-based device and the mCherry output.<br />
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Exemplary, the graph below on the right shows the positive control, induced and uninduced at OD<sub>600</sub>=0.7 followed by 16 h incubation at 25 °C. Clearly visible are eGFP and mCherry fluorescence in the induced samples. The uninduced control showed no fluorescence at all, demonstrating the PBad promoter to be tight and providing very low basal transcription, what is a major advantage for the screening system. This newly designed screening approach renders the characterization of PoPS-based devices in general and switches in particular easy and robust. The low basal transcription furthermore fulfills one of the most important requirements for the designed switches, since output transmitters may only be produced in presence of an input transmitter. This helps to avoid strong "background" noise, which would extremely harden the successful interconnection of several switches. <br />
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[[Image:TUM2010_PosControlklein.JPG|200px||thumb|left|Bacteria containing positive control]]<br />
[[Image:TUM2010_graphPosControl1.png|355px|thumb|center|Emission spectra of induced (green/red) and uninduced(black) positive control BBa_K494002 ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
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Due to the time limitations of the iGEM completion we had to focus our efforts on few switches after designing the screening system. Relying on the functionality of systems occurring in nature, we choose the [[Team:TU_Munich/Modeling#Switch|HisTerm]] as well as the [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Both switches are based on known natural [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|attenuators]]. Testing synthetic and none-naturally switchable terminators in vivo are goals for future work.<br />
Delorme et al. reported the His-Terminator to be a remarkable effective Terminator with more than 99% termination efficiency.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup> The exemplary measurement below on the right confirms the high terminator efficiency. In fact, we could not detect any mCherry fluorescence in any cells containing the [[Team:TU_Munich/Modeling#Switch|HisTerm]]. Even induction of the corresponding signal transmitter RNA via IPTG did not alter the Terminator efficiency. Again time was the limiting factor and prevented us from testing more than one corresponding transmitter, although the [[Team:TU_Munich/Modeling| Modeling]] highly suggested the necessarily of finding an optimized transmitter length. Thus, the results are insufficient either to prove or to disprove the functionality of the [[Team:TU_Munich/Modeling#Switch|HisTerm]] or our concept in general.<br />
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[[Image:TUM2010_HisSwitchklein.JPG|200px|thumb|left|Bacteria containing HisTerm]][[Image:TUM2010_HisSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing HisTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
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Attaining only 90% terminator efficiency, the natural Trp [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|Attenuator]] is known be less effective than the [[Team:TU_Munich/Modeling#Switch|HisTerm]].<sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup> The graph on the right depicts our designed [[Team:TU_Munich/Modeling#Switch|TrpTerm]] characteristic efficiency of about 40 %, notably below the natural standard. Allowing 60% transcription in the “off” state excludes the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] from possible candidates for a scalable network of logic gates, due to the mentioned required "yes or no" function (see [[Team:TU_Munich/Project#Implementation| Implementation and how to connect Biobricks]]). Thus the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] is inoperative as intended, but may still be useful in other contexts. Similar to the [[Team:TU_Munich/Modeling#Switch|HisTerm]], the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] also did not react to the induction of the corresponding signal. Under circumstances, termination efficiencies altered by the transmitter are on a low range and not resolvable within observed 40% basal transcription. <br />
<br><br />
[[Image:TUM2010_TrpSwitchklein.JPG|200px|thumb|left|Bacteria containing TrpTerm]][[Image:TUM2010_TrpSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing TrpTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
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Making use of our improved screening system we also carried out some ''in vivo'' kinetic measurements in addition to the end-point measurements above. In contrast to the ''in vitro'' experiments we did not obtain significant results for the characterization of our switches. As the switching process is many times faster than protein synthesis our ''in vivo'' kinetics include the synthesis of mCherry as well as its maturation. Therefore we centered our attention on end-point experiments. For more information browse the [[Team:TU_Munich/Lab#Lab_Book|lab book]]. <br><br />
Considering our ''in vivo'' measurements, neither of the tested switches showed any effect regarding the signal induction. But due to the small number of tested switches and signals this can hardly be regarded as disprove of concept. In particular in light of the recent findings by Sooncheol proving antitermination in principle using a T7 system.<sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup><br />
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==''in vitro'' Screening==<br />
To minimize the amount of disturbing factors we decided to countercheck our ''in vivo'' results with a set of ''in vitro'' measurements. While the ''in vitro'' systems are no doubt much less complex than living cells, the work with these set-ups proved to be quite as difficult.<br />
Just as with the ''in vivo'' measurements we could prove our switching system neither right nor wrong, leaving enough work for future iGEM teams.<br />
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===''in vitro translation''===<br />
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Beside optimization of the reporter proteins in use, the major problem occuring in the experiments was the low capacity of the kit. The signal intensity was very low, which made it difficult to observe any signal intensity alterations, so no conclusion could be drawn from these measurements.<br />
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===''in vitro'' transcription===<br />
We used two completely independent ''in vitro'' systems: Using ''E.coli'' RNA Polymerase we analyzed the His and Trp switches that had already been tested ''in vivo''. In a second set-up, we used the well-established T7 RNA Polymerase and switch based on the T7 terminator as well as several signal sequences.<br />
<br />
====T7 System====<br />
In contradiction to the results of Kang and coworkers and other groups, in our ''in vitro'' set-up the T7 terminator did not seem to terminate at all. The negative control (Promoter_Terminator_malachite binding aptamer) showed a similar increase in fluorescence as the positive control (Promoter_random sequence_malachite binding aptamer). <br />
[[Image:TUM2010_T7Result1.png|360px||thumb|left|''in vitro'' transcription measurement of T7 terminator with no signal(upper left), nonsense signal (upper right) and two different designed signals (below)]]<br />
[[Image:TUM2010_T7Result3.png|360px||thumb|right|''in vitro'' transcription measurement of positive control(upper left and T7 terminator with three different designed signals (remaining traces)]]<br />
Furthermore denaturing Polyacrylamide Gel Electrophoresis (PAGE) confirmed that there was no observeable termination of transcription. The addition of a signaling sequence led to a significantly lower increase in fluorescence, which can be attributed to the fact that both DNA sequences, switch and signal, compete for RNA Polymerases.<br />
However, there is almost no difference between the designed signals and random sequences, which is not a big surprise since there can be no antitermination if the terminator itself does not work.<br><br />
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Possible explanations for the contradiction between our results and those of Kang and coworkers might be the experimental set-up and the RNA Polymerases we used. Different variants of T7 RNA Polymerase might respond in different ways to terminator structures, and the termination might be influenced by the presence or absence of cofactors, depending on the purification methods used in producing the Polymerase.<br><br><br />
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This set-up offers a lot of possible experiments for the future, which we would have loved to conduct with a just a bit more time...<br />
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====''E.coli'' System====<br />
<br />
Compared to the T7 System, the ''E. coli'' RPO system produced poor increases in fluorescence, indicating little RNA synthesis. It was shown that the presence of a terminator decreases, as expected, the production of downstream RNA. This result was also confirmed by denaturing PAGE. However, due to the poor changes in fluorescence we were not able to actually characterize the behaviour of our switches ''in vitro'', and the small RNA concentrations did not allow a quantitative interpretation of our gels. A major problem with this method was the low concentration of the ordered Polymerase resulting in a much weaker overall signal as comparable measurements using the T7 Polymerase. <br><br><br />
In future experiments we might try to work with smaller volumes in order to reach higher concentration of RPO and of the synthesized RNA molecules, so measuring in 96 well plate readers might be a good choice. <br />
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==Software==<br />
Although we could not show the full functionality of bioLOGICS in the lab we still want to demonstrate the potential of our approach. Hence we implemented the idea behind our logic gates in a program which illustrates how bioLOGCIS theoretically would allow the construction of complex information processing networks interconnecting BioBricks. For further details take a look at our [[Team:TU Munich/Software|Software page]].<br />
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=References=<br />
<html><a name="ref1"></a></html>[1] http://partsregistry.org/cgi/partsdb/Statistics.cgi<br />
<html><a name="ref2"></a></html>[2] https://2009.igem.org/Team:Imperial_College_London/M1 encapsulation<br />
<html><a name="ref3"></a></html>[3] https://2009.igem.org/Team:TUDelft<br />
<html><a name="ref4"></a></html>[4] https://2008.igem.org/Team:Heidelberg<br />
<html><a name="ref5"></a></html>[5] Maung Nyan Win and Christina D. Smolke, Science Oct. 2008 Vol. 322. no. 5900, pp. 456 - 460<br />
<html><a name="ref6"></a></html>[6] Lu, T.K., A.S. Khalil, and J.J. Collins, Next-generation synthetic gene networks. Nature biotechnology, 2009. 27(12): p. 1139-1150. <br />
<html><a name="ref7"></a></html>[7] Schaller, R.R., Moore's law: past, present and future. Spectrum, IEEE, 2002. 34(6): p. 52-59.<br />
<html><a name="ref8"></a></html>[8] von Mering, C., et al., Comparative assessment of large-scale data sets of protein–protein interactions. Nature, 2002. 417(6887): p. 399-403.<br />
<html><a name="ref9"></a></html>[9] Mandal, M. and R.R. Breaker, Gene regulation by riboswitches. Nature Reviews Molecular Cell Biology, 2004. 5(6): p. 451-463. <br />
<html><a name="ref10"></a></html>[10] Benner, S.A. and A.M. Sismour, Synthetic biology. Nature Reviews Genetics, 2005. 6(7): p. 533-543.<br />
<html><a name="ref11"></a></html>[11] Beaudry, A. and G. Joyce, Directed evolution of an RNA enzyme. Science, 1992. 257(5070): p. 635-641.<br />
<html><a name="ref12"></a></html>[12] Delorme, Ehrlich and Renault, Regulation of Expression of the Lactococcus lactis Histidine Operon. Journal of Bacteriology, Apr. 1999, p. 2026–2037<br />
<html><a name="ref13"></a></html>[13] Trun and Trempy(2003): Fundamental Bacterial Genetics, Wiley-Blackwell, Chapter 12 <br />
<html><a name="ref14"></a></html>[14]Sooncheol Lee, Huong Minh Nguyen and Changwon Kang, Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator. Nucleic Acids Research, 2010, 1–9<br />
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<!-- The idea behind our project is to change the way BioBricks have been used up to now. Over the years, many receptors and signals have been constructed as BioBricks during the annual iGEM competition, but still it is not possible to interconnect these Bricks in a complex biological network resuting in a cell, that is able to respond to its environment giving differenciated responses depending on the input signals. (Beispiel: cambridge hat das gemacht, xx dies, aber eine zelle kann nicht beides...<br><br />
We plan to create biological switches, that can function as locial gates inside a cell. Our switches rely on RNA/RNA-interactions, regulating transcriptional termination. This is a major advance of our concept, as regular switches rely on complex regulation including proteins and/or metabolites. Thus, our switches shall offer a greater robustness and their behaviour should be easier to predict. [[switch|Read more]] (hier sollte noch das hochskalieren erwähnt werden...<br><br />
These switches can further be used to build up a logical network inside a bacterial cell, enabling every scientist to connect as many functionalities (in form of BioBricks) as designated. We plan to offer simulation on each specifically designed network.<br />
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<br><br>Over the years, many teams participating in the iGEM competition spent their time on constructing receptors and systems to detect a certain input that a variety of gorgeous oppurtunities is available so far.[[Image:TUM2010 network.png|thumb|300 px|right|Our visioon: A logic network inside the cell]] Nevertheless, until now it is not possible to link all those functionalities and build up a network giving differenciated responses to several of those input signals, where the molecular response depends on the complex composition of the environment a cell faces. We would like to offer this possibility to everyone.<br />
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The logic network we want to apply will be based on devices, that can be easily upscaled and therefor offer the chance to build networks of any wanted complexicity. Our devices rely on pure RNA/RNA interactions and thus their behaviour is well predictable.<br />
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The concept we rely on for our design of RNA-switches is based on the principle of [https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation/ '''attenuation'''].<br />
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= Experiments =<br />
We designed several experiments to test our switches, all of them based on fluorescence measurements. We designed experiment setting for measurements ''in vivo'' as well as ''in vitro''. Our ''in vitro'' measurements relied on two different experiment set-ups. While the first was based on a commercial ''E. coli''-lysate, the latter was reporting on a transcriptional level only, eliminating most of the possible side-effects one could expect in the complex behaviour of a living cell or cell-lysate. [[Experiments_main|Read more]]<br />
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= Results =<br />
We ...blablabla<br />
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Text that will present our results...<br />
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'''bioLOGICS: Logical RNA-Devices Enabling BioBrick-Network Formation'''<br />
'''Abstract'''<br />
Among the goals of iGEM is the creation of synthetic biological parts and their utilization to achieve novel features and behavior in biological systems. The emphasis of our project is put on this latter, "systems" aspect of iGEM. More precisely, we aim at the development and experimental demonstration of a scalable approach for the realization of logical functions in vivo.<br />
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By developing a computational biological network based on RNA logical devices we will offer everyone the opportunity to 'program' their own cells with individual AND/OR/NOT connections between BioBricks of their choice. Thereby, BioBricks can finally fulfill their original assignment as biological parts that can be connected in many different ways. We will achieve this by engineering simple and easy-to-handle switches based on predictable RNA/RNA-interactions regulating transcriptional termination. These switches represent a complete set of logical functions and are capable of forming arbitrarily complex networks.<br />
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== The Experiments ==<br />
===Fluorescent proteins as reporter===<br />
<div align="justify"><br />
Our initial idea to prove our concept of antitermination was to use flourescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks ''in vivo'' as well as ''in vitro'', the latter using a translation kit based on e.coli lysate. <br>We decided to use the flourescent proteins GFP and RFP, as their spectra do not overlap and we would not measure any FRET. We would use GFP fluorescence as internal control and RFP fluorescence as signal to detect termination/antitermination by our switch we cloned in between the coding sequences of the proteins. Both protein sequences are under the control of one (L-arabinose induced) promoter.<br />
[[Image:TUM2010_gfprfp_schalter_klein.gif|center|our idea]]<br />
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When measuring the termination of our BioBricks and the antitermination by their corresponding signal-RNA, we should be able to observe an increasing RFP emission compared to the GFP emission upon induced signal-RNA production in the cells/in the kit:<br><br />
[[Image:TUM2010_Expected_emission_spextra.png|center|our idea]]<br />
Wiith these measurements, it should also be possible to observe differences in efficiency of termination as well as antitermination between our designed switches. <br><br><br />
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===Measurements with the malachite green aptamer as reporter===<br />
A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramaticly (about 3000 times) upon binding of a specific RNA-aptamer.<br><br>#<br />
[[Image:TUM2010_Malachitgruen-2.png|500px|center|our idea]]<br />
---concept to be desribed, as well as literature---<br />
<ref>refs</ref><br />
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To study the switches on the transcriptional level gives the advantage, that we would have less interferences and possible artefacts. Also, we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems.<br><br />
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[[Image:TUM2010_Malachit_emission.png|200px|thumb|left|Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA]]We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.<br>Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level.<br />
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Results <br />
==Flourescent proteins==<br />
Unfortunatly, we had to change the reporter construct two times during our experiments as several problems occured in our measurements:<br><br><br />
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===First Try: based on the measurement plasmid pSB1A10===<br />
At the beginning, we decided to use the reporter plasmid [http://partsregistry.org/Part:pSB1A10 pSB1A10] from the registry. It consists of the fluorescent proteins eGFP and mRFP1. Each sequence includes a ribosome binding site and a stop-codon; the two genes are divided by a cloning side including the BioBrick cleavage sites.[[Image:ScreeningPlasmid1.0.PNG|300px|thumb|right|pSB1A10]]<br> In front of the eGFP sequence, the plasmid includes an arabinose-inducable promoter. The plasmid also contains an ampicilline resistence.<br><br />
We cloned our switches into the cloning site of the measurement plasmid and used an empty cloning site as control; our signal-RNAs we cloned into the [http://partsregistry.org/Part:pSB1K3 pSB1K3] vector, together with the BioBricks R0011 (Lac promoter) and B0014 (double terminator of transcription). Afterwards, we cut pSB1K3 with Aat2 and Pst1 and pSB1A10 with Nsi1 and Aat2 and ligated those fragments of each plasmid that contained our Bricks to get a Monsterplasmid.<br>We had to do so, as both plasmids contain the same ori mechanism. In addition, having both the switch and the signal RNA transcribed from the same plasmid gives us a high local concentration of the signal, once its transcription is induced.<br><br />
[[Image:TUM2010_Messplasmid_nr1.png|left|the measurement plasmid]]<br />
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We transformed BL21(DE3) cells with the plasmid. We set up cultures, induced the arabinose promoter and measured the GFP and mRFP1 excitation/emission spectra within time.<br><br><br />
Unfortunatly, we were ot able to detect any RFP signal, not even in the positive control with no switch in between the GFP/RFP sequences.<br><br><br />
From these experiments, we concluded, that the mRNA of the RFP variant used was instable and rapidly degraded by RNases, so the RFP was not synthesized in the cells. This was also the conclusion from XXX...<br />
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As solution to this, we decided to design a measurement plasmid ourselves:<br><br />
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===Second Try: A measurement plasmid of our own design===<br />
To design our own plasmid to overcome the problems that occurred in our first try gave us tghe possibility to overcome several other problems:<br><br />
#<br />
#<br />
#<br />
[[Image:TUM2010_Construct_no2.png|400px|our construct|center]]<br />
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===Third Try: One promoter for each protein===<br />
We decided to use the measuremnt plasmid we developed in our second try but to clone another L-arabinose induced promoter into the plasmid, in front of our switch followed by the mCherry sequence.<br><br />
<br>[[Image:TUM2010_Construct_no3.png|left|400px|Construct #3]]In this way, we still can use GFP fluorescence as internal control, because both protein transcription is under the control of a promoter of identical design.<br />
Though we are still not able to tell exactly why our previous measurements did not work, but with this construct we measured the first time fluorescence of the mCherry protein in our positive control.<br />
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{{:Team:TU_Munich/Templates/End}}</div>Hartlmuellerhttp://2010.igem.org/Team:TU_Munich/ProjectTeam:TU Munich/Project2010-10-28T03:22:04Z<p>Hartlmueller: /* From switches towards bioLOGICS logic gates */</p>
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<center><font size="5pt" color="#000000">'''bioLOGICS'''</font><font size="4pt" color="#000000">: Logical RNA-Devices Enabling BioBrick-Network Formation</font></center><hr color="black"><br><br />
= Vision=<br />
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Until today, 13.628 biobrick sequences<sup>[[Team:TU_Munich/Project#ref1|&#91;1&#93;]]</sup> have been submitted to partsregistry, thereof 102 reporter units and 12 signaling bricks.<br />
Since then, people are trying to arrange these single biological building blocks in such a manner that allows producing special biotechnological products (metabolic engineering), developing biological sensory circuits (biosensors) and even giving microorganisms the ability to react on multiple environmental factors and serve both as disease indicator and drug. These examples and further promising ideas were implemented on previous iGEM-competitions.<sup>[[Team:TU_Munich/Project#ref2|&#91;2&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref3|&#91;3&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref4|&#91;4&#93;]]</sup> <br><br><br />
The idea of combining the outcome of several iGEM competitions to construct complex synthetic biological systems falls at the last hurdle - the fact, that each team uses a different principle how to access and functionally connect the respectively used biobricks. For example, it is a major challenge to create a system that uses several sensoring BioBricks from different iGEM-teams which in turn regulates reportering BioBricks from various teams. In order to combine and fully take advantage of these promising projects, our vision is to develop an adapter that allows interconnecting arbitrary biobricks on a functional level. Such a system easily allows to setup sensor-reporter circuits and interconnect them to complete biological chips... A further step towards artificial cells.<br><br><br />
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Generally speaking, the above adapter has to meet the following requirements:<br />
*'''Universality'''<br />
:The adapter has to be compatible to as many BioBricks as possible. This objective will guarantee that a large number of BioBricks can be connected.<br />
*'''Scalability'''<br />
:Once the basic design of the system is established, the construction of the system is supposed to be automated in silico. This way it will be possible to create an adapter connecting a large amount of BioBricks.<br />
*'''Biological orthogonality'''<br />
:Interference with cellular components has to be as low as possible in order to avoid unwanted and perturbing side effects.<br />
*'''Logic'''<br />
:The adapter is supposed to not only associate different BioBricks, but to functionally connect BioBricks in a precisely determined manner (including operations such as AND/OR/NOT).<br />
<br><br />
Several biological logic units, devices and circuits have been developed so far<sup>[[Team:TU_Munich/Project#ref5|&#91;5&#93;]]</sup>, but to our knowledge, none was shown to meet all requirements listed above.<br />
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=Implementation=<br />
To functionally connect BioBricks, there are several possibilities including genetic switches, riboswitches and direct protein-protein interactions. We investigated several hypothetically principles, and decided to focus our practical work on the development of a RNA-RNA interaction-based switch. These switches are capable of changing between two states, a state of antitermination and termination, and make use of highly-specific RNA-RNA interaction. In principle such a switch can fulfill all requirements mentioned previously. The following text clarifies how these switches work in detail.<br />
==How to connect BioBricks==<br />
Our adapter is a system, that activates or disables BioBricks (output BioBricks) in response to the presence of other Biobricks (input Biobricks). Our approach uses a molecular network to put this into practice and consists of four major elements:<br />
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{|<br />
|-<br />
|[[Image:Networks.png|center|thumb|730px|The general principle how different inputs can be connect to various outputs. For details see text.<br>Inputs (such as proteins or small molecules) are indicated on the left side. blue lines represent transmitter molecules whereas organe lines present logic gates. The type of logic gate is indicated. Green lines indicate transmitter RNA that can function as mRNA and consequently generate any output gene (indicated on the very right).]]<br />
|}<br />
In order to connect different BioBricks, our network requires four major types of components:<br />
*Input elements<br />
*Transmitter molecules<br />
*Logic gates<br />
*Output elements<br />
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{{:Team:TU_Munich/Templates/InfoBoxStart}}'''Computer vs. molecular network - and our approach'''<br><br />
Logic gates in a molecular network are often compared to transistors used in a computer, where billions of transistors are incorporated<sup>[[Team:TU_Munich/Project#ref7|&#91;7&#93;]]</sup>. The main advantage on a computer chip is, all transistors share the same functional principle, and only the way connecting them in a special sequence allows specific addressing of only a subset of other transistors by an input. However, spatially fixed connections of molecular logic gates are not possible in a living cell. The "wiring" within a cell relies on the specific interaction between transmitter molecule and their corresponding logic gates, for example implemented by protein-protein/ligand-protein interactions or specific ligand-riboswitch interactions.<sup>[[Team:TU_Munich/Project#ref8|&#91;8&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref9|&#91;9&#93;]]</sup> As a result, in a cell, each occurring logic gate ("transistor") has to be different, at least in a special recognition site<sup>[[Team:TU_Munich/Project#ref10|&#91;10&#93;]]</sup> - for example like different transcription factors, recognizing different DNA-sites. Thanks to evolution, nature easily can invent a new transistor for each task - science achieves this only on a limited scale, and producing synthetic molecular logic gates artificially by either rational or evolutionary protein or riboswitch engineering, is limited to small circuits so far<sup>[[Team:TU_Munich/Project#ref11|&#91;11&#93;]]</sup>. Our project aims to establish a molecular switch as close as possible to a electronic transistor, thus sharing the same functional principle for all logic gates. At the same time, we want to design a easily exchangeable recognition site, which can individually be designed by everyone! {{:Team:TU_Munich/Templates/InfoBoxEnd}}<br />
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These elements can be combined to build up a molecular network (see illustration). Each input molecule (such as a BioBrick) produces a unique transmitter molecule. All transmitters belong to the same type of molecule and share a common design. However, each transmitter molecule can only interact and activate a certain subset of logic gates. In other words, logic gates have to recognize as well as bind the corresponding transmitter molecules and are capable of producing a new output transmitter molecule. Depending on the type of the logic gate (AND, OR or NOT<sup>[[Team:TU_Munich/Project#ref6|&#91;6&#93;]]</sup>), an output transmitter is only created if both input transmitter molecules are present (AND), at least one of two input transmitters is present (OR) or if no input transmitter is present at all (NOT). Once a logic gate has produced a new output transmitter, these transmitters can in turn address another subset ("layer") of logic gates. In theory many layers of logic gates can be connected this way allowing the creation of large networks. Until this step, various transmitter molecules might have been produced. But in order to create a Biobrick output, the last layer of logic gates finally generates transmitter molecules that will not active logic gates, but will rather interact with the cell metabolism to produce a BioBrick response. In other words, the last layer of transmitter molecules is capable of regulating BioBrick formation.<br />
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Summarizing, the network establishes a connection between input BioBricks and output BioBricks in a functional manner.<br />
Having addressed the basic layout of the molecular network, the next step is to determine what type of molecules can perform the required functions. We decided to use RNA, both as transmitter molecules and for constructing logic gates. Several advantages result from the utilization of RNA as the central element:<br />
*During the last years, many Biobricks were designed that are sensitive to various chemicals and substances. These BioBricks often function as a transcription factor that binds to a specific DNA sequence and consequently would be capable to produce a specific transmitter RNA molecule. Thus, in principle each BioBrick which involves transcription can be integrated in our network.<br />
*Since all logic gates are capable of producing transmitter RNA, they can also produce functional mRNA encoding any protein. This means, each BioBrick consisting of protein or RNA can be produced as an output of our network.<br />
*If RNA forms both, the transmitter molecule and the logic gates, they can specifically interact by RNA-RNA interaction, which is highly predictable compared to protein interactions. This allows to generate a library of transmitters and gates ''in silico''. Such a library is essential for the creation of large networks.<br />
*RNA production is fast and energy saving for a cell. Consequently, operating a network that only produces RNA rather than proteins will also be faster and more efficient for the host cell. Since our logic gates are based on transcription, translation and resource consuming protein production will only be required at the very last step. <br />
*As the half-time of RNA can be rather short, transmitter RNA will not accumulate within the cell and it is therefore less likely for the system to become saturated.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Design and functional principle of logic gates==<br />
The concept introduced above provides a framework that can potentially serve as an universal adapter between different BioBricks. However, the [[Team:TU_Munich/Glossary#logic gate | logic gates]] have not been specified more precisely so far. This will be done in the following section.<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, our logic gates are to possess the following characteristics:<br />
*Logic gates, such as AND, OR and NOT, have to be implemented by RNA-interaction based principles (see [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]).<br />
*All logic gates have to recognize their corresponding [[Team:TU_Munich/Glossary#Transmitter (bioLOGICS)| transmitter RNAs]] and, in response, produce an output transmitter molecule.<br />
*Logic gates should follow a basic design rule, in such a way, that their creation can be automated ''in silico''.<br />
*The response efficiency of a logic gate toward a transmitter molecule should be comparable for all logic gates to provide calculable robustness and sensitivity. This will ensure comparable molecular concentrations and functionality of large networks.<br />
*The system has to be designed for ''in vivo'' utilization at the first place. As a reference we always assumed a temperature of 37 °C and an ''E. coli'' environment.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}} <br />
In order to build logic gates for our bioLOGICS system we will first create a simple switch. A switch can be activated by one transmitter RNA and produce an output transmitter RNA. In contrast to a logic gate, a switch does not perform logic operations. However by combining switches, logic gates can be created. The following text will first describe how the developed switch works and secondly, how logic gates such as AND/OR/NOT can be created using these switches.<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Read more{{:Team:TU Munich/Templates/ToggleBoxStart2}}<br />
[[Image:toggle_switch.png|500px|thumb|center|id="hideOnReadMore"|'''A''' The basic structure of a bioLOGICS switch (left) and a transmitter molecule (right).<br>'''B'''The process of switching. See the text in the close-by "Read more" section for details.<br>Rectangles present the composition of our functional units on the level of DNA. Fringed lines represent RNA produced by RNA polymerase. The stem loop structure depicts the switchable terminator. Terminator and target site are illustrated in blue and turquoise, respectively. Recognition sites are indicated in different colors, in this case red for the input transmitter and green for the output transmitter.Each switch and or later logical unit has to be flanked by a promotor and another constitutive terminator, to allow RNA-production by RNA-polymerase in a proper way. ]]<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===Switch===<br />
[[Image:TUM2010_switch-and-transmitter.jpg|550px|right|thumb|The basic strcutrue of a switch (left) and a transmitter RNA (right). See text for details.]]<br />
Roughly speaking, a switch can be regarded as an enhanced switchable transcriptional terminator. The enhancement can be described easier by dividing a switch into its functional components: <br />
*'''Target site'''<br><br />
:The target site is the functional core element of our switches, allowing a shift between an "on" and "off" state. Since we work on the level of RNA-production (transcription), a "switchable" transcriptional terminator is suitable for this purpose. By allowing or preventing formation of a transcriptional terminator, that is by switching between termination and antitermination it is possible to represent an "off" and an "on" state, respectively. Therefore, the target site is the 5' ending of the terminator and is required for a stable terminator formation. It should be noted that this principle was also observed in nature.<br />
:To highlight and illustrate the functional principle of our switches, only the part of the terminator which is involved in interacting with a transmitter molecule and which is responsible for shifting between "on" and "off" state is called target site. The remaining terminator sequence is called terminator in the following, even if both, target site and terminator build up the terminator structure occurring in nature. <br />
:The important aspect of our switches is the fact that all switches will hold the same identical target site. Therefore having found one functional "switchable" terminator, will allow almost unlimited upscaling since this terminator can be used for a large library of switches. This is the main difference to previous works done on this field, which always required developing a new shifting principle for each switch.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup> Beside this scalability, this principle provides a comparable on/off shifting rate (responds function) for all switches, avoiding complex fine tuning of molecular networks.<br />
:To sum it up, the target site, allows to switch between an "on" and "off" state. But so far, the switch is not capable of performing specific interaction with transmitter molecules. This is where the recognition site comes into play.<br />
*'''Recognition site'''<br />
:The recognition site defines which transmitter molecule can actually interact with the switch. Therefore, a unique recognition site is generated for each switch and is positioned right upstream of the target site. In principle the recognition can be any random sequence as long as it remains unique within the molecular network.<br />
Summing up, the recognition site allows a specific interaction between switches and transmitter molecules. Once this interaction is formed, an interaction between the transmitter and the target will actually switch the state of the terminator. This allows the specific arrangement and interconnection of numerous of these switches by transmitter molecules, without changing the target site. Comparable to wires connecting many identical transistors, our target site remains the same.<br />
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===Transmitter RNA´s===<br />
As desccribed above, transmitter RNAs are the input and output of bioLOGICS switches (compare [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]). These transmitters are short ssRNA molecules representing the "trigger" to shift switches between the "on" and "off" state. To fulfill this role, they need to posses the following properties:<br />
*A transmitter may only interact with certain switches. That is, a transmitter has to find the corresponding recognition site of a switch.<br />
*Once an interaction is established between a transmitter and a switch, a transmitter has to be capable of changing the secondary structure of a terminator and thus cause antitermination.<br />
Again, these two properties are fulfilled by two components of the transmitter:<br />
*'''Identity site'''<br />
:This site is capable of forcing an interaction between the transmitter and the switch. Therefore it is complementary to the recognition site of this switch. As the recognition site is unique within a network, so is the identity site. However, the single identity site is not capable of changing the state of the switch. That is were the trigger site comes into play.<br />
*'''Trigger site'''<br />
:Once an interaction is created by the identity site, the trigger site is capable of actually shifting the switch since it is complementary to the target site of the switch. To fulfill this role, it is placed upstream at the 5' end of the identity site. As the target site is the same for all switches, the trigger site is the same for all signals. Therefore it is important, that similar to the identity site, a trigger site cannot function on its own. That is, a single trigger site cannot shift the state of a switch without the help of an identity site.<br />
<br />
Summing up, we applied the principle introduced for the switches to the transmitter molecules. In contrast to previous approaches on this field <sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup>, we introduced the described synthetic trigger site in such a manner that it is not able to change the state of the terminator on its own, but only in combination with the identity site. So the challenge is to arrange and optimize these elementary building blocks thermodynamically, that a trigger site is only able to switch in combination with its respective identity site. This was done by ''in silico'' design using [[TU Munich/Glossary#NUPACK| NUPACK]], presented in section [[TU Munich/Modeling#in silico design based on thermodynamic calculations| in silico design]].<br />
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===Putting it all together: the switching process===<br />
[[Image:TUM2010_switching-process.jpg|550px|right|thumb|The basic structure of a switch (left) and a transmitter RNA (right). See text for details.]]The functional principle of the designed switches is illustrated in the figure. The switch is positioned on DNA upstream of a desired output transmitter. So in the absence of a triggering transmitter molecule, transcription will be canceled by the formation of a RNA stem loop in the nascent RNA-chain. This will cause the RNA polymerase to stop transcription and fall off the DNA and consequently no output RNA will be produced. This process only relies on [[Team:TU_Munich/Glossary#Termination| rho-independent termination]].<br />
On the other hand, in the presence of a [[Team:TU_Munich/Project#RNA_transmitters | input transmitter]], this small functional RNA inhibits the stem loop formation by complementary base-pairing and hence avoids termination of transcription. In detail, the identity site (red part on transmitter) binds the recognition site (red part on switch) and serves as [[Team:TU_Munich/Glossary#Toehold|toehold]], which will thermodynamically allow the trigger site (turquoise part on transmitter) to perform a [[Team:TU_Munich/Glossary#Strand Displacement| strand displacement]] and open up the stem loop structure. Consequently the polymerase can read all the way through and form the output RNA.<br>Summing up, we use this concept to create a switch that can be toggled by a transmitter RNA molecule and in response, is able to produce another transmitter RNA.<br />
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<br />
===From switches towards bioLOGICS logic gates===<br />
As described, each switch can be accessed by a specific RNA-transmitter molecule, representing the input. In turn, another RNA-transmitter molecule will be produced if the switch shifts its state. This output transmitter of one switch can serve as input transmitter for the next switch by meaningful selection and design of the respective recognition sites. This easily allows arranging several switches in specific sequences and faulty wiring - the corner stone of a logical network.<br><br />
To ease the building of logical networks we want to create a switch capable of Boolean logics, a common mathematical principle fundamental for computational science. Since AND/OR/NOT are basic logic operations which can be implemented with the presented switches, all remaining operations (such as XOR, NAND, ...) can be expressed by these three operators according to laws of boolean logics.<br />
Creating logic gates is achieved by combining two switches in two different ways, as illustrated below.<br />
{|<br />
|-<br />
| *AND gate<br />
:An AND gate can be constrcuted by positioning two switches right next to each other. For the output transmitter to be created, both input transmitter have to be present.<br />
|[[Image:AND2.png|500px|thumb|center|Combining two switches in series creates a logic AND gate.]] <br />
|-<br />
| *OR gate<br />
:An OR gate is created by utilizing two independent switches sharing the same output transmitter. If each one of both switches is activated, an output transmitter is generated. Therefore, one input transmitter is enough to produce an output transmitter.<br />
|[[Image:OR2.png|500px|thumb|center|Combining two switches in parallel creates a logic OR gate.]]<br />
|-<br />
| *NOT is more complex to explain. In principle, it consists only of one switch which contains its respective signal molecule intrinsic, so via intramolecular interaction, antitermination is the initial state. The signal is intrinsically of the same components as usual to allow interconnection with other logic gates.<br />
|-<br />
|[[Image:NOT2.png|500px|thumb|center]]<br />
|}<br />
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<br />
==Network construction==<br />
Designing complex biological networks based on either traditional protein engineering or our new bioLOGICS is still a complex task. We developed a software which allows the fast construction of a bioLOGICS based networks. <br><br />
To read more about this, look at our [https://2010.igem.org/Team:TU_Munich/Software Software page]<br />
<br />
=Our Objective=<br />
Putting the implementation described above into practice, will be a major challenge. For this year's iGEM competition our goal is to do the first step: design and build a switch that can be toggled by a RNA molecule. To be precise, we want to apply the design rules of our switch to modify a transcription terminator in such a way that it interacts with a second RNA molecule and, as a result, is no longer capable of forming a stem loop. This objective will require intensive ''in silico'' designing and modeling of switches based on different terminators and their corresponding transmitters. In connection to this theoretical part, we also have to test and verify the switches. For this step, we establish custom-made assays, ''in vitro'' and ''in vivo''.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Once the objective mentioned above is accomplished, these basic RNA/RNA-interactions have to be modified in such a manner that the described identity/trigger site pattern for the transmitter and the complementary recognition/target site switch composition has to be established. The most important requirement is to is to optimize these modules that the transmitter is only able to switches specifically, meaning only in the presence of both, identity AND trigger site. <br />
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Once the objective mentioned above is accomplished, the creation of an OR gate will be rather simple since it only requires two switches. However the creation of an AND or NOT gate and optimizing the logic gates to improve their responds function will remain the goal of future work. Also the creation of small networks and the correct integration of BioBricks as input and output molecules will be future challenges. Furthermore, we wanted to rather focus on the development and the testing of our structural design of the switches, rather than developing a variety of new BioBricks.<br />
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==''In silico'' design==<br />
As described above, our switches are based on certain design rules. However, there still are different structural parameters that need to be tested and optimized (length of recognition site and target site, choice of terminator, etc.).<br />
We used [[Team:TU_Munich/Project#in silico design |''in silico'' design]] and [[Team:TU_Munich/Modeling| modeling]]) to test different parameters. Furthermore we tried to use the [[Team:TU_Munich/Glossary#Antitermination|antitermination principle]] observed in nature, such as [[Team:TU_Munich/Glossary#Attenuation| attenuation]] in ''E. coli'' or [[Team:TU_Munich/Glossary#Tiny Abortive RNA´s| tiny abortive RNA´s]] of T7-phage.<br />
==Evaluation and Measurements==<br />
To evaluate the functionality of our molecular switches, we first had to establish several assays. Therefore, we improved an existing [[Team:TU_Munich/Lab#In vivo Measurements |''in vivo'' assay]] and developed an [[Team:TU_Munich/Lab#In vitro Transcription | ''in vitro'' assay]] for this purpose. For more information please refer to the [[Team:TU_Munich/Lab | lab]] section.<br />
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<br><br />
Summarizing, the main challenges are <br />
* to find a suitable terminator construct and design a complementary trigger unit, which is only functional in combination with a specificity site - meaning an optimization of the '''thermodynamically parameters''' (see[[Team:TU_Munich/Project#in silico design| in silico design]])<br />
* to investigate whether the transmitter/switch interaction reaction is on a timescale to be competitive to terminator formation - meaning an comparison of '''kinetic parameters''' (see [[Team:TU_Munich/Modeling|Modeling page]])<br />
* to proof antitermination can be also be caused by synthetically RNA-interaction (see [[Team:TU_Munich/Glossary#Antitermination| Antitermination in nature]] and [[Team:TU_Munich/Project#Results| ''in vivo'' and ''in vitro'' measurements]] )<br />
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=Results=<br />
Every network starts with a basic unit. While our declared aim is to enable networks allowing fine-tuning of gene expression beyond the regular on/off, exploring such an on/off switch/signal pair is the first step towards a functional network. We constructed several units and tested their efficiency, robustness and reproducibility ''in vivo'', ''in vitro'' and ''in silico''. Furthermore we developed a software which allows easy constructions of networks based on our designed logic gates. Conclusive elaboration of a few first RNA-based logic units is the major contribution of our iGEM team.<br />
<br />
==in silico Design of Switching and Trigger Unit==<br />
As described on the [[Team:TU_Munich/Project | project]] page, one key aspect of our switches is the idea, that a [[Team:TU_Munich/Glossary#Transmitter_(bioLOGICS) | RNA transmitter molecule]] is capable to shift the state of a switch only if its [[Team:TU_Munich/Glossary#Trigger_Site_(bioLOGICS) | trigger site]] is present and its [[Team:TU_Munich/Glossary#Identity_Site_(bioLOGICS) | identity site]] corresponds to the [[Team:TU_Munich/Glossary#Recognition_Site_(bioLOGICS) | recognition site]] of the [[Team:TU_Munich/Glossary#Switch_(bioLOGICS) | switch]]. We successfully constructed several switches and their corresponding transmitter RNA ''in silico'' on a thermodynamical basis. We modified different transcriptional terminators in such a way, that the formation of the terminator was prevented by a transmitter molecule. As desired, this only occured if the transmitter molecule contained both, a trigger and an identity site. Analogously, we were able to design and verify a NOT gate using the same thermodynical approach.<br />
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==Diffusion and RNA Folding Dynamics==<br />
We estimated the diffusion time for our constructs and modeled the folding dynamics of our bioLOGICS switches including the switching process with a stochastic RNA folding program. We were able to provide better insight in their folding dynamics and proved that they are able to interrupt termination. We also optimized the switches and the corresponding signals. Furthermore, we combined the switches what resulted in a logic gate. See our [[Team:TU Munich/Modeling|Modeling page]] for further details.<br />
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==''in vivo'' Functionality Screening==<br />
Since our logic gates are intended to function in living cells, ''in vivo'' measurements were essential. In a set of experiments we concentrated on two different switches based on known [[Team:TU_Munich/Glossary#Attenuation|attenuators]] from nature: the [[Team:TU_Munich/Modeling#Switch|HisTerm]] and [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Focusing on fluorescent proteins for quantifiable input and output we designed a functional and robust screening system. For greater detail see [[Team:TU_Munich/Lab#Experiment_Design|Experimental Design]]. Unfortunately, setting up a working screening system failed twice. Only in redesigning and improving the screening plasmid pSB1A10 we succeeded, but lost precious time.<br />
<br />
Ultimately, the two switches displayed remarkable differences in their terminator efficiency, but neither of them responded to their corresponding signal. However, screening one transmitter signal does not disprove the basic working principle of our system. Limited by time, we hope for future teams to take up our work and to use our improved test system that we submitted to the parts registry, for performing successful in vivo measurement.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Considering the high complexity of ''in vivo'' measurements compared to other experimental challenges, a robust and easy to handle test system for [[Team:TU_Munich/Glossary#PoPS-based devices| PoPS-based devices]] is desirable. As described in [[Team:TU_Munich/Lab#Experiment_Design|Experimental design]], we used fluorescent proteins: RFP or mCherry to measure the amount of produced output and eGFP for normalization. Our first attempt, using the screening plasmid pSB1A10, yielded no interpretable results. Switching the fluorescent protein to mCherry did not work either, but after several experimental setups we determined a transcriptional problem causing no reporter protein expression regardless of the inserted part. Thereby we demonstrated the screening plasmid pSB1A10 to be [[Team:TU_Munich/Biobricks#Falsification| malfunctioning]]. <br />
Finally a new design based on pSB1A10 lead to a functional and robust screening system (compare [[Team:TU_Munich/Parts#Screening system: Backbone BBa_K494001| Screening system: Backbone BBa_K494001]]). A second promoter with identical induction properties inside the BioBrick cloning site enforces transcription of the PoPS-based device and the mCherry output.<br />
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Exemplary, the graph below on the right shows the positive control, induced and uninduced at OD<sub>600</sub>=0.7 followed by 16 h incubation at 25 °C. Clearly visible are eGFP and mCherry fluorescence in the induced samples. The uninduced control showed no fluorescence at all, demonstrating the PBad promoter to be tight and providing very low basal transcription, what is a major advantage for the screening system. This newly designed screening approach renders the characterization of PoPS-based devices in general and switches in particular easy and robust. The low basal transcription furthermore fulfills one of the most important requirements for the designed switches, since output transmitters may only be produced in presence of an input transmitter. This helps to avoid strong "background" noise, which would extremely harden the successful interconnection of several switches. <br />
<br><br />
[[Image:TUM2010_PosControlklein.JPG|200px||thumb|left|Bacteria containing positive control]]<br />
[[Image:TUM2010_graphPosControl1.png|355px|thumb|center|Emission spectra of induced (green/red) and uninduced(black) positive control BBa_K494002 ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
Due to the time limitations of the iGEM completion we had to focus our efforts on few switches after designing the screening system. Relying on the functionality of systems occurring in nature, we choose the [[Team:TU_Munich/Modeling#Switch|HisTerm]] as well as the [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Both switches are based on known natural [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|attenuators]]. Testing synthetic and none-naturally switchable terminators in vivo are goals for future work.<br />
Delorme et al. reported the His-Terminator to be a remarkable effective Terminator with more than 99% termination efficiency.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup> The exemplary measurement below on the right confirms the high terminator efficiency. In fact, we could not detect any mCherry fluorescence in any cells containing the [[Team:TU_Munich/Modeling#Switch|HisTerm]]. Even induction of the corresponding signal transmitter RNA via IPTG did not alter the Terminator efficiency. Again time was the limiting factor and prevented us from testing more than one corresponding transmitter, although the [[Team:TU_Munich/Modeling| Modeling]] highly suggested the necessarily of finding an optimized transmitter length. Thus, the results are insufficient either to prove or to disprove the functionality of the [[Team:TU_Munich/Modeling#Switch|HisTerm]] or our concept in general.<br />
<br><br />
[[Image:TUM2010_HisSwitchklein.JPG|200px|thumb|left|Bacteria containing HisTerm]][[Image:TUM2010_HisSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing HisTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
<br />
Attaining only 90% terminator efficiency, the natural Trp [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|Attenuator]] is known be less effective than the [[Team:TU_Munich/Modeling#Switch|HisTerm]].<sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup> The graph on the right depicts our designed [[Team:TU_Munich/Modeling#Switch|TrpTerm]] characteristic efficiency of about 40 %, notably below the natural standard. Allowing 60% transcription in the “off” state excludes the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] from possible candidates for a scalable network of logic gates, due to the mentioned required "yes or no" function (see [[Team:TU_Munich/Project#Implementation| Implementation and how to connect Biobricks]]). Thus the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] is inoperative as intended, but may still be useful in other contexts. Similar to the [[Team:TU_Munich/Modeling#Switch|HisTerm]], the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] also did not react to the induction of the corresponding signal. Under circumstances, termination efficiencies altered by the transmitter are on a low range and not resolvable within observed 40% basal transcription. <br />
<br><br />
[[Image:TUM2010_TrpSwitchklein.JPG|200px|thumb|left|Bacteria containing TrpTerm]][[Image:TUM2010_TrpSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing TrpTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
<br />
Making use of our improved screening system we also carried out some ''in vivo'' kinetic measurements in addition to the end-point measurements above. In contrast to the ''in vitro'' experiments we did not obtain significant results for the characterization of our switches. As the switching process is many times faster than protein synthesis our ''in vivo'' kinetics include the synthesis of mCherry as well as its maturation. Therefore we centered our attention on end-point experiments. For more information browse the [[Team:TU_Munich/Lab#Lab_Book|lab book]]. <br><br />
Considering our ''in vivo'' measurements, neither of the tested switches showed any effect regarding the signal induction. But due to the small number of tested switches and signals this can hardly be regarded as disprove of concept. In particular in light of the recent findings by Sooncheol proving antitermination in principle using a T7 system.<sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup><br />
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<br />
==''in vitro'' Screening==<br />
To minimize the amount of disturbing factors we decided to countercheck our ''in vivo'' results with a set of ''in vitro'' measurements. While the ''in vitro'' systems are no doubt much less complex than living cells, the work with these set-ups proved to be quite as difficult.<br />
Just as with the ''in vivo'' measurements we could prove our switching system neither right nor wrong, leaving enough work for future iGEM teams.<br />
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===''in vitro translation''===<br />
<br />
Beside optimization of the reporter proteins in use, the major problem occuring in the experiments was the low capacity of the kit. The signal intensity was very low, which made it difficult to observe any signal intensity alterations, so no conclusion could be drawn from these measurements.<br />
<br />
===''in vitro'' transcription===<br />
We used two completely independent ''in vitro'' systems: Using ''E.coli'' RNA Polymerase we analyzed the His and Trp switches that had already been tested ''in vivo''. In a second set-up, we used the well-established T7 RNA Polymerase and switch based on the T7 terminator as well as several signal sequences.<br />
<br />
====T7 System====<br />
In contradiction to the results of Kang and coworkers and other groups, in our ''in vitro'' set-up the T7 terminator did not seem to terminate at all. The negative control (Promoter_Terminator_malachite binding aptamer) showed a similar increase in fluorescence as the positive control (Promoter_random sequence_malachite binding aptamer). <br />
[[Image:TUM2010_T7Result1.png|360px||thumb|left|''in vitro'' transcription measurement of T7 terminator with no signal(upper left), nonsense signal (upper right) and two different designed signals (below)]]<br />
[[Image:TUM2010_T7Result3.png|360px||thumb|right|''in vitro'' transcription measurement of positive control(upper left and T7 terminator with three different designed signals (remaining traces)]]<br />
Furthermore denaturing Polyacrylamide Gel Electrophoresis (PAGE) confirmed that there was no observeable termination of transcription. The addition of a signaling sequence led to a significantly lower increase in fluorescence, which can be attributed to the fact that both DNA sequences, switch and signal, compete for RNA Polymerases.<br />
However, there is almost no difference between the designed signals and random sequences, which is not a big surprise since there can be no antitermination if the terminator itself does not work.<br><br />
<br />
Possible explanations for the contradiction between our results and those of Kang and coworkers might be the experimental set-up and the RNA Polymerases we used. Different variants of T7 RNA Polymerase might respond in different ways to terminator structures, and the termination might be influenced by the presence or absence of cofactors, depending on the purification methods used in producing the Polymerase.<br><br><br />
<br />
This set-up offers a lot of possible experiments for the future, which we would have loved to conduct with a just a bit more time...<br />
<br />
====''E.coli'' System====<br />
<br />
Compared to the T7 System, the ''E. coli'' RPO system produced poor increases in fluorescence, indicating little RNA synthesis. It was shown that the presence of a terminator decreases, as expected, the production of downstream RNA. This result was also confirmed by denaturing PAGE. However, due to the poor changes in fluorescence we were not able to actually characterize the behaviour of our switches ''in vitro'', and the small RNA concentrations did not allow a quantitative interpretation of our gels. A major problem with this method was the low concentration of the ordered Polymerase resulting in a much weaker overall signal as comparable measurements using the T7 Polymerase. <br><br><br />
In future experiments we might try to work with smaller volumes in order to reach higher concentration of RPO and of the synthesized RNA molecules, so measuring in 96 well plate readers might be a good choice. <br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Software==<br />
Although we could not show the full functionality of bioLOGICS in the lab we still want to demonstrate the potential of our approach. Hence we implemented the idea behind our logic gates in a program which illustrates how bioLOGCIS theoretically would allow the construction of complex information processing networks interconnecting BioBricks. For further details take a look at our [[Team:TU Munich/Software|Software page]].<br />
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<br />
<br />
=References=<br />
<html><a name="ref1"></a></html>[1] http://partsregistry.org/cgi/partsdb/Statistics.cgi<br />
<html><a name="ref2"></a></html>[2] https://2009.igem.org/Team:Imperial_College_London/M1 encapsulation<br />
<html><a name="ref3"></a></html>[3] https://2009.igem.org/Team:TUDelft<br />
<html><a name="ref4"></a></html>[4] https://2008.igem.org/Team:Heidelberg<br />
<html><a name="ref5"></a></html>[5] Maung Nyan Win and Christina D. Smolke, Science Oct. 2008 Vol. 322. no. 5900, pp. 456 - 460<br />
<html><a name="ref6"></a></html>[6] Lu, T.K., A.S. Khalil, and J.J. Collins, Next-generation synthetic gene networks. Nature biotechnology, 2009. 27(12): p. 1139-1150. <br />
<html><a name="ref7"></a></html>[7] Schaller, R.R., Moore's law: past, present and future. Spectrum, IEEE, 2002. 34(6): p. 52-59.<br />
<html><a name="ref8"></a></html>[8] von Mering, C., et al., Comparative assessment of large-scale data sets of protein–protein interactions. Nature, 2002. 417(6887): p. 399-403.<br />
<html><a name="ref9"></a></html>[9] Mandal, M. and R.R. Breaker, Gene regulation by riboswitches. Nature Reviews Molecular Cell Biology, 2004. 5(6): p. 451-463. <br />
<html><a name="ref10"></a></html>[10] Benner, S.A. and A.M. Sismour, Synthetic biology. Nature Reviews Genetics, 2005. 6(7): p. 533-543.<br />
<html><a name="ref11"></a></html>[11] Beaudry, A. and G. Joyce, Directed evolution of an RNA enzyme. Science, 1992. 257(5070): p. 635-641.<br />
<html><a name="ref12"></a></html>[12] Delorme, Ehrlich and Renault, Regulation of Expression of the Lactococcus lactis Histidine Operon. Journal of Bacteriology, Apr. 1999, p. 2026–2037<br />
<html><a name="ref13"></a></html>[13] Trun and Trempy(2003): Fundamental Bacterial Genetics, Wiley-Blackwell, Chapter 12 <br />
<html><a name="ref14"></a></html>[14]Sooncheol Lee, Huong Minh Nguyen and Changwon Kang, Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator. Nucleic Acids Research, 2010, 1–9<br />
<br />
<!-- The idea behind our project is to change the way BioBricks have been used up to now. Over the years, many receptors and signals have been constructed as BioBricks during the annual iGEM competition, but still it is not possible to interconnect these Bricks in a complex biological network resuting in a cell, that is able to respond to its environment giving differenciated responses depending on the input signals. (Beispiel: cambridge hat das gemacht, xx dies, aber eine zelle kann nicht beides...<br><br />
We plan to create biological switches, that can function as locial gates inside a cell. Our switches rely on RNA/RNA-interactions, regulating transcriptional termination. This is a major advance of our concept, as regular switches rely on complex regulation including proteins and/or metabolites. Thus, our switches shall offer a greater robustness and their behaviour should be easier to predict. [[switch|Read more]] (hier sollte noch das hochskalieren erwähnt werden...<br><br />
These switches can further be used to build up a logical network inside a bacterial cell, enabling every scientist to connect as many functionalities (in form of BioBricks) as designated. We plan to offer simulation on each specifically designed network.<br />
<br />
<br><br>Over the years, many teams participating in the iGEM competition spent their time on constructing receptors and systems to detect a certain input that a variety of gorgeous oppurtunities is available so far.[[Image:TUM2010 network.png|thumb|300 px|right|Our visioon: A logic network inside the cell]] Nevertheless, until now it is not possible to link all those functionalities and build up a network giving differenciated responses to several of those input signals, where the molecular response depends on the complex composition of the environment a cell faces. We would like to offer this possibility to everyone.<br />
<br><br />
The logic network we want to apply will be based on devices, that can be easily upscaled and therefor offer the chance to build networks of any wanted complexicity. Our devices rely on pure RNA/RNA interactions and thus their behaviour is well predictable.<br />
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The concept we rely on for our design of RNA-switches is based on the principle of [https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation/ '''attenuation'''].<br />
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= Experiments =<br />
We designed several experiments to test our switches, all of them based on fluorescence measurements. We designed experiment setting for measurements ''in vivo'' as well as ''in vitro''. Our ''in vitro'' measurements relied on two different experiment set-ups. While the first was based on a commercial ''E. coli''-lysate, the latter was reporting on a transcriptional level only, eliminating most of the possible side-effects one could expect in the complex behaviour of a living cell or cell-lysate. [[Experiments_main|Read more]]<br />
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= Results =<br />
We ...blablabla<br />
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Text that will present our results...<br />
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= thing to move =<br />
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'''bioLOGICS: Logical RNA-Devices Enabling BioBrick-Network Formation'''<br />
'''Abstract'''<br />
Among the goals of iGEM is the creation of synthetic biological parts and their utilization to achieve novel features and behavior in biological systems. The emphasis of our project is put on this latter, "systems" aspect of iGEM. More precisely, we aim at the development and experimental demonstration of a scalable approach for the realization of logical functions in vivo.<br />
<br />
By developing a computational biological network based on RNA logical devices we will offer everyone the opportunity to 'program' their own cells with individual AND/OR/NOT connections between BioBricks of their choice. Thereby, BioBricks can finally fulfill their original assignment as biological parts that can be connected in many different ways. We will achieve this by engineering simple and easy-to-handle switches based on predictable RNA/RNA-interactions regulating transcriptional termination. These switches represent a complete set of logical functions and are capable of forming arbitrarily complex networks.<br />
<br />
== The Experiments ==<br />
===Fluorescent proteins as reporter===<br />
<div align="justify"><br />
Our initial idea to prove our concept of antitermination was to use flourescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks ''in vivo'' as well as ''in vitro'', the latter using a translation kit based on e.coli lysate. <br>We decided to use the flourescent proteins GFP and RFP, as their spectra do not overlap and we would not measure any FRET. We would use GFP fluorescence as internal control and RFP fluorescence as signal to detect termination/antitermination by our switch we cloned in between the coding sequences of the proteins. Both protein sequences are under the control of one (L-arabinose induced) promoter.<br />
[[Image:TUM2010_gfprfp_schalter_klein.gif|center|our idea]]<br />
<br><br />
When measuring the termination of our BioBricks and the antitermination by their corresponding signal-RNA, we should be able to observe an increasing RFP emission compared to the GFP emission upon induced signal-RNA production in the cells/in the kit:<br><br />
[[Image:TUM2010_Expected_emission_spextra.png|center|our idea]]<br />
Wiith these measurements, it should also be possible to observe differences in efficiency of termination as well as antitermination between our designed switches. <br><br><br />
<br />
===Measurements with the malachite green aptamer as reporter===<br />
A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramaticly (about 3000 times) upon binding of a specific RNA-aptamer.<br><br>#<br />
[[Image:TUM2010_Malachitgruen-2.png|500px|center|our idea]]<br />
---concept to be desribed, as well as literature---<br />
<ref>refs</ref><br />
<br><br><br />
To study the switches on the transcriptional level gives the advantage, that we would have less interferences and possible artefacts. Also, we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems.<br><br />
<br />
[[Image:TUM2010_Malachit_emission.png|200px|thumb|left|Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA]]We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.<br>Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level.<br />
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<br><br><br><br><br><br><br><br><br><br><br />
<br />
Results <br />
==Flourescent proteins==<br />
Unfortunatly, we had to change the reporter construct two times during our experiments as several problems occured in our measurements:<br><br><br />
<br />
===First Try: based on the measurement plasmid pSB1A10===<br />
At the beginning, we decided to use the reporter plasmid [http://partsregistry.org/Part:pSB1A10 pSB1A10] from the registry. It consists of the fluorescent proteins eGFP and mRFP1. Each sequence includes a ribosome binding site and a stop-codon; the two genes are divided by a cloning side including the BioBrick cleavage sites.[[Image:ScreeningPlasmid1.0.PNG|300px|thumb|right|pSB1A10]]<br> In front of the eGFP sequence, the plasmid includes an arabinose-inducable promoter. The plasmid also contains an ampicilline resistence.<br><br />
We cloned our switches into the cloning site of the measurement plasmid and used an empty cloning site as control; our signal-RNAs we cloned into the [http://partsregistry.org/Part:pSB1K3 pSB1K3] vector, together with the BioBricks R0011 (Lac promoter) and B0014 (double terminator of transcription). Afterwards, we cut pSB1K3 with Aat2 and Pst1 and pSB1A10 with Nsi1 and Aat2 and ligated those fragments of each plasmid that contained our Bricks to get a Monsterplasmid.<br>We had to do so, as both plasmids contain the same ori mechanism. In addition, having both the switch and the signal RNA transcribed from the same plasmid gives us a high local concentration of the signal, once its transcription is induced.<br><br />
[[Image:TUM2010_Messplasmid_nr1.png|left|the measurement plasmid]]<br />
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We transformed BL21(DE3) cells with the plasmid. We set up cultures, induced the arabinose promoter and measured the GFP and mRFP1 excitation/emission spectra within time.<br><br><br />
Unfortunatly, we were ot able to detect any RFP signal, not even in the positive control with no switch in between the GFP/RFP sequences.<br><br><br />
From these experiments, we concluded, that the mRNA of the RFP variant used was instable and rapidly degraded by RNases, so the RFP was not synthesized in the cells. This was also the conclusion from XXX...<br />
<br><br><br />
As solution to this, we decided to design a measurement plasmid ourselves:<br><br />
<hr width="300"><br />
<br><br />
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===Second Try: A measurement plasmid of our own design===<br />
To design our own plasmid to overcome the problems that occurred in our first try gave us tghe possibility to overcome several other problems:<br><br />
#<br />
#<br />
#<br />
[[Image:TUM2010_Construct_no2.png|400px|our construct|center]]<br />
<br />
===Third Try: One promoter for each protein===<br />
We decided to use the measuremnt plasmid we developed in our second try but to clone another L-arabinose induced promoter into the plasmid, in front of our switch followed by the mCherry sequence.<br><br />
<br>[[Image:TUM2010_Construct_no3.png|left|400px|Construct #3]]In this way, we still can use GFP fluorescence as internal control, because both protein transcription is under the control of a promoter of identical design.<br />
Though we are still not able to tell exactly why our previous measurements did not work, but with this construct we measured the first time fluorescence of the mCherry protein in our positive control.<br />
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{{:Team:TU_Munich/Templates/End}}</div>Hartlmuellerhttp://2010.igem.org/Team:TU_Munich/ProjectTeam:TU Munich/Project2010-10-28T03:08:32Z<p>Hartlmueller: /* Outlook */</p>
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<center><font size="5pt" color="#000000">'''bioLOGICS'''</font><font size="4pt" color="#000000">: Logical RNA-Devices Enabling BioBrick-Network Formation</font></center><hr color="black"><br><br />
= Vision=<br />
<br />
Until today, 13.628 biobrick sequences<sup>[[Team:TU_Munich/Project#ref1|&#91;1&#93;]]</sup> have been submitted to partsregistry, thereof 102 reporter units and 12 signaling bricks.<br />
Since then, people are trying to arrange these single biological building blocks in such a manner that allows producing special biotechnological products (metabolic engineering), developing biological sensory circuits (biosensors) and even giving microorganisms the ability to react on multiple environmental factors and serve both as disease indicator and drug. These examples and further promising ideas were implemented on previous iGEM-competitions.<sup>[[Team:TU_Munich/Project#ref2|&#91;2&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref3|&#91;3&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref4|&#91;4&#93;]]</sup> <br><br><br />
The idea of combining the outcome of several iGEM competitions to construct complex synthetic biological systems falls at the last hurdle - the fact, that each team uses a different principle how to access and functionally connect the respectively used biobricks. For example, it is a major challenge to create a system that uses several sensoring BioBricks from different iGEM-teams which in turn regulates reportering BioBricks from various teams. In order to combine and fully take advantage of these promising projects, our vision is to develop an adapter that allows interconnecting arbitrary biobricks on a functional level. Such a system easily allows to setup sensor-reporter circuits and interconnect them to complete biological chips... A further step towards artificial cells.<br><br><br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, the above adapter has to meet the following requirements:<br />
*'''Universality'''<br />
:The adapter has to be compatible to as many BioBricks as possible. This objective will guarantee that a large number of BioBricks can be connected.<br />
*'''Scalability'''<br />
:Once the basic design of the system is established, the construction of the system is supposed to be automated in silico. This way it will be possible to create an adapter connecting a large amount of BioBricks.<br />
*'''Biological orthogonality'''<br />
:Interference with cellular components has to be as low as possible in order to avoid unwanted and perturbing side effects.<br />
*'''Logic'''<br />
:The adapter is supposed to not only associate different BioBricks, but to functionally connect BioBricks in a precisely determined manner (including operations such as AND/OR/NOT).<br />
<br><br />
Several biological logic units, devices and circuits have been developed so far<sup>[[Team:TU_Munich/Project#ref5|&#91;5&#93;]]</sup>, but to our knowledge, none was shown to meet all requirements listed above.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
=Implementation=<br />
To functionally connect BioBricks, there are several possibilities including genetic switches, riboswitches and direct protein-protein interactions. We investigated several hypothetically principles, and decided to focus our practical work on the development of a RNA-RNA interaction-based switch. These switches are capable of changing between two states, a state of antitermination and termination, and make use of highly-specific RNA-RNA interaction. In principle such a switch can fulfill all requirements mentioned previously. The following text clarifies how these switches work in detail.<br />
==How to connect BioBricks==<br />
Our adapter is a system, that activates or disables BioBricks (output BioBricks) in response to the presence of other Biobricks (input Biobricks). Our approach uses a molecular network to put this into practice and consists of four major elements:<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
<br><br />
{|<br />
|-<br />
|[[Image:Networks.png|center|thumb|730px|The general principle how different inputs can be connect to various outputs. For details see text.<br>Inputs (such as proteins or small molecules) are indicated on the left side. blue lines represent transmitter molecules whereas organe lines present logic gates. The type of logic gate is indicated. Green lines indicate transmitter RNA that can function as mRNA and consequently generate any output gene (indicated on the very right).]]<br />
|}<br />
In order to connect different BioBricks, our network requires four major types of components:<br />
*Input elements<br />
*Transmitter molecules<br />
*Logic gates<br />
*Output elements<br />
<br />
{{:Team:TU_Munich/Templates/InfoBoxStart}}'''Computer vs. molecular network - and our approach'''<br><br />
Logic gates in a molecular network are often compared to transistors used in a computer, where billions of transistors are incorporated<sup>[[Team:TU_Munich/Project#ref7|&#91;7&#93;]]</sup>. The main advantage on a computer chip is, all transistors share the same functional principle, and only the way connecting them in a special sequence allows specific addressing of only a subset of other transistors by an input. However, spatially fixed connections of molecular logic gates are not possible in a living cell. The "wiring" within a cell relies on the specific interaction between transmitter molecule and their corresponding logic gates, for example implemented by protein-protein/ligand-protein interactions or specific ligand-riboswitch interactions.<sup>[[Team:TU_Munich/Project#ref8|&#91;8&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref9|&#91;9&#93;]]</sup> As a result, in a cell, each occurring logic gate ("transistor") has to be different, at least in a special recognition site<sup>[[Team:TU_Munich/Project#ref10|&#91;10&#93;]]</sup> - for example like different transcription factors, recognizing different DNA-sites. Thanks to evolution, nature easily can invent a new transistor for each task - science achieves this only on a limited scale, and producing synthetic molecular logic gates artificially by either rational or evolutionary protein or riboswitch engineering, is limited to small circuits so far<sup>[[Team:TU_Munich/Project#ref11|&#91;11&#93;]]</sup>. Our project aims to establish a molecular switch as close as possible to a electronic transistor, thus sharing the same functional principle for all logic gates. At the same time, we want to design a easily exchangeable recognition site, which can individually be designed by everyone! {{:Team:TU_Munich/Templates/InfoBoxEnd}}<br />
<br />
These elements can be combined to build up a molecular network (see illustration). Each input molecule (such as a BioBrick) produces a unique transmitter molecule. All transmitters belong to the same type of molecule and share a common design. However, each transmitter molecule can only interact and activate a certain subset of logic gates. In other words, logic gates have to recognize as well as bind the corresponding transmitter molecules and are capable of producing a new output transmitter molecule. Depending on the type of the logic gate (AND, OR or NOT<sup>[[Team:TU_Munich/Project#ref6|&#91;6&#93;]]</sup>), an output transmitter is only created if both input transmitter molecules are present (AND), at least one of two input transmitters is present (OR) or if no input transmitter is present at all (NOT). Once a logic gate has produced a new output transmitter, these transmitters can in turn address another subset ("layer") of logic gates. In theory many layers of logic gates can be connected this way allowing the creation of large networks. Until this step, various transmitter molecules might have been produced. But in order to create a Biobrick output, the last layer of logic gates finally generates transmitter molecules that will not active logic gates, but will rather interact with the cell metabolism to produce a BioBrick response. In other words, the last layer of transmitter molecules is capable of regulating BioBrick formation.<br />
<br />
<br />
Summarizing, the network establishes a connection between input BioBricks and output BioBricks in a functional manner.<br />
Having addressed the basic layout of the molecular network, the next step is to determine what type of molecules can perform the required functions. We decided to use RNA, both as transmitter molecules and for constructing logic gates. Several advantages result from the utilization of RNA as the central element:<br />
*During the last years, many Biobricks were designed that are sensitive to various chemicals and substances. These BioBricks often function as a transcription factor that binds to a specific DNA sequence and consequently would be capable to produce a specific transmitter RNA molecule. Thus, in principle each BioBrick which involves transcription can be integrated in our network.<br />
*Since all logic gates are capable of producing transmitter RNA, they can also produce functional mRNA encoding any protein. This means, each BioBrick consisting of protein or RNA can be produced as an output of our network.<br />
*If RNA forms both, the transmitter molecule and the logic gates, they can specifically interact by RNA-RNA interaction, which is highly predictable compared to protein interactions. This allows to generate a library of transmitters and gates ''in silico''. Such a library is essential for the creation of large networks.<br />
*RNA production is fast and energy saving for a cell. Consequently, operating a network that only produces RNA rather than proteins will also be faster and more efficient for the host cell. Since our logic gates are based on transcription, translation and resource consuming protein production will only be required at the very last step. <br />
*As the half-time of RNA can be rather short, transmitter RNA will not accumulate within the cell and it is therefore less likely for the system to become saturated.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Design and functional principle of logic gates==<br />
The concept introduced above provides a framework that can potentially serve as an universal adapter between different BioBricks. However, the [[Team:TU_Munich/Glossary#logic gate | logic gates]] have not been specified more precisely so far. This will be done in the following section.<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, our logic gates are to possess the following characteristics:<br />
*Logic gates, such as AND, OR and NOT, have to be implemented by RNA-interaction based principles (see [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]).<br />
*All logic gates have to recognize their corresponding [[Team:TU_Munich/Glossary#Transmitter (bioLOGICS)| transmitter RNAs]] and, in response, produce an output transmitter molecule.<br />
*Logic gates should follow a basic design rule, in such a way, that their creation can be automated ''in silico''.<br />
*The response efficiency of a logic gate toward a transmitter molecule should be comparable for all logic gates to provide calculable robustness and sensitivity. This will ensure comparable molecular concentrations and functionality of large networks.<br />
*The system has to be designed for ''in vivo'' utilization at the first place. As a reference we always assumed a temperature of 37 °C and an ''E. coli'' environment.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}} <br />
In order to build logic gates for our bioLOGICS system we will first create a simple switch. A switch can be activated by one transmitter RNA and produce an output transmitter RNA. In contrast to a logic gate, a switch does not perform logic operations. However by combining switches, logic gates can be created. The following text will first describe how the developed switch works and secondly, how logic gates such as AND/OR/NOT can be created using these switches.<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Read more{{:Team:TU Munich/Templates/ToggleBoxStart2}}<br />
[[Image:toggle_switch.png|500px|thumb|center|id="hideOnReadMore"|'''A''' The basic structure of a bioLOGICS switch (left) and a transmitter molecule (right).<br>'''B'''The process of switching. See the text in the close-by "Read more" section for details.<br>Rectangles present the composition of our functional units on the level of DNA. Fringed lines represent RNA produced by RNA polymerase. The stem loop structure depicts the switchable terminator. Terminator and target site are illustrated in blue and turquoise, respectively. Recognition sites are indicated in different colors, in this case red for the input transmitter and green for the output transmitter.Each switch and or later logical unit has to be flanked by a promotor and another constitutive terminator, to allow RNA-production by RNA-polymerase in a proper way. ]]<br />
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===Switch===<br />
[[Image:TUM2010_switch-and-transmitter.jpg|550px|right|thumb|The basic strcutrue of a switch (left) and a transmitter RNA (right). See text for details.]]<br />
Roughly speaking, a switch can be regarded as an enhanced switchable transcriptional terminator. The enhancement can be described easier by dividing a switch into its functional components: <br />
*'''Target site'''<br><br />
:The target site is the functional core element of our switches, allowing a shift between an "on" and "off" state. Since we work on the level of RNA-production (transcription), a "switchable" transcriptional terminator is suitable for this purpose. By allowing or preventing formation of a transcriptional terminator, that is by switching between termination and antitermination it is possible to represent an "off" and an "on" state, respectively. Therefore, the target site is the 5' ending of the terminator and is required for a stable terminator formation. It should be noted that this principle was also observed in nature.<br />
:To highlight and illustrate the functional principle of our switches, only the part of the terminator which is involved in interacting with a transmitter molecule and which is responsible for shifting between "on" and "off" state is called target site. The remaining terminator sequence is called terminator in the following, even if both, target site and terminator build up the terminator structure occurring in nature. <br />
:The important aspect of our switches is the fact that all switches will hold the same identical target site. Therefore having found one functional "switchable" terminator, will allow almost unlimited upscaling since this terminator can be used for a large library of switches. This is the main difference to previous works done on this field, which always required developing a new shifting principle for each switch.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup> Beside this scalability, this principle provides a comparable on/off shifting rate (responds function) for all switches, avoiding complex fine tuning of molecular networks.<br />
:To sum it up, the target site, allows to switch between an "on" and "off" state. But so far, the switch is not capable of performing specific interaction with transmitter molecules. This is where the recognition site comes into play.<br />
*'''Recognition site'''<br />
:The recognition site defines which transmitter molecule can actually interact with the switch. Therefore, a unique recognition site is generated for each switch and is positioned right upstream of the target site. In principle the recognition can be any random sequence as long as it remains unique within the molecular network.<br />
Summing up, the recognition site allows a specific interaction between switches and transmitter molecules. Once this interaction is formed, an interaction between the transmitter and the target will actually switch the state of the terminator. This allows the specific arrangement and interconnection of numerous of these switches by transmitter molecules, without changing the target site. Comparable to wires connecting many identical transistors, our target site remains the same.<br />
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===Transmitter RNA´s===<br />
As desccribed above, transmitter RNAs are the input and output of bioLOGICS switches (compare [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]). These transmitters are short ssRNA molecules representing the "trigger" to shift switches between the "on" and "off" state. To fulfill this role, they need to posses the following properties:<br />
*A transmitter may only interact with certain switches. That is, a transmitter has to find the corresponding recognition site of a switch.<br />
*Once an interaction is established between a transmitter and a switch, a transmitter has to be capable of changing the secondary structure of a terminator and thus cause antitermination.<br />
Again, these two properties are fulfilled by two components of the transmitter:<br />
*'''Identity site'''<br />
:This site is capable of forcing an interaction between the transmitter and the switch. Therefore it is complementary to the recognition site of this switch. As the recognition site is unique within a network, so is the identity site. However, the single identity site is not capable of changing the state of the switch. That is were the trigger site comes into play.<br />
*'''Trigger site'''<br />
:Once an interaction is created by the identity site, the trigger site is capable of actually shifting the switch since it is complementary to the target site of the switch. To fulfill this role, it is placed upstream at the 5' end of the identity site. As the target site is the same for all switches, the trigger site is the same for all signals. Therefore it is important, that similar to the identity site, a trigger site cannot function on its own. That is, a single trigger site cannot shift the state of a switch without the help of an identity site.<br />
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Summing up, we applied the principle introduced for the switches to the transmitter molecules. In contrast to previous approaches on this field <sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup>, we introduced the described synthetic trigger site in such a manner that it is not able to change the state of the terminator on its own, but only in combination with the identity site. So the challenge is to arrange and optimize these elementary building blocks thermodynamically, that a trigger site is only able to switch in combination with its respective identity site. This was done by ''in silico'' design using [[TU Munich/Glossary#NUPACK| NUPACK]], presented in section [[TU Munich/Modeling#in silico design based on thermodynamic calculations| in silico design]].<br />
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===Putting it all together: the switching process===<br />
[[Image:TUM2010_switching-process.jpg|550px|right|thumb|The basic structure of a switch (left) and a transmitter RNA (right). See text for details.]]The functional principle of the designed switches is illustrated in the figure. The switch is positioned on DNA upstream of a desired output transmitter. So in the absence of a triggering transmitter molecule, transcription will be canceled by the formation of a RNA stem loop in the nascent RNA-chain. This will cause the RNA polymerase to stop transcription and fall off the DNA and consequently no output RNA will be produced. This process only relies on [[Team:TU_Munich/Glossary#Termination| rho-independent termination]].<br />
On the other hand, in the presence of a [[Team:TU_Munich/Project#RNA_transmitters | input transmitter]], this small functional RNA inhibits the stem loop formation by complementary base-pairing and hence avoids termination of transcription. In detail, the identity site (red part on transmitter) binds the recognition site (red part on switch) and serves as [[Team:TU_Munich/Glossary#Toehold|toehold]], which will thermodynamically allow the trigger site (turquoise part on transmitter) to perform a [[Team:TU_Munich/Glossary#Strand Displacement| strand displacement]] and open up the stem loop structure. Consequently the polymerase can read all the way through and form the output RNA.<br>Summing up, we use this concept to create a switch that can be toggled by a transmitter RNA molecule and in response, is able to produce another transmitter RNA.<br />
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===From switches towards bioLOGICS logic gates===<br />
As described, each switch can be accessed by a specific RNA-transmitter molecule, illustrating the input. In turn, another RNA-transmitter molecule will be produced if the switch shifts its state. This output transmitter of one switch can serve as input transmitter for the next switch by meaningful selection and design of the respective recognition sites. This easily allows arranging several switches in specific sequences and faulty wiring - the corner stone of a logical network.<br />
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To ease the building of logical networks, applying mathematical logics, e.g. Boolean logics like in computational science would be worthwhile. It is possible to establish general Boolean operators with our switches and thus build "logical modules". <br />
Since AND/OR/NOT are the most simple logic operations which can be implemented with the presented switches, and all remaining operations can be expressed by these three operators according to laws of boolean logics, we exemplary designed them.<br />
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{|<br />
|-<br />
| *AND consists of a parallel circuit of two switches<br />
|-<br />
|[[Image:AND2.png|500px|thumb|center]] <br />
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| *OR is implemented by connecting two switches in series<br />
|-<br />
|[[Image:OR2.png|500px|thumb|center]]<br />
|-<br />
| *NOT is more complex to explain. In principle, it consists only of one switch which contains its respective signal molecule intrinsic, so via intramolecular interaction, antitermination is the initial state. The signal is intrinsically of the same components as usual to allow interconnection with other logic gates.<br />
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|[[Image:NOT2.png|500px|thumb|center]]<br />
|}<br />
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==Network construction==<br />
Designing complex biological networks based on either traditional protein engineering or our new bioLOGICS is still a complex task. We developed a software which allows the fast construction of a bioLOGICS based networks. <br><br />
To read more about this, look at our [https://2010.igem.org/Team:TU_Munich/Software Software page]<br />
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=Our Objective=<br />
Putting the implementation described above into practice, will be a major challenge. For this year's iGEM competition our goal is to do the first step: design and build a switch that can be toggled by a RNA molecule. To be precise, we want to apply the design rules of our switch to modify a transcription terminator in such a way that it interacts with a second RNA molecule and, as a result, is no longer capable of forming a stem loop. This objective will require intensive ''in silico'' designing and modeling of switches based on different terminators and their corresponding transmitters. In connection to this theoretical part, we also have to test and verify the switches. For this step, we establish custom-made assays, ''in vitro'' and ''in vivo''.<br />
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Once the objective mentioned above is accomplished, these basic RNA/RNA-interactions have to be modified in such a manner that the described identity/trigger site pattern for the transmitter and the complementary recognition/target site switch composition has to be established. The most important requirement is to is to optimize these modules that the transmitter is only able to switches specifically, meaning only in the presence of both, identity AND trigger site. <br />
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Once the objective mentioned above is accomplished, the creation of an OR gate will be rather simple since it only requires two switches. However the creation of an AND or NOT gate and optimizing the logic gates to improve their responds function will remain the goal of future work. Also the creation of small networks and the correct integration of BioBricks as input and output molecules will be future challenges. Furthermore, we wanted to rather focus on the development and the testing of our structural design of the switches, rather than developing a variety of new BioBricks.<br />
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==''In silico'' design==<br />
As described above, our switches are based on certain design rules. However, there still are different structural parameters that need to be tested and optimized (length of recognition site and target site, choice of terminator, etc.).<br />
We used [[Team:TU_Munich/Project#in silico design |''in silico'' design]] and [[Team:TU_Munich/Modeling| modeling]]) to test different parameters. Furthermore we tried to use the [[Team:TU_Munich/Glossary#Antitermination|antitermination principle]] observed in nature, such as [[Team:TU_Munich/Glossary#Attenuation| attenuation]] in ''E. coli'' or [[Team:TU_Munich/Glossary#Tiny Abortive RNA´s| tiny abortive RNA´s]] of T7-phage.<br />
==Evaluation and Measurements==<br />
To evaluate the functionality of our molecular switches, we first had to establish several assays. Therefore, we improved an existing [[Team:TU_Munich/Lab#In vivo Measurements |''in vivo'' assay]] and developed an [[Team:TU_Munich/Lab#In vitro Transcription | ''in vitro'' assay]] for this purpose. For more information please refer to the [[Team:TU_Munich/Lab | lab]] section.<br />
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Summarizing, the main challenges are <br />
* to find a suitable terminator construct and design a complementary trigger unit, which is only functional in combination with a specificity site - meaning an optimization of the '''thermodynamically parameters''' (see[[Team:TU_Munich/Project#in silico design| in silico design]])<br />
* to investigate whether the transmitter/switch interaction reaction is on a timescale to be competitive to terminator formation - meaning an comparison of '''kinetic parameters''' (see [[Team:TU_Munich/Modeling|Modeling page]])<br />
* to proof antitermination can be also be caused by synthetically RNA-interaction (see [[Team:TU_Munich/Glossary#Antitermination| Antitermination in nature]] and [[Team:TU_Munich/Project#Results| ''in vivo'' and ''in vitro'' measurements]] )<br />
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=Results=<br />
Every network starts with a basic unit. While our declared aim is to enable networks allowing fine-tuning of gene expression beyond the regular on/off, exploring such an on/off switch/signal pair is the first step towards a functional network. We constructed several units and tested their efficiency, robustness and reproducibility ''in vivo'', ''in vitro'' and ''in silico''. Furthermore we developed a software which allows easy constructions of networks based on our designed logic gates. Conclusive elaboration of a few first RNA-based logic units is the major contribution of our iGEM team.<br />
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==in silico Design of Switching and Trigger Unit==<br />
As described on the [[Team:TU_Munich/Project | project]] page, one key aspect of our switches is the idea, that a [[Team:TU_Munich/Glossary#Transmitter_(bioLOGICS) | RNA transmitter molecule]] is capable to shift the state of a switch only if its [[Team:TU_Munich/Glossary#Trigger_Site_(bioLOGICS) | trigger site]] is present and its [[Team:TU_Munich/Glossary#Identity_Site_(bioLOGICS) | identity site]] corresponds to the [[Team:TU_Munich/Glossary#Recognition_Site_(bioLOGICS) | recognition site]] of the [[Team:TU_Munich/Glossary#Switch_(bioLOGICS) | switch]]. We successfully constructed several switches and their corresponding transmitter RNA ''in silico'' on a thermodynamical basis. We modified different transcriptional terminators in such a way, that the formation of the terminator was prevented by a transmitter molecule. As desired, this only occured if the transmitter molecule contained both, a trigger and an identity site. Analogously, we were able to design and verify a NOT gate using the same thermodynical approach.<br />
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==Diffusion and RNA Folding Dynamics==<br />
We estimated the diffusion time for our constructs and modeled the folding dynamics of our bioLOGICS switches including the switching process with a stochastic RNA folding program. We were able to provide better insight in their folding dynamics and proved that they are able to interrupt termination. We also optimized the switches and the corresponding signals. Furthermore, we combined the switches what resulted in a logic gate. See our [[Team:TU Munich/Modeling|Modeling page]] for further details.<br />
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==''in vivo'' Functionality Screening==<br />
Since our logic gates are intended to function in living cells, ''in vivo'' measurements were essential. In a set of experiments we concentrated on two different switches based on known [[Team:TU_Munich/Glossary#Attenuation|attenuators]] from nature: the [[Team:TU_Munich/Modeling#Switch|HisTerm]] and [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Focusing on fluorescent proteins for quantifiable input and output we designed a functional and robust screening system. For greater detail see [[Team:TU_Munich/Lab#Experiment_Design|Experimental Design]]. Unfortunately, setting up a working screening system failed twice. Only in redesigning and improving the screening plasmid pSB1A10 we succeeded, but lost precious time.<br />
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Ultimately, the two switches displayed remarkable differences in their terminator efficiency, but neither of them responded to their corresponding signal. However, screening one transmitter signal does not disprove the basic working principle of our system. Limited by time, we hope for future teams to take up our work and to use our improved test system that we submitted to the parts registry, for performing successful in vivo measurement.<br />
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Considering the high complexity of ''in vivo'' measurements compared to other experimental challenges, a robust and easy to handle test system for [[Team:TU_Munich/Glossary#PoPS-based devices| PoPS-based devices]] is desirable. As described in [[Team:TU_Munich/Lab#Experiment_Design|Experimental design]], we used fluorescent proteins: RFP or mCherry to measure the amount of produced output and eGFP for normalization. Our first attempt, using the screening plasmid pSB1A10, yielded no interpretable results. Switching the fluorescent protein to mCherry did not work either, but after several experimental setups we determined a transcriptional problem causing no reporter protein expression regardless of the inserted part. Thereby we demonstrated the screening plasmid pSB1A10 to be [[Team:TU_Munich/Biobricks#Falsification| malfunctioning]]. <br />
Finally a new design based on pSB1A10 lead to a functional and robust screening system (compare [[Team:TU_Munich/Parts#Screening system: Backbone BBa_K494001| Screening system: Backbone BBa_K494001]]). A second promoter with identical induction properties inside the BioBrick cloning site enforces transcription of the PoPS-based device and the mCherry output.<br />
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Exemplary, the graph below on the right shows the positive control, induced and uninduced at OD<sub>600</sub>=0.7 followed by 16 h incubation at 25 °C. Clearly visible are eGFP and mCherry fluorescence in the induced samples. The uninduced control showed no fluorescence at all, demonstrating the PBad promoter to be tight and providing very low basal transcription, what is a major advantage for the screening system. This newly designed screening approach renders the characterization of PoPS-based devices in general and switches in particular easy and robust. The low basal transcription furthermore fulfills one of the most important requirements for the designed switches, since output transmitters may only be produced in presence of an input transmitter. This helps to avoid strong "background" noise, which would extremely harden the successful interconnection of several switches. <br />
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[[Image:TUM2010_PosControlklein.JPG|200px||thumb|left|Bacteria containing positive control]]<br />
[[Image:TUM2010_graphPosControl1.png|355px|thumb|center|Emission spectra of induced (green/red) and uninduced(black) positive control BBa_K494002 ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
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Due to the time limitations of the iGEM completion we had to focus our efforts on few switches after designing the screening system. Relying on the functionality of systems occurring in nature, we choose the [[Team:TU_Munich/Modeling#Switch|HisTerm]] as well as the [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Both switches are based on known natural [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|attenuators]]. Testing synthetic and none-naturally switchable terminators in vivo are goals for future work.<br />
Delorme et al. reported the His-Terminator to be a remarkable effective Terminator with more than 99% termination efficiency.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup> The exemplary measurement below on the right confirms the high terminator efficiency. In fact, we could not detect any mCherry fluorescence in any cells containing the [[Team:TU_Munich/Modeling#Switch|HisTerm]]. Even induction of the corresponding signal transmitter RNA via IPTG did not alter the Terminator efficiency. Again time was the limiting factor and prevented us from testing more than one corresponding transmitter, although the [[Team:TU_Munich/Modeling| Modeling]] highly suggested the necessarily of finding an optimized transmitter length. Thus, the results are insufficient either to prove or to disprove the functionality of the [[Team:TU_Munich/Modeling#Switch|HisTerm]] or our concept in general.<br />
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[[Image:TUM2010_HisSwitchklein.JPG|200px|thumb|left|Bacteria containing HisTerm]][[Image:TUM2010_HisSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing HisTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
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Attaining only 90% terminator efficiency, the natural Trp [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|Attenuator]] is known be less effective than the [[Team:TU_Munich/Modeling#Switch|HisTerm]].<sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup> The graph on the right depicts our designed [[Team:TU_Munich/Modeling#Switch|TrpTerm]] characteristic efficiency of about 40 %, notably below the natural standard. Allowing 60% transcription in the “off” state excludes the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] from possible candidates for a scalable network of logic gates, due to the mentioned required "yes or no" function (see [[Team:TU_Munich/Project#Implementation| Implementation and how to connect Biobricks]]). Thus the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] is inoperative as intended, but may still be useful in other contexts. Similar to the [[Team:TU_Munich/Modeling#Switch|HisTerm]], the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] also did not react to the induction of the corresponding signal. Under circumstances, termination efficiencies altered by the transmitter are on a low range and not resolvable within observed 40% basal transcription. <br />
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[[Image:TUM2010_TrpSwitchklein.JPG|200px|thumb|left|Bacteria containing TrpTerm]][[Image:TUM2010_TrpSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing TrpTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
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Making use of our improved screening system we also carried out some ''in vivo'' kinetic measurements in addition to the end-point measurements above. In contrast to the ''in vitro'' experiments we did not obtain significant results for the characterization of our switches. As the switching process is many times faster than protein synthesis our ''in vivo'' kinetics include the synthesis of mCherry as well as its maturation. Therefore we centered our attention on end-point experiments. For more information browse the [[Team:TU_Munich/Lab#Lab_Book|lab book]]. <br><br />
Considering our ''in vivo'' measurements, neither of the tested switches showed any effect regarding the signal induction. But due to the small number of tested switches and signals this can hardly be regarded as disprove of concept. In particular in light of the recent findings by Sooncheol proving antitermination in principle using a T7 system.<sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup><br />
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==''in vitro'' Screening==<br />
To minimize the amount of disturbing factors we decided to countercheck our ''in vivo'' results with a set of ''in vitro'' measurements. While the ''in vitro'' systems are no doubt much less complex than living cells, the work with these set-ups proved to be quite as difficult.<br />
Just as with the ''in vivo'' measurements we could prove our switching system neither right nor wrong, leaving enough work for future iGEM teams.<br />
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===''in vitro translation''===<br />
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Beside optimization of the reporter proteins in use, the major problem occuring in the experiments was the low capacity of the kit. The signal intensity was very low, which made it difficult to observe any signal intensity alterations, so no conclusion could be drawn from these measurements.<br />
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===''in vitro'' transcription===<br />
We used two completely independent ''in vitro'' systems: Using ''E.coli'' RNA Polymerase we analyzed the His and Trp switches that had already been tested ''in vivo''. In a second set-up, we used the well-established T7 RNA Polymerase and switch based on the T7 terminator as well as several signal sequences.<br />
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====T7 System====<br />
In contradiction to the results of Kang and coworkers and other groups, in our ''in vitro'' set-up the T7 terminator did not seem to terminate at all. The negative control (Promoter_Terminator_malachite binding aptamer) showed a similar increase in fluorescence as the positive control (Promoter_random sequence_malachite binding aptamer). <br />
[[Image:TUM2010_T7Result1.png|360px||thumb|left|''in vitro'' transcription measurement of T7 terminator with no signal(upper left), nonsense signal (upper right) and two different designed signals (below)]]<br />
[[Image:TUM2010_T7Result3.png|360px||thumb|right|''in vitro'' transcription measurement of positive control(upper left and T7 terminator with three different designed signals (remaining traces)]]<br />
Furthermore denaturing Polyacrylamide Gel Electrophoresis (PAGE) confirmed that there was no observeable termination of transcription. The addition of a signaling sequence led to a significantly lower increase in fluorescence, which can be attributed to the fact that both DNA sequences, switch and signal, compete for RNA Polymerases.<br />
However, there is almost no difference between the designed signals and random sequences, which is not a big surprise since there can be no antitermination if the terminator itself does not work.<br><br />
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Possible explanations for the contradiction between our results and those of Kang and coworkers might be the experimental set-up and the RNA Polymerases we used. Different variants of T7 RNA Polymerase might respond in different ways to terminator structures, and the termination might be influenced by the presence or absence of cofactors, depending on the purification methods used in producing the Polymerase.<br><br><br />
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This set-up offers a lot of possible experiments for the future, which we would have loved to conduct with a just a bit more time...<br />
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====''E.coli'' System====<br />
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Compared to the T7 System, the ''E. coli'' RPO system produced poor increases in fluorescence, indicating little RNA synthesis. It was shown that the presence of a terminator decreases, as expected, the production of downstream RNA. This result was also confirmed by denaturing PAGE. However, due to the poor changes in fluorescence we were not able to actually characterize the behaviour of our switches ''in vitro'', and the small RNA concentrations did not allow a quantitative interpretation of our gels. A major problem with this method was the low concentration of the ordered Polymerase resulting in a much weaker overall signal as comparable measurements using the T7 Polymerase. <br><br><br />
In future experiments we might try to work with smaller volumes in order to reach higher concentration of RPO and of the synthesized RNA molecules, so measuring in 96 well plate readers might be a good choice. <br />
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==Software==<br />
Although we could not show the full functionality of bioLOGICS in the lab we still want to demonstrate the potential of our approach. Hence we implemented the idea behind our logic gates in a program which illustrates how bioLOGCIS theoretically would allow the construction of complex information processing networks interconnecting BioBricks. For further details take a look at our [[Team:TU Munich/Software|Software page]].<br />
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=References=<br />
<html><a name="ref1"></a></html>[1] http://partsregistry.org/cgi/partsdb/Statistics.cgi<br />
<html><a name="ref2"></a></html>[2] https://2009.igem.org/Team:Imperial_College_London/M1 encapsulation<br />
<html><a name="ref3"></a></html>[3] https://2009.igem.org/Team:TUDelft<br />
<html><a name="ref4"></a></html>[4] https://2008.igem.org/Team:Heidelberg<br />
<html><a name="ref5"></a></html>[5] Maung Nyan Win and Christina D. Smolke, Science Oct. 2008 Vol. 322. no. 5900, pp. 456 - 460<br />
<html><a name="ref6"></a></html>[6] http://en.wikipedia.org/wiki/Logic_gate#Symbols<br />
<html><a name="ref6"></a></html>[7] http://en.wikipedia.org/wiki/Moore's_law<br />
<html><a name="ref6"></a></html>[8] http://en.wikipedia.org/wiki/Protein_interaction<br />
<html><a name="ref6"></a></html>[9] http://en.wikipedia.org/wiki/Riboswitch<br />
<html><a name="ref6"></a></html>[10] http://en.wikipedia.org/wiki/Binding_sites + http://en.wikipedia.org/wiki/Recognition_site<br />
<html><a name="ref6"></a></html>[11] irgend ein damn review über directed evolution and so on<br />
<html><a name="ref12"></a></html>[12] Delorme, Ehrlich and Renault, Regulation of Expression of the Lactococcus lactis Histidine Operon. Journal of Bacteriology, Apr. 1999, p. 2026–2037<br />
<html><a name="ref13"></a></html>[13] Trun and Trempy(2003): Fundamental Bacterial Genetics, Wiley-Blackwell, Chapter 12 <br />
<html><a name="ref14"></a></html>[14]Sooncheol Lee, Huong Minh Nguyen and Changwon Kang, Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator. Nucleic Acids Research, 2010, 1–9<br />
<html><a name="ref6"></a></html>[15] <br />
<html><a name="ref6"></a></html>[16]<br />
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<!-- The idea behind our project is to change the way BioBricks have been used up to now. Over the years, many receptors and signals have been constructed as BioBricks during the annual iGEM competition, but still it is not possible to interconnect these Bricks in a complex biological network resuting in a cell, that is able to respond to its environment giving differenciated responses depending on the input signals. (Beispiel: cambridge hat das gemacht, xx dies, aber eine zelle kann nicht beides...<br><br />
We plan to create biological switches, that can function as locial gates inside a cell. Our switches rely on RNA/RNA-interactions, regulating transcriptional termination. This is a major advance of our concept, as regular switches rely on complex regulation including proteins and/or metabolites. Thus, our switches shall offer a greater robustness and their behaviour should be easier to predict. [[switch|Read more]] (hier sollte noch das hochskalieren erwähnt werden...<br><br />
These switches can further be used to build up a logical network inside a bacterial cell, enabling every scientist to connect as many functionalities (in form of BioBricks) as designated. We plan to offer simulation on each specifically designed network.<br />
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<br><br>Over the years, many teams participating in the iGEM competition spent their time on constructing receptors and systems to detect a certain input that a variety of gorgeous oppurtunities is available so far.[[Image:TUM2010 network.png|thumb|300 px|right|Our visioon: A logic network inside the cell]] Nevertheless, until now it is not possible to link all those functionalities and build up a network giving differenciated responses to several of those input signals, where the molecular response depends on the complex composition of the environment a cell faces. We would like to offer this possibility to everyone.<br />
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The logic network we want to apply will be based on devices, that can be easily upscaled and therefor offer the chance to build networks of any wanted complexicity. Our devices rely on pure RNA/RNA interactions and thus their behaviour is well predictable.<br />
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The concept we rely on for our design of RNA-switches is based on the principle of [https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation/ '''attenuation'''].<br />
<br />
= Experiments =<br />
We designed several experiments to test our switches, all of them based on fluorescence measurements. We designed experiment setting for measurements ''in vivo'' as well as ''in vitro''. Our ''in vitro'' measurements relied on two different experiment set-ups. While the first was based on a commercial ''E. coli''-lysate, the latter was reporting on a transcriptional level only, eliminating most of the possible side-effects one could expect in the complex behaviour of a living cell or cell-lysate. [[Experiments_main|Read more]]<br />
<br />
= Results =<br />
We ...blablabla<br />
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= thing to move =<br />
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'''bioLOGICS: Logical RNA-Devices Enabling BioBrick-Network Formation'''<br />
'''Abstract'''<br />
Among the goals of iGEM is the creation of synthetic biological parts and their utilization to achieve novel features and behavior in biological systems. The emphasis of our project is put on this latter, "systems" aspect of iGEM. More precisely, we aim at the development and experimental demonstration of a scalable approach for the realization of logical functions in vivo.<br />
<br />
By developing a computational biological network based on RNA logical devices we will offer everyone the opportunity to 'program' their own cells with individual AND/OR/NOT connections between BioBricks of their choice. Thereby, BioBricks can finally fulfill their original assignment as biological parts that can be connected in many different ways. We will achieve this by engineering simple and easy-to-handle switches based on predictable RNA/RNA-interactions regulating transcriptional termination. These switches represent a complete set of logical functions and are capable of forming arbitrarily complex networks.<br />
<br />
== The Experiments ==<br />
===Fluorescent proteins as reporter===<br />
<div align="justify"><br />
Our initial idea to prove our concept of antitermination was to use flourescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks ''in vivo'' as well as ''in vitro'', the latter using a translation kit based on e.coli lysate. <br>We decided to use the flourescent proteins GFP and RFP, as their spectra do not overlap and we would not measure any FRET. We would use GFP fluorescence as internal control and RFP fluorescence as signal to detect termination/antitermination by our switch we cloned in between the coding sequences of the proteins. Both protein sequences are under the control of one (L-arabinose induced) promoter.<br />
[[Image:TUM2010_gfprfp_schalter_klein.gif|center|our idea]]<br />
<br><br />
When measuring the termination of our BioBricks and the antitermination by their corresponding signal-RNA, we should be able to observe an increasing RFP emission compared to the GFP emission upon induced signal-RNA production in the cells/in the kit:<br><br />
[[Image:TUM2010_Expected_emission_spextra.png|center|our idea]]<br />
Wiith these measurements, it should also be possible to observe differences in efficiency of termination as well as antitermination between our designed switches. <br><br><br />
<br />
===Measurements with the malachite green aptamer as reporter===<br />
A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramaticly (about 3000 times) upon binding of a specific RNA-aptamer.<br><br>#<br />
[[Image:TUM2010_Malachitgruen-2.png|500px|center|our idea]]<br />
---concept to be desribed, as well as literature---<br />
<ref>refs</ref><br />
<br><br><br />
To study the switches on the transcriptional level gives the advantage, that we would have less interferences and possible artefacts. Also, we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems.<br><br />
<br />
[[Image:TUM2010_Malachit_emission.png|200px|thumb|left|Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA]]We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.<br>Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
Results <br />
==Flourescent proteins==<br />
Unfortunatly, we had to change the reporter construct two times during our experiments as several problems occured in our measurements:<br><br><br />
<br />
===First Try: based on the measurement plasmid pSB1A10===<br />
At the beginning, we decided to use the reporter plasmid [http://partsregistry.org/Part:pSB1A10 pSB1A10] from the registry. It consists of the fluorescent proteins eGFP and mRFP1. Each sequence includes a ribosome binding site and a stop-codon; the two genes are divided by a cloning side including the BioBrick cleavage sites.[[Image:ScreeningPlasmid1.0.PNG|300px|thumb|right|pSB1A10]]<br> In front of the eGFP sequence, the plasmid includes an arabinose-inducable promoter. The plasmid also contains an ampicilline resistence.<br><br />
We cloned our switches into the cloning site of the measurement plasmid and used an empty cloning site as control; our signal-RNAs we cloned into the [http://partsregistry.org/Part:pSB1K3 pSB1K3] vector, together with the BioBricks R0011 (Lac promoter) and B0014 (double terminator of transcription). Afterwards, we cut pSB1K3 with Aat2 and Pst1 and pSB1A10 with Nsi1 and Aat2 and ligated those fragments of each plasmid that contained our Bricks to get a Monsterplasmid.<br>We had to do so, as both plasmids contain the same ori mechanism. In addition, having both the switch and the signal RNA transcribed from the same plasmid gives us a high local concentration of the signal, once its transcription is induced.<br><br />
[[Image:TUM2010_Messplasmid_nr1.png|left|the measurement plasmid]]<br />
<br />
We transformed BL21(DE3) cells with the plasmid. We set up cultures, induced the arabinose promoter and measured the GFP and mRFP1 excitation/emission spectra within time.<br><br><br />
Unfortunatly, we were ot able to detect any RFP signal, not even in the positive control with no switch in between the GFP/RFP sequences.<br><br><br />
From these experiments, we concluded, that the mRNA of the RFP variant used was instable and rapidly degraded by RNases, so the RFP was not synthesized in the cells. This was also the conclusion from XXX...<br />
<br><br><br />
As solution to this, we decided to design a measurement plasmid ourselves:<br><br />
<hr width="300"><br />
<br><br />
<br />
===Second Try: A measurement plasmid of our own design===<br />
To design our own plasmid to overcome the problems that occurred in our first try gave us tghe possibility to overcome several other problems:<br><br />
#<br />
#<br />
#<br />
[[Image:TUM2010_Construct_no2.png|400px|our construct|center]]<br />
<br />
===Third Try: One promoter for each protein===<br />
We decided to use the measuremnt plasmid we developed in our second try but to clone another L-arabinose induced promoter into the plasmid, in front of our switch followed by the mCherry sequence.<br><br />
<br>[[Image:TUM2010_Construct_no3.png|left|400px|Construct #3]]In this way, we still can use GFP fluorescence as internal control, because both protein transcription is under the control of a promoter of identical design.<br />
Though we are still not able to tell exactly why our previous measurements did not work, but with this construct we measured the first time fluorescence of the mCherry protein in our positive control.<br />
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{{:Team:TU_Munich/Templates/End}}</div>Hartlmuellerhttp://2010.igem.org/Team:TU_Munich/ProjectTeam:TU Munich/Project2010-10-28T03:06:08Z<p>Hartlmueller: /* in silico Design of Switching and Trigger Unit */</p>
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<center><font size="5pt" color="#000000">'''bioLOGICS'''</font><font size="4pt" color="#000000">: Logical RNA-Devices Enabling BioBrick-Network Formation</font></center><hr color="black"><br><br />
= Vision=<br />
<br />
Until today, 13.628 biobrick sequences<sup>[[Team:TU_Munich/Project#ref1|&#91;1&#93;]]</sup> have been submitted to partsregistry, thereof 102 reporter units and 12 signaling bricks.<br />
Since then, people are trying to arrange these single biological building blocks in such a manner that allows producing special biotechnological products (metabolic engineering), developing biological sensory circuits (biosensors) and even giving microorganisms the ability to react on multiple environmental factors and serve both as disease indicator and drug. These examples and further promising ideas were implemented on previous iGEM-competitions.<sup>[[Team:TU_Munich/Project#ref2|&#91;2&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref3|&#91;3&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref4|&#91;4&#93;]]</sup> <br><br><br />
The idea of combining the outcome of several iGEM competitions to construct complex synthetic biological systems falls at the last hurdle - the fact, that each team uses a different principle how to access and functionally connect the respectively used biobricks. For example, it is a major challenge to create a system that uses several sensoring BioBricks from different iGEM-teams which in turn regulates reportering BioBricks from various teams. In order to combine and fully take advantage of these promising projects, our vision is to develop an adapter that allows interconnecting arbitrary biobricks on a functional level. Such a system easily allows to setup sensor-reporter circuits and interconnect them to complete biological chips... A further step towards artificial cells.<br><br><br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, the above adapter has to meet the following requirements:<br />
*'''Universality'''<br />
:The adapter has to be compatible to as many BioBricks as possible. This objective will guarantee that a large number of BioBricks can be connected.<br />
*'''Scalability'''<br />
:Once the basic design of the system is established, the construction of the system is supposed to be automated in silico. This way it will be possible to create an adapter connecting a large amount of BioBricks.<br />
*'''Biological orthogonality'''<br />
:Interference with cellular components has to be as low as possible in order to avoid unwanted and perturbing side effects.<br />
*'''Logic'''<br />
:The adapter is supposed to not only associate different BioBricks, but to functionally connect BioBricks in a precisely determined manner (including operations such as AND/OR/NOT).<br />
<br><br />
Several biological logic units, devices and circuits have been developed so far<sup>[[Team:TU_Munich/Project#ref5|&#91;5&#93;]]</sup>, but to our knowledge, none was shown to meet all requirements listed above.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
=Implementation=<br />
To functionally connect BioBricks, there are several possibilities including genetic switches, riboswitches and direct protein-protein interactions. We investigated several hypothetically principles, and decided to focus our practical work on the development of a RNA-RNA interaction-based switch. These switches are capable of changing between two states, a state of antitermination and termination, and make use of highly-specific RNA-RNA interaction. In principle such a switch can fulfill all requirements mentioned previously. The following text clarifies how these switches work in detail.<br />
==How to connect BioBricks==<br />
Our adapter is a system, that activates or disables BioBricks (output BioBricks) in response to the presence of other Biobricks (input Biobricks). Our approach uses a molecular network to put this into practice and consists of four major elements:<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
<br><br />
{|<br />
|-<br />
|[[Image:Networks.png|center|thumb|730px|The general principle how different inputs can be connect to various outputs. For details see text.<br>Inputs (such as proteins or small molecules) are indicated on the left side. blue lines represent transmitter molecules whereas organe lines present logic gates. The type of logic gate is indicated. Green lines indicate transmitter RNA that can function as mRNA and consequently generate any output gene (indicated on the very right).]]<br />
|}<br />
In order to connect different BioBricks, our network requires four major types of components:<br />
*Input elements<br />
*Transmitter molecules<br />
*Logic gates<br />
*Output elements<br />
<br />
{{:Team:TU_Munich/Templates/InfoBoxStart}}'''Computer vs. molecular network - and our approach'''<br><br />
Logic gates in a molecular network are often compared to transistors used in a computer, where billions of transistors are incorporated<sup>[[Team:TU_Munich/Project#ref7|&#91;7&#93;]]</sup>. The main advantage on a computer chip is, all transistors share the same functional principle, and only the way connecting them in a special sequence allows specific addressing of only a subset of other transistors by an input. However, spatially fixed connections of molecular logic gates are not possible in a living cell. The "wiring" within a cell relies on the specific interaction between transmitter molecule and their corresponding logic gates, for example implemented by protein-protein/ligand-protein interactions or specific ligand-riboswitch interactions.<sup>[[Team:TU_Munich/Project#ref8|&#91;8&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref9|&#91;9&#93;]]</sup> As a result, in a cell, each occurring logic gate ("transistor") has to be different, at least in a special recognition site<sup>[[Team:TU_Munich/Project#ref10|&#91;10&#93;]]</sup> - for example like different transcription factors, recognizing different DNA-sites. Thanks to evolution, nature easily can invent a new transistor for each task - science achieves this only on a limited scale, and producing synthetic molecular logic gates artificially by either rational or evolutionary protein or riboswitch engineering, is limited to small circuits so far<sup>[[Team:TU_Munich/Project#ref11|&#91;11&#93;]]</sup>. Our project aims to establish a molecular switch as close as possible to a electronic transistor, thus sharing the same functional principle for all logic gates. At the same time, we want to design a easily exchangeable recognition site, which can individually be designed by everyone! {{:Team:TU_Munich/Templates/InfoBoxEnd}}<br />
<br />
These elements can be combined to build up a molecular network (see illustration). Each input molecule (such as a BioBrick) produces a unique transmitter molecule. All transmitters belong to the same type of molecule and share a common design. However, each transmitter molecule can only interact and activate a certain subset of logic gates. In other words, logic gates have to recognize as well as bind the corresponding transmitter molecules and are capable of producing a new output transmitter molecule. Depending on the type of the logic gate (AND, OR or NOT<sup>[[Team:TU_Munich/Project#ref6|&#91;6&#93;]]</sup>), an output transmitter is only created if both input transmitter molecules are present (AND), at least one of two input transmitters is present (OR) or if no input transmitter is present at all (NOT). Once a logic gate has produced a new output transmitter, these transmitters can in turn address another subset ("layer") of logic gates. In theory many layers of logic gates can be connected this way allowing the creation of large networks. Until this step, various transmitter molecules might have been produced. But in order to create a Biobrick output, the last layer of logic gates finally generates transmitter molecules that will not active logic gates, but will rather interact with the cell metabolism to produce a BioBrick response. In other words, the last layer of transmitter molecules is capable of regulating BioBrick formation.<br />
<br />
<br />
Summarizing, the network establishes a connection between input BioBricks and output BioBricks in a functional manner.<br />
Having addressed the basic layout of the molecular network, the next step is to determine what type of molecules can perform the required functions. We decided to use RNA, both as transmitter molecules and for constructing logic gates. Several advantages result from the utilization of RNA as the central element:<br />
*During the last years, many Biobricks were designed that are sensitive to various chemicals and substances. These BioBricks often function as a transcription factor that binds to a specific DNA sequence and consequently would be capable to produce a specific transmitter RNA molecule. Thus, in principle each BioBrick which involves transcription can be integrated in our network.<br />
*Since all logic gates are capable of producing transmitter RNA, they can also produce functional mRNA encoding any protein. This means, each BioBrick consisting of protein or RNA can be produced as an output of our network.<br />
*If RNA forms both, the transmitter molecule and the logic gates, they can specifically interact by RNA-RNA interaction, which is highly predictable compared to protein interactions. This allows to generate a library of transmitters and gates ''in silico''. Such a library is essential for the creation of large networks.<br />
*RNA production is fast and energy saving for a cell. Consequently, operating a network that only produces RNA rather than proteins will also be faster and more efficient for the host cell. Since our logic gates are based on transcription, translation and resource consuming protein production will only be required at the very last step. <br />
*As the half-time of RNA can be rather short, transmitter RNA will not accumulate within the cell and it is therefore less likely for the system to become saturated.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Design and functional principle of logic gates==<br />
The concept introduced above provides a framework that can potentially serve as an universal adapter between different BioBricks. However, the [[Team:TU_Munich/Glossary#logic gate | logic gates]] have not been specified more precisely so far. This will be done in the following section.<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, our logic gates are to possess the following characteristics:<br />
*Logic gates, such as AND, OR and NOT, have to be implemented by RNA-interaction based principles (see [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]).<br />
*All logic gates have to recognize their corresponding [[Team:TU_Munich/Glossary#Transmitter (bioLOGICS)| transmitter RNAs]] and, in response, produce an output transmitter molecule.<br />
*Logic gates should follow a basic design rule, in such a way, that their creation can be automated ''in silico''.<br />
*The response efficiency of a logic gate toward a transmitter molecule should be comparable for all logic gates to provide calculable robustness and sensitivity. This will ensure comparable molecular concentrations and functionality of large networks.<br />
*The system has to be designed for ''in vivo'' utilization at the first place. As a reference we always assumed a temperature of 37 °C and an ''E. coli'' environment.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}} <br />
In order to build logic gates for our bioLOGICS system we will first create a simple switch. A switch can be activated by one transmitter RNA and produce an output transmitter RNA. In contrast to a logic gate, a switch does not perform logic operations. However by combining switches, logic gates can be created. The following text will first describe how the developed switch works and secondly, how logic gates such as AND/OR/NOT can be created using these switches.<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Read more{{:Team:TU Munich/Templates/ToggleBoxStart2}}<br />
[[Image:toggle_switch.png|500px|thumb|center|id="hideOnReadMore"|'''A''' The basic structure of a bioLOGICS switch (left) and a transmitter molecule (right).<br>'''B'''The process of switching. See the text in the close-by "Read more" section for details.<br>Rectangles present the composition of our functional units on the level of DNA. Fringed lines represent RNA produced by RNA polymerase. The stem loop structure depicts the switchable terminator. Terminator and target site are illustrated in blue and turquoise, respectively. Recognition sites are indicated in different colors, in this case red for the input transmitter and green for the output transmitter.Each switch and or later logical unit has to be flanked by a promotor and another constitutive terminator, to allow RNA-production by RNA-polymerase in a proper way. ]]<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===Switch===<br />
[[Image:TUM2010_switch-and-transmitter.jpg|550px|right|thumb|The basic strcutrue of a switch (left) and a transmitter RNA (right). See text for details.]]<br />
Roughly speaking, a switch can be regarded as an enhanced switchable transcriptional terminator. The enhancement can be described easier by dividing a switch into its functional components: <br />
*'''Target site'''<br><br />
:The target site is the functional core element of our switches, allowing a shift between an "on" and "off" state. Since we work on the level of RNA-production (transcription), a "switchable" transcriptional terminator is suitable for this purpose. By allowing or preventing formation of a transcriptional terminator, that is by switching between termination and antitermination it is possible to represent an "off" and an "on" state, respectively. Therefore, the target site is the 5' ending of the terminator and is required for a stable terminator formation. It should be noted that this principle was also observed in nature.<br />
:To highlight and illustrate the functional principle of our switches, only the part of the terminator which is involved in interacting with a transmitter molecule and which is responsible for shifting between "on" and "off" state is called target site. The remaining terminator sequence is called terminator in the following, even if both, target site and terminator build up the terminator structure occurring in nature. <br />
:The important aspect of our switches is the fact that all switches will hold the same identical target site. Therefore having found one functional "switchable" terminator, will allow almost unlimited upscaling since this terminator can be used for a large library of switches. This is the main difference to previous works done on this field, which always required developing a new shifting principle for each switch.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup> Beside this scalability, this principle provides a comparable on/off shifting rate (responds function) for all switches, avoiding complex fine tuning of molecular networks.<br />
:To sum it up, the target site, allows to switch between an "on" and "off" state. But so far, the switch is not capable of performing specific interaction with transmitter molecules. This is where the recognition site comes into play.<br />
*'''Recognition site'''<br />
:The recognition site defines which transmitter molecule can actually interact with the switch. Therefore, a unique recognition site is generated for each switch and is positioned right upstream of the target site. In principle the recognition can be any random sequence as long as it remains unique within the molecular network.<br />
Summing up, the recognition site allows a specific interaction between switches and transmitter molecules. Once this interaction is formed, an interaction between the transmitter and the target will actually switch the state of the terminator. This allows the specific arrangement and interconnection of numerous of these switches by transmitter molecules, without changing the target site. Comparable to wires connecting many identical transistors, our target site remains the same.<br />
<br><br />
<br />
===Transmitter RNA´s===<br />
As desccribed above, transmitter RNAs are the input and output of bioLOGICS switches (compare [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]). These transmitters are short ssRNA molecules representing the "trigger" to shift switches between the "on" and "off" state. To fulfill this role, they need to posses the following properties:<br />
*A transmitter may only interact with certain switches. That is, a transmitter has to find the corresponding recognition site of a switch.<br />
*Once an interaction is established between a transmitter and a switch, a transmitter has to be capable of changing the secondary structure of a terminator and thus cause antitermination.<br />
Again, these two properties are fulfilled by two components of the transmitter:<br />
*'''Identity site'''<br />
:This site is capable of forcing an interaction between the transmitter and the switch. Therefore it is complementary to the recognition site of this switch. As the recognition site is unique within a network, so is the identity site. However, the single identity site is not capable of changing the state of the switch. That is were the trigger site comes into play.<br />
*'''Trigger site'''<br />
:Once an interaction is created by the identity site, the trigger site is capable of actually shifting the switch since it is complementary to the target site of the switch. To fulfill this role, it is placed upstream at the 5' end of the identity site. As the target site is the same for all switches, the trigger site is the same for all signals. Therefore it is important, that similar to the identity site, a trigger site cannot function on its own. That is, a single trigger site cannot shift the state of a switch without the help of an identity site.<br />
<br />
Summing up, we applied the principle introduced for the switches to the transmitter molecules. In contrast to previous approaches on this field <sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup>, we introduced the described synthetic trigger site in such a manner that it is not able to change the state of the terminator on its own, but only in combination with the identity site. So the challenge is to arrange and optimize these elementary building blocks thermodynamically, that a trigger site is only able to switch in combination with its respective identity site. This was done by ''in silico'' design using [[TU Munich/Glossary#NUPACK| NUPACK]], presented in section [[TU Munich/Modeling#in silico design based on thermodynamic calculations| in silico design]].<br />
<br />
<br><br />
<br />
===Putting it all together: the switching process===<br />
[[Image:TUM2010_switching-process.jpg|550px|right|thumb|The basic structure of a switch (left) and a transmitter RNA (right). See text for details.]]The functional principle of the designed switches is illustrated in the figure. The switch is positioned on DNA upstream of a desired output transmitter. So in the absence of a triggering transmitter molecule, transcription will be canceled by the formation of a RNA stem loop in the nascent RNA-chain. This will cause the RNA polymerase to stop transcription and fall off the DNA and consequently no output RNA will be produced. This process only relies on [[Team:TU_Munich/Glossary#Termination| rho-independent termination]].<br />
On the other hand, in the presence of a [[Team:TU_Munich/Project#RNA_transmitters | input transmitter]], this small functional RNA inhibits the stem loop formation by complementary base-pairing and hence avoids termination of transcription. In detail, the identity site (red part on transmitter) binds the recognition site (red part on switch) and serves as [[Team:TU_Munich/Glossary#Toehold|toehold]], which will thermodynamically allow the trigger site (turquoise part on transmitter) to perform a [[Team:TU_Munich/Glossary#Strand Displacement| strand displacement]] and open up the stem loop structure. Consequently the polymerase can read all the way through and form the output RNA.<br>Summing up, we use this concept to create a switch that can be toggled by a transmitter RNA molecule and in response, is able to produce another transmitter RNA.<br />
<br><br />
<br><br />
<br><br />
<br />
===From switches towards bioLOGICS logic gates===<br />
As described, each switch can be accessed by a specific RNA-transmitter molecule, illustrating the input. In turn, another RNA-transmitter molecule will be produced if the switch shifts its state. This output transmitter of one switch can serve as input transmitter for the next switch by meaningful selection and design of the respective recognition sites. This easily allows arranging several switches in specific sequences and faulty wiring - the corner stone of a logical network.<br />
<br />
To ease the building of logical networks, applying mathematical logics, e.g. Boolean logics like in computational science would be worthwhile. It is possible to establish general Boolean operators with our switches and thus build "logical modules". <br />
Since AND/OR/NOT are the most simple logic operations which can be implemented with the presented switches, and all remaining operations can be expressed by these three operators according to laws of boolean logics, we exemplary designed them.<br />
<br />
{|<br />
|-<br />
| *AND consists of a parallel circuit of two switches<br />
|-<br />
|[[Image:AND2.png|500px|thumb|center]] <br />
|-<br />
| *OR is implemented by connecting two switches in series<br />
|-<br />
|[[Image:OR2.png|500px|thumb|center]]<br />
|-<br />
| *NOT is more complex to explain. In principle, it consists only of one switch which contains its respective signal molecule intrinsic, so via intramolecular interaction, antitermination is the initial state. The signal is intrinsically of the same components as usual to allow interconnection with other logic gates.<br />
|-<br />
|[[Image:NOT2.png|500px|thumb|center]]<br />
|}<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Network construction==<br />
Designing complex biological networks based on either traditional protein engineering or our new bioLOGICS is still a complex task. We developed a software which allows the fast construction of a bioLOGICS based networks. <br><br />
To read more about this, look at our [https://2010.igem.org/Team:TU_Munich/Software Software page]<br />
<br />
=Our Objective=<br />
Putting the implementation described above into practice, will be a major challenge. For this year's iGEM competition our goal is to do the first step: design and build a switch that can be toggled by a RNA molecule. To be precise, we want to apply the design rules of our switch to modify a transcription terminator in such a way that it interacts with a second RNA molecule and, as a result, is no longer capable of forming a stem loop. This objective will require intensive ''in silico'' designing and modeling of switches based on different terminators and their corresponding transmitters. In connection to this theoretical part, we also have to test and verify the switches. For this step, we establish custom-made assays, ''in vitro'' and ''in vivo''.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Once the objective mentioned above is accomplished, these basic RNA/RNA-interactions have to be modified in such a manner that the described identity/trigger site pattern for the transmitter and the complementary recognition/target site switch composition has to be established. The most important requirement is to is to optimize these modules that the transmitter is only able to switches specifically, meaning only in the presence of both, identity AND trigger site. <br />
<br><br />
Once the objective mentioned above is accomplished, the creation of an OR gate will be rather simple since it only requires two switches. However the creation of an AND or NOT gate and optimizing the logic gates to improve their responds function will remain the goal of future work. Also the creation of small networks and the correct integration of BioBricks as input and output molecules will be future challenges. Furthermore, we wanted to rather focus on the development and the testing of our structural design of the switches, rather than developing a variety of new BioBricks.<br />
<br />
==''In silico'' design==<br />
As described above, our switches are based on certain design rules. However, there still are different structural parameters that need to be tested and optimized (length of recognition site and target site, choice of terminator, etc.).<br />
We used [[Team:TU_Munich/Project#in silico design |''in silico'' design]] and [[Team:TU_Munich/Modeling| modeling]]) to test different parameters. Furthermore we tried to use the [[Team:TU_Munich/Glossary#Antitermination|antitermination principle]] observed in nature, such as [[Team:TU_Munich/Glossary#Attenuation| attenuation]] in ''E. coli'' or [[Team:TU_Munich/Glossary#Tiny Abortive RNA´s| tiny abortive RNA´s]] of T7-phage.<br />
==Evaluation and Measurements==<br />
To evaluate the functionality of our molecular switches, we first had to establish several assays. Therefore, we improved an existing [[Team:TU_Munich/Lab#In vivo Measurements |''in vivo'' assay]] and developed an [[Team:TU_Munich/Lab#In vitro Transcription | ''in vitro'' assay]] for this purpose. For more information please refer to the [[Team:TU_Munich/Lab | lab]] section.<br />
<br><br />
<br><br />
Summarizing, the main challenges are <br />
* to find a suitable terminator construct and design a complementary trigger unit, which is only functional in combination with a specificity site - meaning an optimization of the '''thermodynamically parameters''' (see[[Team:TU_Munich/Project#in silico design| in silico design]])<br />
* to investigate whether the transmitter/switch interaction reaction is on a timescale to be competitive to terminator formation - meaning an comparison of '''kinetic parameters''' (see [[Team:TU_Munich/Modeling|Modeling page]])<br />
* to proof antitermination can be also be caused by synthetically RNA-interaction (see [[Team:TU_Munich/Glossary#Antitermination| Antitermination in nature]] and [[Team:TU_Munich/Project#Results| ''in vivo'' and ''in vitro'' measurements]] )<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
=Results=<br />
Every network starts with a basic unit. While our declared aim is to enable networks allowing fine-tuning of gene expression beyond the regular on/off, exploring such an on/off switch/signal pair is the first step towards a functional network. We constructed several units and tested their efficiency, robustness and reproducibility ''in vivo'', ''in vitro'' and ''in silico''. Furthermore we developed a software which allows easy constructions of networks based on our designed logic gates. Conclusive elaboration of a few first RNA-based logic units is the major contribution of our iGEM team.<br />
<br />
==in silico Design of Switching and Trigger Unit==<br />
As described on the [[Team:TU_Munich/Project | project]] page, one key aspect of our switches is the idea, that a [[Team:TU_Munich/Glossary#Transmitter_(bioLOGICS) | RNA transmitter molecule]] is capable to shift the state of a switch only if its [[Team:TU_Munich/Glossary#Trigger_Site_(bioLOGICS) | trigger site]] is present and its [[Team:TU_Munich/Glossary#Identity_Site_(bioLOGICS) | identity site]] corresponds to the [[Team:TU_Munich/Glossary#Recognition_Site_(bioLOGICS) | recognition site]] of the [[Team:TU_Munich/Glossary#Switch_(bioLOGICS) | switch]]. We successfully constructed several switches and their corresponding transmitter RNA ''in silico'' on a thermodynamical basis. We modified different transcriptional terminators in such a way, that the formation of the terminator was prevented by a transmitter molecule. As desired, this only occured if the transmitter molecule contained both, a trigger and an identity site. Analogously, we were able to design and verify a NOT gate using the same thermodynical approach.<br />
<br />
==Diffusion and RNA Folding Dynamics==<br />
We estimated the diffusion time for our constructs and modeled the folding dynamics of our bioLOGICS switches including the switching process with a stochastic RNA folding program. We were able to provide better insight in their folding dynamics and proved that they are able to interrupt termination. We also optimized the switches and the corresponding signals. Furthermore, we combined the switches what resulted in a logic gate. See our [[Team:TU Munich/Modeling|Modeling page]] for further details.<br />
<br />
==''in vivo'' Functionality Screening==<br />
Since our logic gates are intended to function in living cells, ''in vivo'' measurements were essential. In a set of experiments we concentrated on two different switches based on known [[Team:TU_Munich/Glossary#Attenuation|attenuators]] from nature: the [[Team:TU_Munich/Modeling#Switch|HisTerm]] and [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Focusing on fluorescent proteins for quantifiable input and output we designed a functional and robust screening system. For greater detail see [[Team:TU_Munich/Lab#Experiment_Design|Experimental Design]]. Unfortunately, setting up a working screening system failed twice. Only in redesigning and improving the screening plasmid pSB1A10 we succeeded, but lost precious time.<br />
<br />
Ultimately, the two switches displayed remarkable differences in their terminator efficiency, but neither of them responded to their corresponding signal. However, screening one transmitter signal does not disprove the basic working principle of our system. Limited by time, we hope for future teams to take up our work and to use our improved test system that we submitted to the parts registry, for performing successful in vivo measurement.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Considering the high complexity of ''in vivo'' measurements compared to other experimental challenges, a robust and easy to handle test system for [[Team:TU_Munich/Glossary#PoPS-based devices| PoPS-based devices]] is desirable. As described in [[Team:TU_Munich/Lab#Experiment_Design|Experimental design]], we used fluorescent proteins: RFP or mCherry to measure the amount of produced output and eGFP for normalization. Our first attempt, using the screening plasmid pSB1A10, yielded no interpretable results. Switching the fluorescent protein to mCherry did not work either, but after several experimental setups we determined a transcriptional problem causing no reporter protein expression regardless of the inserted part. Thereby we demonstrated the screening plasmid pSB1A10 to be [[Team:TU_Munich/Biobricks#Falsification| malfunctioning]]. <br />
Finally a new design based on pSB1A10 lead to a functional and robust screening system (compare [[Team:TU_Munich/Parts#Screening system: Backbone BBa_K494001| Screening system: Backbone BBa_K494001]]). A second promoter with identical induction properties inside the BioBrick cloning site enforces transcription of the PoPS-based device and the mCherry output.<br />
<br />
Exemplary, the graph below on the right shows the positive control, induced and uninduced at OD<sub>600</sub>=0.7 followed by 16 h incubation at 25 °C. Clearly visible are eGFP and mCherry fluorescence in the induced samples. The uninduced control showed no fluorescence at all, demonstrating the PBad promoter to be tight and providing very low basal transcription, what is a major advantage for the screening system. This newly designed screening approach renders the characterization of PoPS-based devices in general and switches in particular easy and robust. The low basal transcription furthermore fulfills one of the most important requirements for the designed switches, since output transmitters may only be produced in presence of an input transmitter. This helps to avoid strong "background" noise, which would extremely harden the successful interconnection of several switches. <br />
<br><br />
[[Image:TUM2010_PosControlklein.JPG|200px||thumb|left|Bacteria containing positive control]]<br />
[[Image:TUM2010_graphPosControl1.png|355px|thumb|center|Emission spectra of induced (green/red) and uninduced(black) positive control BBa_K494002 ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
Due to the time limitations of the iGEM completion we had to focus our efforts on few switches after designing the screening system. Relying on the functionality of systems occurring in nature, we choose the [[Team:TU_Munich/Modeling#Switch|HisTerm]] as well as the [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Both switches are based on known natural [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|attenuators]]. Testing synthetic and none-naturally switchable terminators in vivo are goals for future work.<br />
Delorme et al. reported the His-Terminator to be a remarkable effective Terminator with more than 99% termination efficiency.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup> The exemplary measurement below on the right confirms the high terminator efficiency. In fact, we could not detect any mCherry fluorescence in any cells containing the [[Team:TU_Munich/Modeling#Switch|HisTerm]]. Even induction of the corresponding signal transmitter RNA via IPTG did not alter the Terminator efficiency. Again time was the limiting factor and prevented us from testing more than one corresponding transmitter, although the [[Team:TU_Munich/Modeling| Modeling]] highly suggested the necessarily of finding an optimized transmitter length. Thus, the results are insufficient either to prove or to disprove the functionality of the [[Team:TU_Munich/Modeling#Switch|HisTerm]] or our concept in general.<br />
<br><br />
[[Image:TUM2010_HisSwitchklein.JPG|200px|thumb|left|Bacteria containing HisTerm]][[Image:TUM2010_HisSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing HisTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
<br />
Attaining only 90% terminator efficiency, the natural Trp [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|Attenuator]] is known be less effective than the [[Team:TU_Munich/Modeling#Switch|HisTerm]].<sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup> The graph on the right depicts our designed [[Team:TU_Munich/Modeling#Switch|TrpTerm]] characteristic efficiency of about 40 %, notably below the natural standard. Allowing 60% transcription in the “off” state excludes the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] from possible candidates for a scalable network of logic gates, due to the mentioned required "yes or no" function (see [[Team:TU_Munich/Project#Implementation| Implementation and how to connect Biobricks]]). Thus the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] is inoperative as intended, but may still be useful in other contexts. Similar to the [[Team:TU_Munich/Modeling#Switch|HisTerm]], the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] also did not react to the induction of the corresponding signal. Under circumstances, termination efficiencies altered by the transmitter are on a low range and not resolvable within observed 40% basal transcription. <br />
<br><br />
[[Image:TUM2010_TrpSwitchklein.JPG|200px|thumb|left|Bacteria containing TrpTerm]][[Image:TUM2010_TrpSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing TrpTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
<br />
Making use of our improved screening system we also carried out some ''in vivo'' kinetic measurements in addition to the end-point measurements above. In contrast to the ''in vitro'' experiments we did not obtain significant results for the characterization of our switches. As the switching process is many times faster than protein synthesis our ''in vivo'' kinetics include the synthesis of mCherry as well as its maturation. Therefore we centered our attention on end-point experiments. For more information browse the [[Team:TU_Munich/Lab#Lab_Book|lab book]]. <br><br />
Considering our ''in vivo'' measurements, neither of the tested switches showed any effect regarding the signal induction. But due to the small number of tested switches and signals this can hardly be regarded as disprove of concept. In particular in light of the recent findings by Sooncheol proving antitermination in principle using a T7 system.<sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup><br />
<br />
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<br />
==''in vitro'' Screening==<br />
To minimize the amount of disturbing factors we decided to countercheck our ''in vivo'' results with a set of ''in vitro'' measurements. While the ''in vitro'' systems are no doubt much less complex than living cells, the work with these set-ups proved to be quite as difficult.<br />
Just as with the ''in vivo'' measurements we could prove our switching system neither right nor wrong, leaving enough work for future iGEM teams.<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
===''in vitro translation''===<br />
<br />
Beside optimization of the reporter proteins in use, the major problem occuring in the experiments was the low capacity of the kit. The signal intensity was very low, which made it difficult to observe any signal intensity alterations, so no conclusion could be drawn from these measurements.<br />
<br />
===''in vitro'' transcription===<br />
We used two completely independent ''in vitro'' systems: Using ''E.coli'' RNA Polymerase we analyzed the His and Trp switches that had already been tested ''in vivo''. In a second set-up, we used the well-established T7 RNA Polymerase and switch based on the T7 terminator as well as several signal sequences.<br />
<br />
====T7 System====<br />
In contradiction to the results of Kang and coworkers and other groups, in our ''in vitro'' set-up the T7 terminator did not seem to terminate at all. The negative control (Promoter_Terminator_malachite binding aptamer) showed a similar increase in fluorescence as the positive control (Promoter_random sequence_malachite binding aptamer). <br />
[[Image:TUM2010_T7Result1.png|360px||thumb|left|''in vitro'' transcription measurement of T7 terminator with no signal(upper left), nonsense signal (upper right) and two different designed signals (below)]]<br />
[[Image:TUM2010_T7Result3.png|360px||thumb|right|''in vitro'' transcription measurement of positive control(upper left and T7 terminator with three different designed signals (remaining traces)]]<br />
Furthermore denaturing Polyacrylamide Gel Electrophoresis (PAGE) confirmed that there was no observeable termination of transcription. The addition of a signaling sequence led to a significantly lower increase in fluorescence, which can be attributed to the fact that both DNA sequences, switch and signal, compete for RNA Polymerases.<br />
However, there is almost no difference between the designed signals and random sequences, which is not a big surprise since there can be no antitermination if the terminator itself does not work.<br><br />
<br />
Possible explanations for the contradiction between our results and those of Kang and coworkers might be the experimental set-up and the RNA Polymerases we used. Different variants of T7 RNA Polymerase might respond in different ways to terminator structures, and the termination might be influenced by the presence or absence of cofactors, depending on the purification methods used in producing the Polymerase.<br><br><br />
<br />
This set-up offers a lot of possible experiments for the future, which we would have loved to conduct with a just a bit more time...<br />
<br />
====''E.coli'' System====<br />
<br />
Compared to the T7 System, the ''E. coli'' RPO system produced poor increases in fluorescence, indicating little RNA synthesis. It was shown that the presence of a terminator decreases, as expected, the production of downstream RNA. This result was also confirmed by denaturing PAGE. However, due to the poor changes in fluorescence we were not able to actually characterize the behaviour of our switches ''in vitro'', and the small RNA concentrations did not allow a quantitative interpretation of our gels. A major problem with this method was the low concentration of the ordered Polymerase resulting in a much weaker overall signal as comparable measurements using the T7 Polymerase. <br><br><br />
In future experiments we might try to work with smaller volumes in order to reach higher concentration of RPO and of the synthesized RNA molecules, so measuring in 96 well plate readers might be a good choice. <br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Software==<br />
Although we could not show the full functionality of bioLOGICS in the lab we still want to demonstrate the potential of our approach. Hence we implemented the idea behind our logic gates in a program which illustrates how bioLOGCIS theoretically would allow the construction of complex information processing networks interconnecting BioBricks. For further details take a look at our [[Team:TU Munich/Software|Software page]].<br />
<br />
<br />
=Outlook=<br />
...<br />
future plans will also work with [[Team:TU_Munich/Glossary#Synthetic Terminator| Synthetic Terminators]], which might retrieve additional informations on what drives the process of Termination<br />
...<br />
<br />
=References=<br />
<html><a name="ref1"></a></html>[1] http://partsregistry.org/cgi/partsdb/Statistics.cgi<br />
<html><a name="ref2"></a></html>[2] https://2009.igem.org/Team:Imperial_College_London/M1 encapsulation<br />
<html><a name="ref3"></a></html>[3] https://2009.igem.org/Team:TUDelft<br />
<html><a name="ref4"></a></html>[4] https://2008.igem.org/Team:Heidelberg<br />
<html><a name="ref5"></a></html>[5] Maung Nyan Win and Christina D. Smolke, Science Oct. 2008 Vol. 322. no. 5900, pp. 456 - 460<br />
<html><a name="ref6"></a></html>[6] http://en.wikipedia.org/wiki/Logic_gate#Symbols<br />
<html><a name="ref6"></a></html>[7] http://en.wikipedia.org/wiki/Moore's_law<br />
<html><a name="ref6"></a></html>[8] http://en.wikipedia.org/wiki/Protein_interaction<br />
<html><a name="ref6"></a></html>[9] http://en.wikipedia.org/wiki/Riboswitch<br />
<html><a name="ref6"></a></html>[10] http://en.wikipedia.org/wiki/Binding_sites + http://en.wikipedia.org/wiki/Recognition_site<br />
<html><a name="ref6"></a></html>[11] irgend ein damn review über directed evolution and so on<br />
<html><a name="ref12"></a></html>[12] Delorme, Ehrlich and Renault, Regulation of Expression of the Lactococcus lactis Histidine Operon. Journal of Bacteriology, Apr. 1999, p. 2026–2037<br />
<html><a name="ref13"></a></html>[13] Trun and Trempy(2003): Fundamental Bacterial Genetics, Wiley-Blackwell, Chapter 12 <br />
<html><a name="ref14"></a></html>[14]Sooncheol Lee, Huong Minh Nguyen and Changwon Kang, Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator. Nucleic Acids Research, 2010, 1–9<br />
<html><a name="ref6"></a></html>[15] <br />
<html><a name="ref6"></a></html>[16]<br />
<br />
<!-- The idea behind our project is to change the way BioBricks have been used up to now. Over the years, many receptors and signals have been constructed as BioBricks during the annual iGEM competition, but still it is not possible to interconnect these Bricks in a complex biological network resuting in a cell, that is able to respond to its environment giving differenciated responses depending on the input signals. (Beispiel: cambridge hat das gemacht, xx dies, aber eine zelle kann nicht beides...<br><br />
We plan to create biological switches, that can function as locial gates inside a cell. Our switches rely on RNA/RNA-interactions, regulating transcriptional termination. This is a major advance of our concept, as regular switches rely on complex regulation including proteins and/or metabolites. Thus, our switches shall offer a greater robustness and their behaviour should be easier to predict. [[switch|Read more]] (hier sollte noch das hochskalieren erwähnt werden...<br><br />
These switches can further be used to build up a logical network inside a bacterial cell, enabling every scientist to connect as many functionalities (in form of BioBricks) as designated. We plan to offer simulation on each specifically designed network.<br />
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<br><br>Over the years, many teams participating in the iGEM competition spent their time on constructing receptors and systems to detect a certain input that a variety of gorgeous oppurtunities is available so far.[[Image:TUM2010 network.png|thumb|300 px|right|Our visioon: A logic network inside the cell]] Nevertheless, until now it is not possible to link all those functionalities and build up a network giving differenciated responses to several of those input signals, where the molecular response depends on the complex composition of the environment a cell faces. We would like to offer this possibility to everyone.<br />
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The logic network we want to apply will be based on devices, that can be easily upscaled and therefor offer the chance to build networks of any wanted complexicity. Our devices rely on pure RNA/RNA interactions and thus their behaviour is well predictable.<br />
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The concept we rely on for our design of RNA-switches is based on the principle of [https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation/ '''attenuation'''].<br />
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= Experiments =<br />
We designed several experiments to test our switches, all of them based on fluorescence measurements. We designed experiment setting for measurements ''in vivo'' as well as ''in vitro''. Our ''in vitro'' measurements relied on two different experiment set-ups. While the first was based on a commercial ''E. coli''-lysate, the latter was reporting on a transcriptional level only, eliminating most of the possible side-effects one could expect in the complex behaviour of a living cell or cell-lysate. [[Experiments_main|Read more]]<br />
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= Results =<br />
We ...blablabla<br />
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Text that will present our results...<br />
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= thing to move =<br />
<br />
'''bioLOGICS: Logical RNA-Devices Enabling BioBrick-Network Formation'''<br />
'''Abstract'''<br />
Among the goals of iGEM is the creation of synthetic biological parts and their utilization to achieve novel features and behavior in biological systems. The emphasis of our project is put on this latter, "systems" aspect of iGEM. More precisely, we aim at the development and experimental demonstration of a scalable approach for the realization of logical functions in vivo.<br />
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By developing a computational biological network based on RNA logical devices we will offer everyone the opportunity to 'program' their own cells with individual AND/OR/NOT connections between BioBricks of their choice. Thereby, BioBricks can finally fulfill their original assignment as biological parts that can be connected in many different ways. We will achieve this by engineering simple and easy-to-handle switches based on predictable RNA/RNA-interactions regulating transcriptional termination. These switches represent a complete set of logical functions and are capable of forming arbitrarily complex networks.<br />
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== The Experiments ==<br />
===Fluorescent proteins as reporter===<br />
<div align="justify"><br />
Our initial idea to prove our concept of antitermination was to use flourescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks ''in vivo'' as well as ''in vitro'', the latter using a translation kit based on e.coli lysate. <br>We decided to use the flourescent proteins GFP and RFP, as their spectra do not overlap and we would not measure any FRET. We would use GFP fluorescence as internal control and RFP fluorescence as signal to detect termination/antitermination by our switch we cloned in between the coding sequences of the proteins. Both protein sequences are under the control of one (L-arabinose induced) promoter.<br />
[[Image:TUM2010_gfprfp_schalter_klein.gif|center|our idea]]<br />
<br><br />
When measuring the termination of our BioBricks and the antitermination by their corresponding signal-RNA, we should be able to observe an increasing RFP emission compared to the GFP emission upon induced signal-RNA production in the cells/in the kit:<br><br />
[[Image:TUM2010_Expected_emission_spextra.png|center|our idea]]<br />
Wiith these measurements, it should also be possible to observe differences in efficiency of termination as well as antitermination between our designed switches. <br><br><br />
<br />
===Measurements with the malachite green aptamer as reporter===<br />
A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramaticly (about 3000 times) upon binding of a specific RNA-aptamer.<br><br>#<br />
[[Image:TUM2010_Malachitgruen-2.png|500px|center|our idea]]<br />
---concept to be desribed, as well as literature---<br />
<ref>refs</ref><br />
<br><br><br />
To study the switches on the transcriptional level gives the advantage, that we would have less interferences and possible artefacts. Also, we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems.<br><br />
<br />
[[Image:TUM2010_Malachit_emission.png|200px|thumb|left|Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA]]We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.<br>Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level.<br />
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<br><br><br><br><br><br><br><br><br><br><br />
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Results <br />
==Flourescent proteins==<br />
Unfortunatly, we had to change the reporter construct two times during our experiments as several problems occured in our measurements:<br><br><br />
<br />
===First Try: based on the measurement plasmid pSB1A10===<br />
At the beginning, we decided to use the reporter plasmid [http://partsregistry.org/Part:pSB1A10 pSB1A10] from the registry. It consists of the fluorescent proteins eGFP and mRFP1. Each sequence includes a ribosome binding site and a stop-codon; the two genes are divided by a cloning side including the BioBrick cleavage sites.[[Image:ScreeningPlasmid1.0.PNG|300px|thumb|right|pSB1A10]]<br> In front of the eGFP sequence, the plasmid includes an arabinose-inducable promoter. The plasmid also contains an ampicilline resistence.<br><br />
We cloned our switches into the cloning site of the measurement plasmid and used an empty cloning site as control; our signal-RNAs we cloned into the [http://partsregistry.org/Part:pSB1K3 pSB1K3] vector, together with the BioBricks R0011 (Lac promoter) and B0014 (double terminator of transcription). Afterwards, we cut pSB1K3 with Aat2 and Pst1 and pSB1A10 with Nsi1 and Aat2 and ligated those fragments of each plasmid that contained our Bricks to get a Monsterplasmid.<br>We had to do so, as both plasmids contain the same ori mechanism. In addition, having both the switch and the signal RNA transcribed from the same plasmid gives us a high local concentration of the signal, once its transcription is induced.<br><br />
[[Image:TUM2010_Messplasmid_nr1.png|left|the measurement plasmid]]<br />
<br />
We transformed BL21(DE3) cells with the plasmid. We set up cultures, induced the arabinose promoter and measured the GFP and mRFP1 excitation/emission spectra within time.<br><br><br />
Unfortunatly, we were ot able to detect any RFP signal, not even in the positive control with no switch in between the GFP/RFP sequences.<br><br><br />
From these experiments, we concluded, that the mRNA of the RFP variant used was instable and rapidly degraded by RNases, so the RFP was not synthesized in the cells. This was also the conclusion from XXX...<br />
<br><br><br />
As solution to this, we decided to design a measurement plasmid ourselves:<br><br />
<hr width="300"><br />
<br><br />
<br />
===Second Try: A measurement plasmid of our own design===<br />
To design our own plasmid to overcome the problems that occurred in our first try gave us tghe possibility to overcome several other problems:<br><br />
#<br />
#<br />
#<br />
[[Image:TUM2010_Construct_no2.png|400px|our construct|center]]<br />
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===Third Try: One promoter for each protein===<br />
We decided to use the measuremnt plasmid we developed in our second try but to clone another L-arabinose induced promoter into the plasmid, in front of our switch followed by the mCherry sequence.<br><br />
<br>[[Image:TUM2010_Construct_no3.png|left|400px|Construct #3]]In this way, we still can use GFP fluorescence as internal control, because both protein transcription is under the control of a promoter of identical design.<br />
Though we are still not able to tell exactly why our previous measurements did not work, but with this construct we measured the first time fluorescence of the mCherry protein in our positive control.<br />
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{{:Team:TU_Munich/Templates/End}}</div>Hartlmuellerhttp://2010.igem.org/Team:TU_Munich/ProjectTeam:TU Munich/Project2010-10-28T03:02:46Z<p>Hartlmueller: /* in silico Design of Switching and Trigger Unit */</p>
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<center><font size="5pt" color="#000000">'''bioLOGICS'''</font><font size="4pt" color="#000000">: Logical RNA-Devices Enabling BioBrick-Network Formation</font></center><hr color="black"><br><br />
= Vision=<br />
<br />
Until today, 13.628 biobrick sequences<sup>[[Team:TU_Munich/Project#ref1|&#91;1&#93;]]</sup> have been submitted to partsregistry, thereof 102 reporter units and 12 signaling bricks.<br />
Since then, people are trying to arrange these single biological building blocks in such a manner that allows producing special biotechnological products (metabolic engineering), developing biological sensory circuits (biosensors) and even giving microorganisms the ability to react on multiple environmental factors and serve both as disease indicator and drug. These examples and further promising ideas were implemented on previous iGEM-competitions.<sup>[[Team:TU_Munich/Project#ref2|&#91;2&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref3|&#91;3&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref4|&#91;4&#93;]]</sup> <br><br><br />
The idea of combining the outcome of several iGEM competitions to construct complex synthetic biological systems falls at the last hurdle - the fact, that each team uses a different principle how to access and functionally connect the respectively used biobricks. For example, it is a major challenge to create a system that uses several sensoring BioBricks from different iGEM-teams which in turn regulates reportering BioBricks from various teams. In order to combine and fully take advantage of these promising projects, our vision is to develop an adapter that allows interconnecting arbitrary biobricks on a functional level. Such a system easily allows to setup sensor-reporter circuits and interconnect them to complete biological chips... A further step towards artificial cells.<br><br><br />
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Generally speaking, the above adapter has to meet the following requirements:<br />
*'''Universality'''<br />
:The adapter has to be compatible to as many BioBricks as possible. This objective will guarantee that a large number of BioBricks can be connected.<br />
*'''Scalability'''<br />
:Once the basic design of the system is established, the construction of the system is supposed to be automated in silico. This way it will be possible to create an adapter connecting a large amount of BioBricks.<br />
*'''Biological orthogonality'''<br />
:Interference with cellular components has to be as low as possible in order to avoid unwanted and perturbing side effects.<br />
*'''Logic'''<br />
:The adapter is supposed to not only associate different BioBricks, but to functionally connect BioBricks in a precisely determined manner (including operations such as AND/OR/NOT).<br />
<br><br />
Several biological logic units, devices and circuits have been developed so far<sup>[[Team:TU_Munich/Project#ref5|&#91;5&#93;]]</sup>, but to our knowledge, none was shown to meet all requirements listed above.<br />
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<br />
=Implementation=<br />
To functionally connect BioBricks, there are several possibilities including genetic switches, riboswitches and direct protein-protein interactions. We investigated several hypothetically principles, and decided to focus our practical work on the development of a RNA-RNA interaction-based switch. These switches are capable of changing between two states, a state of antitermination and termination, and make use of highly-specific RNA-RNA interaction. In principle such a switch can fulfill all requirements mentioned previously. The following text clarifies how these switches work in detail.<br />
==How to connect BioBricks==<br />
Our adapter is a system, that activates or disables BioBricks (output BioBricks) in response to the presence of other Biobricks (input Biobricks). Our approach uses a molecular network to put this into practice and consists of four major elements:<br />
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{|<br />
|-<br />
|[[Image:Networks.png|center|thumb|730px|The general principle how different inputs can be connect to various outputs. For details see text.<br>Inputs (such as proteins or small molecules) are indicated on the left side. blue lines represent transmitter molecules whereas organe lines present logic gates. The type of logic gate is indicated. Green lines indicate transmitter RNA that can function as mRNA and consequently generate any output gene (indicated on the very right).]]<br />
|}<br />
In order to connect different BioBricks, our network requires four major types of components:<br />
*Input elements<br />
*Transmitter molecules<br />
*Logic gates<br />
*Output elements<br />
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{{:Team:TU_Munich/Templates/InfoBoxStart}}'''Computer vs. molecular network - and our approach'''<br><br />
Logic gates in a molecular network are often compared to transistors used in a computer, where billions of transistors are incorporated<sup>[[Team:TU_Munich/Project#ref7|&#91;7&#93;]]</sup>. The main advantage on a computer chip is, all transistors share the same functional principle, and only the way connecting them in a special sequence allows specific addressing of only a subset of other transistors by an input. However, spatially fixed connections of molecular logic gates are not possible in a living cell. The "wiring" within a cell relies on the specific interaction between transmitter molecule and their corresponding logic gates, for example implemented by protein-protein/ligand-protein interactions or specific ligand-riboswitch interactions.<sup>[[Team:TU_Munich/Project#ref8|&#91;8&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref9|&#91;9&#93;]]</sup> As a result, in a cell, each occurring logic gate ("transistor") has to be different, at least in a special recognition site<sup>[[Team:TU_Munich/Project#ref10|&#91;10&#93;]]</sup> - for example like different transcription factors, recognizing different DNA-sites. Thanks to evolution, nature easily can invent a new transistor for each task - science achieves this only on a limited scale, and producing synthetic molecular logic gates artificially by either rational or evolutionary protein or riboswitch engineering, is limited to small circuits so far<sup>[[Team:TU_Munich/Project#ref11|&#91;11&#93;]]</sup>. Our project aims to establish a molecular switch as close as possible to a electronic transistor, thus sharing the same functional principle for all logic gates. At the same time, we want to design a easily exchangeable recognition site, which can individually be designed by everyone! {{:Team:TU_Munich/Templates/InfoBoxEnd}}<br />
<br />
These elements can be combined to build up a molecular network (see illustration). Each input molecule (such as a BioBrick) produces a unique transmitter molecule. All transmitters belong to the same type of molecule and share a common design. However, each transmitter molecule can only interact and activate a certain subset of logic gates. In other words, logic gates have to recognize as well as bind the corresponding transmitter molecules and are capable of producing a new output transmitter molecule. Depending on the type of the logic gate (AND, OR or NOT<sup>[[Team:TU_Munich/Project#ref6|&#91;6&#93;]]</sup>), an output transmitter is only created if both input transmitter molecules are present (AND), at least one of two input transmitters is present (OR) or if no input transmitter is present at all (NOT). Once a logic gate has produced a new output transmitter, these transmitters can in turn address another subset ("layer") of logic gates. In theory many layers of logic gates can be connected this way allowing the creation of large networks. Until this step, various transmitter molecules might have been produced. But in order to create a Biobrick output, the last layer of logic gates finally generates transmitter molecules that will not active logic gates, but will rather interact with the cell metabolism to produce a BioBrick response. In other words, the last layer of transmitter molecules is capable of regulating BioBrick formation.<br />
<br />
<br />
Summarizing, the network establishes a connection between input BioBricks and output BioBricks in a functional manner.<br />
Having addressed the basic layout of the molecular network, the next step is to determine what type of molecules can perform the required functions. We decided to use RNA, both as transmitter molecules and for constructing logic gates. Several advantages result from the utilization of RNA as the central element:<br />
*During the last years, many Biobricks were designed that are sensitive to various chemicals and substances. These BioBricks often function as a transcription factor that binds to a specific DNA sequence and consequently would be capable to produce a specific transmitter RNA molecule. Thus, in principle each BioBrick which involves transcription can be integrated in our network.<br />
*Since all logic gates are capable of producing transmitter RNA, they can also produce functional mRNA encoding any protein. This means, each BioBrick consisting of protein or RNA can be produced as an output of our network.<br />
*If RNA forms both, the transmitter molecule and the logic gates, they can specifically interact by RNA-RNA interaction, which is highly predictable compared to protein interactions. This allows to generate a library of transmitters and gates ''in silico''. Such a library is essential for the creation of large networks.<br />
*RNA production is fast and energy saving for a cell. Consequently, operating a network that only produces RNA rather than proteins will also be faster and more efficient for the host cell. Since our logic gates are based on transcription, translation and resource consuming protein production will only be required at the very last step. <br />
*As the half-time of RNA can be rather short, transmitter RNA will not accumulate within the cell and it is therefore less likely for the system to become saturated.<br />
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<br />
==Design and functional principle of logic gates==<br />
The concept introduced above provides a framework that can potentially serve as an universal adapter between different BioBricks. However, the [[Team:TU_Munich/Glossary#logic gate | logic gates]] have not been specified more precisely so far. This will be done in the following section.<br />
<br />
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Generally speaking, our logic gates are to possess the following characteristics:<br />
*Logic gates, such as AND, OR and NOT, have to be implemented by RNA-interaction based principles (see [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]).<br />
*All logic gates have to recognize their corresponding [[Team:TU_Munich/Glossary#Transmitter (bioLOGICS)| transmitter RNAs]] and, in response, produce an output transmitter molecule.<br />
*Logic gates should follow a basic design rule, in such a way, that their creation can be automated ''in silico''.<br />
*The response efficiency of a logic gate toward a transmitter molecule should be comparable for all logic gates to provide calculable robustness and sensitivity. This will ensure comparable molecular concentrations and functionality of large networks.<br />
*The system has to be designed for ''in vivo'' utilization at the first place. As a reference we always assumed a temperature of 37 °C and an ''E. coli'' environment.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}} <br />
In order to build logic gates for our bioLOGICS system we will first create a simple switch. A switch can be activated by one transmitter RNA and produce an output transmitter RNA. In contrast to a logic gate, a switch does not perform logic operations. However by combining switches, logic gates can be created. The following text will first describe how the developed switch works and secondly, how logic gates such as AND/OR/NOT can be created using these switches.<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Read more{{:Team:TU Munich/Templates/ToggleBoxStart2}}<br />
[[Image:toggle_switch.png|500px|thumb|center|id="hideOnReadMore"|'''A''' The basic structure of a bioLOGICS switch (left) and a transmitter molecule (right).<br>'''B'''The process of switching. See the text in the close-by "Read more" section for details.<br>Rectangles present the composition of our functional units on the level of DNA. Fringed lines represent RNA produced by RNA polymerase. The stem loop structure depicts the switchable terminator. Terminator and target site are illustrated in blue and turquoise, respectively. Recognition sites are indicated in different colors, in this case red for the input transmitter and green for the output transmitter.Each switch and or later logical unit has to be flanked by a promotor and another constitutive terminator, to allow RNA-production by RNA-polymerase in a proper way. ]]<br />
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===Switch===<br />
[[Image:TUM2010_switch-and-transmitter.jpg|550px|right|thumb|The basic strcutrue of a switch (left) and a transmitter RNA (right). See text for details.]]<br />
Roughly speaking, a switch can be regarded as an enhanced switchable transcriptional terminator. The enhancement can be described easier by dividing a switch into its functional components: <br />
*'''Target site'''<br><br />
:The target site is the functional core element of our switches, allowing a shift between an "on" and "off" state. Since we work on the level of RNA-production (transcription), a "switchable" transcriptional terminator is suitable for this purpose. By allowing or preventing formation of a transcriptional terminator, that is by switching between termination and antitermination it is possible to represent an "off" and an "on" state, respectively. Therefore, the target site is the 5' ending of the terminator and is required for a stable terminator formation. It should be noted that this principle was also observed in nature.<br />
:To highlight and illustrate the functional principle of our switches, only the part of the terminator which is involved in interacting with a transmitter molecule and which is responsible for shifting between "on" and "off" state is called target site. The remaining terminator sequence is called terminator in the following, even if both, target site and terminator build up the terminator structure occurring in nature. <br />
:The important aspect of our switches is the fact that all switches will hold the same identical target site. Therefore having found one functional "switchable" terminator, will allow almost unlimited upscaling since this terminator can be used for a large library of switches. This is the main difference to previous works done on this field, which always required developing a new shifting principle for each switch.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup> Beside this scalability, this principle provides a comparable on/off shifting rate (responds function) for all switches, avoiding complex fine tuning of molecular networks.<br />
:To sum it up, the target site, allows to switch between an "on" and "off" state. But so far, the switch is not capable of performing specific interaction with transmitter molecules. This is where the recognition site comes into play.<br />
*'''Recognition site'''<br />
:The recognition site defines which transmitter molecule can actually interact with the switch. Therefore, a unique recognition site is generated for each switch and is positioned right upstream of the target site. In principle the recognition can be any random sequence as long as it remains unique within the molecular network.<br />
Summing up, the recognition site allows a specific interaction between switches and transmitter molecules. Once this interaction is formed, an interaction between the transmitter and the target will actually switch the state of the terminator. This allows the specific arrangement and interconnection of numerous of these switches by transmitter molecules, without changing the target site. Comparable to wires connecting many identical transistors, our target site remains the same.<br />
<br><br />
<br />
===Transmitter RNA´s===<br />
As desccribed above, transmitter RNAs are the input and output of bioLOGICS switches (compare [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]). These transmitters are short ssRNA molecules representing the "trigger" to shift switches between the "on" and "off" state. To fulfill this role, they need to posses the following properties:<br />
*A transmitter may only interact with certain switches. That is, a transmitter has to find the corresponding recognition site of a switch.<br />
*Once an interaction is established between a transmitter and a switch, a transmitter has to be capable of changing the secondary structure of a terminator and thus cause antitermination.<br />
Again, these two properties are fulfilled by two components of the transmitter:<br />
*'''Identity site'''<br />
:This site is capable of forcing an interaction between the transmitter and the switch. Therefore it is complementary to the recognition site of this switch. As the recognition site is unique within a network, so is the identity site. However, the single identity site is not capable of changing the state of the switch. That is were the trigger site comes into play.<br />
*'''Trigger site'''<br />
:Once an interaction is created by the identity site, the trigger site is capable of actually shifting the switch since it is complementary to the target site of the switch. To fulfill this role, it is placed upstream at the 5' end of the identity site. As the target site is the same for all switches, the trigger site is the same for all signals. Therefore it is important, that similar to the identity site, a trigger site cannot function on its own. That is, a single trigger site cannot shift the state of a switch without the help of an identity site.<br />
<br />
Summing up, we applied the principle introduced for the switches to the transmitter molecules. In contrast to previous approaches on this field <sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup>, we introduced the described synthetic trigger site in such a manner that it is not able to change the state of the terminator on its own, but only in combination with the identity site. So the challenge is to arrange and optimize these elementary building blocks thermodynamically, that a trigger site is only able to switch in combination with its respective identity site. This was done by ''in silico'' design using [[TU Munich/Glossary#NUPACK| NUPACK]], presented in section [[TU Munich/Modeling#in silico design based on thermodynamic calculations| in silico design]].<br />
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<br />
===Putting it all together: the switching process===<br />
[[Image:TUM2010_switching-process.jpg|550px|right|thumb|The basic structure of a switch (left) and a transmitter RNA (right). See text for details.]]The functional principle of the designed switches is illustrated in the figure. The switch is positioned on DNA upstream of a desired output transmitter. So in the absence of a triggering transmitter molecule, transcription will be canceled by the formation of a RNA stem loop in the nascent RNA-chain. This will cause the RNA polymerase to stop transcription and fall off the DNA and consequently no output RNA will be produced. This process only relies on [[Team:TU_Munich/Glossary#Termination| rho-independent termination]].<br />
On the other hand, in the presence of a [[Team:TU_Munich/Project#RNA_transmitters | input transmitter]], this small functional RNA inhibits the stem loop formation by complementary base-pairing and hence avoids termination of transcription. In detail, the identity site (red part on transmitter) binds the recognition site (red part on switch) and serves as [[Team:TU_Munich/Glossary#Toehold|toehold]], which will thermodynamically allow the trigger site (turquoise part on transmitter) to perform a [[Team:TU_Munich/Glossary#Strand Displacement| strand displacement]] and open up the stem loop structure. Consequently the polymerase can read all the way through and form the output RNA.<br>Summing up, we use this concept to create a switch that can be toggled by a transmitter RNA molecule and in response, is able to produce another transmitter RNA.<br />
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<br><br />
<br><br />
<br />
===From switches towards bioLOGICS logic gates===<br />
As described, each switch can be accessed by a specific RNA-transmitter molecule, illustrating the input. In turn, another RNA-transmitter molecule will be produced if the switch shifts its state. This output transmitter of one switch can serve as input transmitter for the next switch by meaningful selection and design of the respective recognition sites. This easily allows arranging several switches in specific sequences and faulty wiring - the corner stone of a logical network.<br />
<br />
To ease the building of logical networks, applying mathematical logics, e.g. Boolean logics like in computational science would be worthwhile. It is possible to establish general Boolean operators with our switches and thus build "logical modules". <br />
Since AND/OR/NOT are the most simple logic operations which can be implemented with the presented switches, and all remaining operations can be expressed by these three operators according to laws of boolean logics, we exemplary designed them.<br />
<br />
{|<br />
|-<br />
| *AND consists of a parallel circuit of two switches<br />
|-<br />
|[[Image:AND2.png|500px|thumb|center]] <br />
|-<br />
| *OR is implemented by connecting two switches in series<br />
|-<br />
|[[Image:OR2.png|500px|thumb|center]]<br />
|-<br />
| *NOT is more complex to explain. In principle, it consists only of one switch which contains its respective signal molecule intrinsic, so via intramolecular interaction, antitermination is the initial state. The signal is intrinsically of the same components as usual to allow interconnection with other logic gates.<br />
|-<br />
|[[Image:NOT2.png|500px|thumb|center]]<br />
|}<br />
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<br />
==Network construction==<br />
Designing complex biological networks based on either traditional protein engineering or our new bioLOGICS is still a complex task. We developed a software which allows the fast construction of a bioLOGICS based networks. <br><br />
To read more about this, look at our [https://2010.igem.org/Team:TU_Munich/Software Software page]<br />
<br />
=Our Objective=<br />
Putting the implementation described above into practice, will be a major challenge. For this year's iGEM competition our goal is to do the first step: design and build a switch that can be toggled by a RNA molecule. To be precise, we want to apply the design rules of our switch to modify a transcription terminator in such a way that it interacts with a second RNA molecule and, as a result, is no longer capable of forming a stem loop. This objective will require intensive ''in silico'' designing and modeling of switches based on different terminators and their corresponding transmitters. In connection to this theoretical part, we also have to test and verify the switches. For this step, we establish custom-made assays, ''in vitro'' and ''in vivo''.<br />
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Once the objective mentioned above is accomplished, these basic RNA/RNA-interactions have to be modified in such a manner that the described identity/trigger site pattern for the transmitter and the complementary recognition/target site switch composition has to be established. The most important requirement is to is to optimize these modules that the transmitter is only able to switches specifically, meaning only in the presence of both, identity AND trigger site. <br />
<br><br />
Once the objective mentioned above is accomplished, the creation of an OR gate will be rather simple since it only requires two switches. However the creation of an AND or NOT gate and optimizing the logic gates to improve their responds function will remain the goal of future work. Also the creation of small networks and the correct integration of BioBricks as input and output molecules will be future challenges. Furthermore, we wanted to rather focus on the development and the testing of our structural design of the switches, rather than developing a variety of new BioBricks.<br />
<br />
==''In silico'' design==<br />
As described above, our switches are based on certain design rules. However, there still are different structural parameters that need to be tested and optimized (length of recognition site and target site, choice of terminator, etc.).<br />
We used [[Team:TU_Munich/Project#in silico design |''in silico'' design]] and [[Team:TU_Munich/Modeling| modeling]]) to test different parameters. Furthermore we tried to use the [[Team:TU_Munich/Glossary#Antitermination|antitermination principle]] observed in nature, such as [[Team:TU_Munich/Glossary#Attenuation| attenuation]] in ''E. coli'' or [[Team:TU_Munich/Glossary#Tiny Abortive RNA´s| tiny abortive RNA´s]] of T7-phage.<br />
==Evaluation and Measurements==<br />
To evaluate the functionality of our molecular switches, we first had to establish several assays. Therefore, we improved an existing [[Team:TU_Munich/Lab#In vivo Measurements |''in vivo'' assay]] and developed an [[Team:TU_Munich/Lab#In vitro Transcription | ''in vitro'' assay]] for this purpose. For more information please refer to the [[Team:TU_Munich/Lab | lab]] section.<br />
<br><br />
<br><br />
Summarizing, the main challenges are <br />
* to find a suitable terminator construct and design a complementary trigger unit, which is only functional in combination with a specificity site - meaning an optimization of the '''thermodynamically parameters''' (see[[Team:TU_Munich/Project#in silico design| in silico design]])<br />
* to investigate whether the transmitter/switch interaction reaction is on a timescale to be competitive to terminator formation - meaning an comparison of '''kinetic parameters''' (see [[Team:TU_Munich/Modeling|Modeling page]])<br />
* to proof antitermination can be also be caused by synthetically RNA-interaction (see [[Team:TU_Munich/Glossary#Antitermination| Antitermination in nature]] and [[Team:TU_Munich/Project#Results| ''in vivo'' and ''in vitro'' measurements]] )<br />
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=Results=<br />
Every network starts with a basic unit. While our declared aim is to enable networks allowing fine-tuning of gene expression beyond the regular on/off, exploring such an on/off switch/signal pair is the first step towards a functional network. We constructed several units and tested their efficiency, robustness and reproducibility ''in vivo'', ''in vitro'' and ''in silico''. Furthermore we developed a software which allows easy constructions of networks based on our designed logic gates. Conclusive elaboration of a few first RNA-based logic units is the major contribution of our iGEM team.<br />
<br />
==in silico Design of Switching and Trigger Unit==<br />
<br />
==Diffusion and RNA Folding Dynamics==<br />
We estimated the diffusion time for our constructs and modeled the folding dynamics of our bioLOGICS switches including the switching process with a stochastic RNA folding program. We were able to provide better insight in their folding dynamics and proved that they are able to interrupt termination. We also optimized the switches and the corresponding signals. Furthermore, we combined the switches what resulted in a logic gate. See our [[Team:TU Munich/Modeling|Modeling page]] for further details.<br />
<br />
==''in vivo'' Functionality Screening==<br />
Since our logic gates are intended to function in living cells, ''in vivo'' measurements were essential. In a set of experiments we concentrated on two different switches based on known [[Team:TU_Munich/Glossary#Attenuation|attenuators]] from nature: the [[Team:TU_Munich/Modeling#Switch|HisTerm]] and [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Focusing on fluorescent proteins for quantifiable input and output we designed a functional and robust screening system. For greater detail see [[Team:TU_Munich/Lab#Experiment_Design|Experimental Design]]. Unfortunately, setting up a working screening system failed twice. Only in redesigning and improving the screening plasmid pSB1A10 we succeeded, but lost precious time.<br />
<br />
Ultimately, the two switches displayed remarkable differences in their terminator efficiency, but neither of them responded to their corresponding signal. However, screening one transmitter signal does not disprove the basic working principle of our system. Limited by time, we hope for future teams to take up our work and to use our improved test system that we submitted to the parts registry, for performing successful in vivo measurement.<br />
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Considering the high complexity of ''in vivo'' measurements compared to other experimental challenges, a robust and easy to handle test system for [[Team:TU_Munich/Glossary#PoPS-based devices| PoPS-based devices]] is desirable. As described in [[Team:TU_Munich/Lab#Experiment_Design|Experimental design]], we used fluorescent proteins: RFP or mCherry to measure the amount of produced output and eGFP for normalization. Our first attempt, using the screening plasmid pSB1A10, yielded no interpretable results. Switching the fluorescent protein to mCherry did not work either, but after several experimental setups we determined a transcriptional problem causing no reporter protein expression regardless of the inserted part. Thereby we demonstrated the screening plasmid pSB1A10 to be [[Team:TU_Munich/Biobricks#Falsification| malfunctioning]]. <br />
Finally a new design based on pSB1A10 lead to a functional and robust screening system (compare [[Team:TU_Munich/Parts#Screening system: Backbone BBa_K494001| Screening system: Backbone BBa_K494001]]). A second promoter with identical induction properties inside the BioBrick cloning site enforces transcription of the PoPS-based device and the mCherry output.<br />
<br />
Exemplary, the graph below on the right shows the positive control, induced and uninduced at OD<sub>600</sub>=0.7 followed by 16 h incubation at 25 °C. Clearly visible are eGFP and mCherry fluorescence in the induced samples. The uninduced control showed no fluorescence at all, demonstrating the PBad promoter to be tight and providing very low basal transcription, what is a major advantage for the screening system. This newly designed screening approach renders the characterization of PoPS-based devices in general and switches in particular easy and robust. The low basal transcription furthermore fulfills one of the most important requirements for the designed switches, since output transmitters may only be produced in presence of an input transmitter. This helps to avoid strong "background" noise, which would extremely harden the successful interconnection of several switches. <br />
<br><br />
[[Image:TUM2010_PosControlklein.JPG|200px||thumb|left|Bacteria containing positive control]]<br />
[[Image:TUM2010_graphPosControl1.png|355px|thumb|center|Emission spectra of induced (green/red) and uninduced(black) positive control BBa_K494002 ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
Due to the time limitations of the iGEM completion we had to focus our efforts on few switches after designing the screening system. Relying on the functionality of systems occurring in nature, we choose the [[Team:TU_Munich/Modeling#Switch|HisTerm]] as well as the [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Both switches are based on known natural [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|attenuators]]. Testing synthetic and none-naturally switchable terminators in vivo are goals for future work.<br />
Delorme et al. reported the His-Terminator to be a remarkable effective Terminator with more than 99% termination efficiency.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup> The exemplary measurement below on the right confirms the high terminator efficiency. In fact, we could not detect any mCherry fluorescence in any cells containing the [[Team:TU_Munich/Modeling#Switch|HisTerm]]. Even induction of the corresponding signal transmitter RNA via IPTG did not alter the Terminator efficiency. Again time was the limiting factor and prevented us from testing more than one corresponding transmitter, although the [[Team:TU_Munich/Modeling| Modeling]] highly suggested the necessarily of finding an optimized transmitter length. Thus, the results are insufficient either to prove or to disprove the functionality of the [[Team:TU_Munich/Modeling#Switch|HisTerm]] or our concept in general.<br />
<br><br />
[[Image:TUM2010_HisSwitchklein.JPG|200px|thumb|left|Bacteria containing HisTerm]][[Image:TUM2010_HisSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing HisTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
<br />
Attaining only 90% terminator efficiency, the natural Trp [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|Attenuator]] is known be less effective than the [[Team:TU_Munich/Modeling#Switch|HisTerm]].<sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup> The graph on the right depicts our designed [[Team:TU_Munich/Modeling#Switch|TrpTerm]] characteristic efficiency of about 40 %, notably below the natural standard. Allowing 60% transcription in the “off” state excludes the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] from possible candidates for a scalable network of logic gates, due to the mentioned required "yes or no" function (see [[Team:TU_Munich/Project#Implementation| Implementation and how to connect Biobricks]]). Thus the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] is inoperative as intended, but may still be useful in other contexts. Similar to the [[Team:TU_Munich/Modeling#Switch|HisTerm]], the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] also did not react to the induction of the corresponding signal. Under circumstances, termination efficiencies altered by the transmitter are on a low range and not resolvable within observed 40% basal transcription. <br />
<br><br />
[[Image:TUM2010_TrpSwitchklein.JPG|200px|thumb|left|Bacteria containing TrpTerm]][[Image:TUM2010_TrpSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing TrpTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
<br />
Making use of our improved screening system we also carried out some ''in vivo'' kinetic measurements in addition to the end-point measurements above. In contrast to the ''in vitro'' experiments we did not obtain significant results for the characterization of our switches. As the switching process is many times faster than protein synthesis our ''in vivo'' kinetics include the synthesis of mCherry as well as its maturation. Therefore we centered our attention on end-point experiments. For more information browse the [[Team:TU_Munich/Lab#Lab_Book|lab book]]. <br><br />
Considering our ''in vivo'' measurements, neither of the tested switches showed any effect regarding the signal induction. But due to the small number of tested switches and signals this can hardly be regarded as disprove of concept. In particular in light of the recent findings by Sooncheol proving antitermination in principle using a T7 system.<sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup><br />
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<br />
==''in vitro'' Screening==<br />
To minimize the amount of disturbing factors we decided to countercheck our ''in vivo'' results with a set of ''in vitro'' measurements. While the ''in vitro'' systems are no doubt much less complex than living cells, the work with these set-ups proved to be quite as difficult.<br />
Just as with the ''in vivo'' measurements we could prove our switching system neither right nor wrong, leaving enough work for future iGEM teams.<br />
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===''in vitro translation''===<br />
<br />
Beside optimization of the reporter proteins in use, the major problem occuring in the experiments was the low capacity of the kit. The signal intensity was very low, which made it difficult to observe any signal intensity alterations, so no conclusion could be drawn from these measurements.<br />
<br />
===''in vitro'' transcription===<br />
We used two completely independent ''in vitro'' systems: Using ''E.coli'' RNA Polymerase we analyzed the His and Trp switches that had already been tested ''in vivo''. In a second set-up, we used the well-established T7 RNA Polymerase and switch based on the T7 terminator as well as several signal sequences.<br />
<br />
====T7 System====<br />
In contradiction to the results of Kang and coworkers and other groups, in our ''in vitro'' set-up the T7 terminator did not seem to terminate at all. The negative control (Promoter_Terminator_malachite binding aptamer) showed a similar increase in fluorescence as the positive control (Promoter_random sequence_malachite binding aptamer). <br />
[[Image:TUM2010_T7Result1.png|360px||thumb|left|''in vitro'' transcription measurement of T7 terminator with no signal(upper left), nonsense signal (upper right) and two different designed signals (below)]]<br />
[[Image:TUM2010_T7Result3.png|360px||thumb|right|''in vitro'' transcription measurement of positive control(upper left and T7 terminator with three different designed signals (remaining traces)]]<br />
Furthermore denaturing Polyacrylamide Gel Electrophoresis (PAGE) confirmed that there was no observeable termination of transcription. The addition of a signaling sequence led to a significantly lower increase in fluorescence, which can be attributed to the fact that both DNA sequences, switch and signal, compete for RNA Polymerases.<br />
However, there is almost no difference between the designed signals and random sequences, which is not a big surprise since there can be no antitermination if the terminator itself does not work.<br><br />
<br />
Possible explanations for the contradiction between our results and those of Kang and coworkers might be the experimental set-up and the RNA Polymerases we used. Different variants of T7 RNA Polymerase might respond in different ways to terminator structures, and the termination might be influenced by the presence or absence of cofactors, depending on the purification methods used in producing the Polymerase.<br><br><br />
<br />
This set-up offers a lot of possible experiments for the future, which we would have loved to conduct with a just a bit more time...<br />
<br />
====''E.coli'' System====<br />
<br />
Compared to the T7 System, the ''E. coli'' RPO system produced poor increases in fluorescence, indicating little RNA synthesis. It was shown that the presence of a terminator decreases, as expected, the production of downstream RNA. This result was also confirmed by denaturing PAGE. However, due to the poor changes in fluorescence we were not able to actually characterize the behaviour of our switches ''in vitro'', and the small RNA concentrations did not allow a quantitative interpretation of our gels. A major problem with this method was the low concentration of the ordered Polymerase resulting in a much weaker overall signal as comparable measurements using the T7 Polymerase. <br><br><br />
In future experiments we might try to work with smaller volumes in order to reach higher concentration of RPO and of the synthesized RNA molecules, so measuring in 96 well plate readers might be a good choice. <br />
<br />
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<br />
==Software==<br />
Although we could not show the full functionality of bioLOGICS in the lab we still want to demonstrate the potential of our approach. Hence we implemented the idea behind our logic gates in a program which illustrates how bioLOGCIS theoretically would allow the construction of complex information processing networks interconnecting BioBricks. For further details take a look at our [[Team:TU Munich/Software|Software page]].<br />
<br />
<br />
=Outlook=<br />
...<br />
future plans will also work with [[Team:TU_Munich/Glossary#Synthetic Terminator| Synthetic Terminators]], which might retrieve additional informations on what drives the process of Termination<br />
...<br />
<br />
=References=<br />
<html><a name="ref1"></a></html>[1] http://partsregistry.org/cgi/partsdb/Statistics.cgi<br />
<html><a name="ref2"></a></html>[2] https://2009.igem.org/Team:Imperial_College_London/M1 encapsulation<br />
<html><a name="ref3"></a></html>[3] https://2009.igem.org/Team:TUDelft<br />
<html><a name="ref4"></a></html>[4] https://2008.igem.org/Team:Heidelberg<br />
<html><a name="ref5"></a></html>[5] Maung Nyan Win and Christina D. Smolke, Science Oct. 2008 Vol. 322. no. 5900, pp. 456 - 460<br />
<html><a name="ref6"></a></html>[6] http://en.wikipedia.org/wiki/Logic_gate#Symbols<br />
<html><a name="ref6"></a></html>[7] http://en.wikipedia.org/wiki/Moore's_law<br />
<html><a name="ref6"></a></html>[8] http://en.wikipedia.org/wiki/Protein_interaction<br />
<html><a name="ref6"></a></html>[9] http://en.wikipedia.org/wiki/Riboswitch<br />
<html><a name="ref6"></a></html>[10] http://en.wikipedia.org/wiki/Binding_sites + http://en.wikipedia.org/wiki/Recognition_site<br />
<html><a name="ref6"></a></html>[11] irgend ein damn review über directed evolution and so on<br />
<html><a name="ref12"></a></html>[12] Delorme, Ehrlich and Renault, Regulation of Expression of the Lactococcus lactis Histidine Operon. Journal of Bacteriology, Apr. 1999, p. 2026–2037<br />
<html><a name="ref13"></a></html>[13] Trun and Trempy(2003): Fundamental Bacterial Genetics, Wiley-Blackwell, Chapter 12 <br />
<html><a name="ref14"></a></html>[14]Sooncheol Lee, Huong Minh Nguyen and Changwon Kang, Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator. Nucleic Acids Research, 2010, 1–9<br />
<html><a name="ref6"></a></html>[15] <br />
<html><a name="ref6"></a></html>[16]<br />
<br />
<!-- The idea behind our project is to change the way BioBricks have been used up to now. Over the years, many receptors and signals have been constructed as BioBricks during the annual iGEM competition, but still it is not possible to interconnect these Bricks in a complex biological network resuting in a cell, that is able to respond to its environment giving differenciated responses depending on the input signals. (Beispiel: cambridge hat das gemacht, xx dies, aber eine zelle kann nicht beides...<br><br />
We plan to create biological switches, that can function as locial gates inside a cell. Our switches rely on RNA/RNA-interactions, regulating transcriptional termination. This is a major advance of our concept, as regular switches rely on complex regulation including proteins and/or metabolites. Thus, our switches shall offer a greater robustness and their behaviour should be easier to predict. [[switch|Read more]] (hier sollte noch das hochskalieren erwähnt werden...<br><br />
These switches can further be used to build up a logical network inside a bacterial cell, enabling every scientist to connect as many functionalities (in form of BioBricks) as designated. We plan to offer simulation on each specifically designed network.<br />
<br />
<br><br>Over the years, many teams participating in the iGEM competition spent their time on constructing receptors and systems to detect a certain input that a variety of gorgeous oppurtunities is available so far.[[Image:TUM2010 network.png|thumb|300 px|right|Our visioon: A logic network inside the cell]] Nevertheless, until now it is not possible to link all those functionalities and build up a network giving differenciated responses to several of those input signals, where the molecular response depends on the complex composition of the environment a cell faces. We would like to offer this possibility to everyone.<br />
<br><br />
The logic network we want to apply will be based on devices, that can be easily upscaled and therefor offer the chance to build networks of any wanted complexicity. Our devices rely on pure RNA/RNA interactions and thus their behaviour is well predictable.<br />
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The concept we rely on for our design of RNA-switches is based on the principle of [https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation/ '''attenuation'''].<br />
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= Experiments =<br />
We designed several experiments to test our switches, all of them based on fluorescence measurements. We designed experiment setting for measurements ''in vivo'' as well as ''in vitro''. Our ''in vitro'' measurements relied on two different experiment set-ups. While the first was based on a commercial ''E. coli''-lysate, the latter was reporting on a transcriptional level only, eliminating most of the possible side-effects one could expect in the complex behaviour of a living cell or cell-lysate. [[Experiments_main|Read more]]<br />
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= Results =<br />
We ...blablabla<br />
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Text that will present our results...<br />
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= thing to move =<br />
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'''bioLOGICS: Logical RNA-Devices Enabling BioBrick-Network Formation'''<br />
'''Abstract'''<br />
Among the goals of iGEM is the creation of synthetic biological parts and their utilization to achieve novel features and behavior in biological systems. The emphasis of our project is put on this latter, "systems" aspect of iGEM. More precisely, we aim at the development and experimental demonstration of a scalable approach for the realization of logical functions in vivo.<br />
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By developing a computational biological network based on RNA logical devices we will offer everyone the opportunity to 'program' their own cells with individual AND/OR/NOT connections between BioBricks of their choice. Thereby, BioBricks can finally fulfill their original assignment as biological parts that can be connected in many different ways. We will achieve this by engineering simple and easy-to-handle switches based on predictable RNA/RNA-interactions regulating transcriptional termination. These switches represent a complete set of logical functions and are capable of forming arbitrarily complex networks.<br />
<br />
== The Experiments ==<br />
===Fluorescent proteins as reporter===<br />
<div align="justify"><br />
Our initial idea to prove our concept of antitermination was to use flourescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks ''in vivo'' as well as ''in vitro'', the latter using a translation kit based on e.coli lysate. <br>We decided to use the flourescent proteins GFP and RFP, as their spectra do not overlap and we would not measure any FRET. We would use GFP fluorescence as internal control and RFP fluorescence as signal to detect termination/antitermination by our switch we cloned in between the coding sequences of the proteins. Both protein sequences are under the control of one (L-arabinose induced) promoter.<br />
[[Image:TUM2010_gfprfp_schalter_klein.gif|center|our idea]]<br />
<br><br />
When measuring the termination of our BioBricks and the antitermination by their corresponding signal-RNA, we should be able to observe an increasing RFP emission compared to the GFP emission upon induced signal-RNA production in the cells/in the kit:<br><br />
[[Image:TUM2010_Expected_emission_spextra.png|center|our idea]]<br />
Wiith these measurements, it should also be possible to observe differences in efficiency of termination as well as antitermination between our designed switches. <br><br><br />
<br />
===Measurements with the malachite green aptamer as reporter===<br />
A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramaticly (about 3000 times) upon binding of a specific RNA-aptamer.<br><br>#<br />
[[Image:TUM2010_Malachitgruen-2.png|500px|center|our idea]]<br />
---concept to be desribed, as well as literature---<br />
<ref>refs</ref><br />
<br><br><br />
To study the switches on the transcriptional level gives the advantage, that we would have less interferences and possible artefacts. Also, we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems.<br><br />
<br />
[[Image:TUM2010_Malachit_emission.png|200px|thumb|left|Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA]]We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.<br>Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level.<br />
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<br><br><br><br><br><br><br><br><br><br><br />
<br />
Results <br />
==Flourescent proteins==<br />
Unfortunatly, we had to change the reporter construct two times during our experiments as several problems occured in our measurements:<br><br><br />
<br />
===First Try: based on the measurement plasmid pSB1A10===<br />
At the beginning, we decided to use the reporter plasmid [http://partsregistry.org/Part:pSB1A10 pSB1A10] from the registry. It consists of the fluorescent proteins eGFP and mRFP1. Each sequence includes a ribosome binding site and a stop-codon; the two genes are divided by a cloning side including the BioBrick cleavage sites.[[Image:ScreeningPlasmid1.0.PNG|300px|thumb|right|pSB1A10]]<br> In front of the eGFP sequence, the plasmid includes an arabinose-inducable promoter. The plasmid also contains an ampicilline resistence.<br><br />
We cloned our switches into the cloning site of the measurement plasmid and used an empty cloning site as control; our signal-RNAs we cloned into the [http://partsregistry.org/Part:pSB1K3 pSB1K3] vector, together with the BioBricks R0011 (Lac promoter) and B0014 (double terminator of transcription). Afterwards, we cut pSB1K3 with Aat2 and Pst1 and pSB1A10 with Nsi1 and Aat2 and ligated those fragments of each plasmid that contained our Bricks to get a Monsterplasmid.<br>We had to do so, as both plasmids contain the same ori mechanism. In addition, having both the switch and the signal RNA transcribed from the same plasmid gives us a high local concentration of the signal, once its transcription is induced.<br><br />
[[Image:TUM2010_Messplasmid_nr1.png|left|the measurement plasmid]]<br />
<br />
We transformed BL21(DE3) cells with the plasmid. We set up cultures, induced the arabinose promoter and measured the GFP and mRFP1 excitation/emission spectra within time.<br><br><br />
Unfortunatly, we were ot able to detect any RFP signal, not even in the positive control with no switch in between the GFP/RFP sequences.<br><br><br />
From these experiments, we concluded, that the mRNA of the RFP variant used was instable and rapidly degraded by RNases, so the RFP was not synthesized in the cells. This was also the conclusion from XXX...<br />
<br><br><br />
As solution to this, we decided to design a measurement plasmid ourselves:<br><br />
<hr width="300"><br />
<br><br />
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===Second Try: A measurement plasmid of our own design===<br />
To design our own plasmid to overcome the problems that occurred in our first try gave us tghe possibility to overcome several other problems:<br><br />
#<br />
#<br />
#<br />
[[Image:TUM2010_Construct_no2.png|400px|our construct|center]]<br />
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===Third Try: One promoter for each protein===<br />
We decided to use the measuremnt plasmid we developed in our second try but to clone another L-arabinose induced promoter into the plasmid, in front of our switch followed by the mCherry sequence.<br><br />
<br>[[Image:TUM2010_Construct_no3.png|left|400px|Construct #3]]In this way, we still can use GFP fluorescence as internal control, because both protein transcription is under the control of a promoter of identical design.<br />
Though we are still not able to tell exactly why our previous measurements did not work, but with this construct we measured the first time fluorescence of the mCherry protein in our positive control.<br />
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{{:Team:TU_Munich/Templates/End}}</div>Hartlmuellerhttp://2010.igem.org/Team:TU_Munich/ProjectTeam:TU Munich/Project2010-10-28T03:00:33Z<p>Hartlmueller: /* Putting it all together: the switching process */</p>
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<center><font size="5pt" color="#000000">'''bioLOGICS'''</font><font size="4pt" color="#000000">: Logical RNA-Devices Enabling BioBrick-Network Formation</font></center><hr color="black"><br><br />
= Vision=<br />
<br />
Until today, 13.628 biobrick sequences<sup>[[Team:TU_Munich/Project#ref1|&#91;1&#93;]]</sup> have been submitted to partsregistry, thereof 102 reporter units and 12 signaling bricks.<br />
Since then, people are trying to arrange these single biological building blocks in such a manner that allows producing special biotechnological products (metabolic engineering), developing biological sensory circuits (biosensors) and even giving microorganisms the ability to react on multiple environmental factors and serve both as disease indicator and drug. These examples and further promising ideas were implemented on previous iGEM-competitions.<sup>[[Team:TU_Munich/Project#ref2|&#91;2&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref3|&#91;3&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref4|&#91;4&#93;]]</sup> <br><br><br />
The idea of combining the outcome of several iGEM competitions to construct complex synthetic biological systems falls at the last hurdle - the fact, that each team uses a different principle how to access and functionally connect the respectively used biobricks. For example, it is a major challenge to create a system that uses several sensoring BioBricks from different iGEM-teams which in turn regulates reportering BioBricks from various teams. In order to combine and fully take advantage of these promising projects, our vision is to develop an adapter that allows interconnecting arbitrary biobricks on a functional level. Such a system easily allows to setup sensor-reporter circuits and interconnect them to complete biological chips... A further step towards artificial cells.<br><br><br />
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Generally speaking, the above adapter has to meet the following requirements:<br />
*'''Universality'''<br />
:The adapter has to be compatible to as many BioBricks as possible. This objective will guarantee that a large number of BioBricks can be connected.<br />
*'''Scalability'''<br />
:Once the basic design of the system is established, the construction of the system is supposed to be automated in silico. This way it will be possible to create an adapter connecting a large amount of BioBricks.<br />
*'''Biological orthogonality'''<br />
:Interference with cellular components has to be as low as possible in order to avoid unwanted and perturbing side effects.<br />
*'''Logic'''<br />
:The adapter is supposed to not only associate different BioBricks, but to functionally connect BioBricks in a precisely determined manner (including operations such as AND/OR/NOT).<br />
<br><br />
Several biological logic units, devices and circuits have been developed so far<sup>[[Team:TU_Munich/Project#ref5|&#91;5&#93;]]</sup>, but to our knowledge, none was shown to meet all requirements listed above.<br />
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<br />
=Implementation=<br />
To functionally connect BioBricks, there are several possibilities including genetic switches, riboswitches and direct protein-protein interactions. We investigated several hypothetically principles, and decided to focus our practical work on the development of a RNA-RNA interaction-based switch. These switches are capable of changing between two states, a state of antitermination and termination, and make use of highly-specific RNA-RNA interaction. In principle such a switch can fulfill all requirements mentioned previously. The following text clarifies how these switches work in detail.<br />
==How to connect BioBricks==<br />
Our adapter is a system, that activates or disables BioBricks (output BioBricks) in response to the presence of other Biobricks (input Biobricks). Our approach uses a molecular network to put this into practice and consists of four major elements:<br />
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<br><br />
{|<br />
|-<br />
|[[Image:Networks.png|center|thumb|730px|The general principle how different inputs can be connect to various outputs. For details see text.<br>Inputs (such as proteins or small molecules) are indicated on the left side. blue lines represent transmitter molecules whereas organe lines present logic gates. The type of logic gate is indicated. Green lines indicate transmitter RNA that can function as mRNA and consequently generate any output gene (indicated on the very right).]]<br />
|}<br />
In order to connect different BioBricks, our network requires four major types of components:<br />
*Input elements<br />
*Transmitter molecules<br />
*Logic gates<br />
*Output elements<br />
<br />
{{:Team:TU_Munich/Templates/InfoBoxStart}}'''Computer vs. molecular network - and our approach'''<br><br />
Logic gates in a molecular network are often compared to transistors used in a computer, where billions of transistors are incorporated<sup>[[Team:TU_Munich/Project#ref7|&#91;7&#93;]]</sup>. The main advantage on a computer chip is, all transistors share the same functional principle, and only the way connecting them in a special sequence allows specific addressing of only a subset of other transistors by an input. However, spatially fixed connections of molecular logic gates are not possible in a living cell. The "wiring" within a cell relies on the specific interaction between transmitter molecule and their corresponding logic gates, for example implemented by protein-protein/ligand-protein interactions or specific ligand-riboswitch interactions.<sup>[[Team:TU_Munich/Project#ref8|&#91;8&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref9|&#91;9&#93;]]</sup> As a result, in a cell, each occurring logic gate ("transistor") has to be different, at least in a special recognition site<sup>[[Team:TU_Munich/Project#ref10|&#91;10&#93;]]</sup> - for example like different transcription factors, recognizing different DNA-sites. Thanks to evolution, nature easily can invent a new transistor for each task - science achieves this only on a limited scale, and producing synthetic molecular logic gates artificially by either rational or evolutionary protein or riboswitch engineering, is limited to small circuits so far<sup>[[Team:TU_Munich/Project#ref11|&#91;11&#93;]]</sup>. Our project aims to establish a molecular switch as close as possible to a electronic transistor, thus sharing the same functional principle for all logic gates. At the same time, we want to design a easily exchangeable recognition site, which can individually be designed by everyone! {{:Team:TU_Munich/Templates/InfoBoxEnd}}<br />
<br />
These elements can be combined to build up a molecular network (see illustration). Each input molecule (such as a BioBrick) produces a unique transmitter molecule. All transmitters belong to the same type of molecule and share a common design. However, each transmitter molecule can only interact and activate a certain subset of logic gates. In other words, logic gates have to recognize as well as bind the corresponding transmitter molecules and are capable of producing a new output transmitter molecule. Depending on the type of the logic gate (AND, OR or NOT<sup>[[Team:TU_Munich/Project#ref6|&#91;6&#93;]]</sup>), an output transmitter is only created if both input transmitter molecules are present (AND), at least one of two input transmitters is present (OR) or if no input transmitter is present at all (NOT). Once a logic gate has produced a new output transmitter, these transmitters can in turn address another subset ("layer") of logic gates. In theory many layers of logic gates can be connected this way allowing the creation of large networks. Until this step, various transmitter molecules might have been produced. But in order to create a Biobrick output, the last layer of logic gates finally generates transmitter molecules that will not active logic gates, but will rather interact with the cell metabolism to produce a BioBrick response. In other words, the last layer of transmitter molecules is capable of regulating BioBrick formation.<br />
<br />
<br />
Summarizing, the network establishes a connection between input BioBricks and output BioBricks in a functional manner.<br />
Having addressed the basic layout of the molecular network, the next step is to determine what type of molecules can perform the required functions. We decided to use RNA, both as transmitter molecules and for constructing logic gates. Several advantages result from the utilization of RNA as the central element:<br />
*During the last years, many Biobricks were designed that are sensitive to various chemicals and substances. These BioBricks often function as a transcription factor that binds to a specific DNA sequence and consequently would be capable to produce a specific transmitter RNA molecule. Thus, in principle each BioBrick which involves transcription can be integrated in our network.<br />
*Since all logic gates are capable of producing transmitter RNA, they can also produce functional mRNA encoding any protein. This means, each BioBrick consisting of protein or RNA can be produced as an output of our network.<br />
*If RNA forms both, the transmitter molecule and the logic gates, they can specifically interact by RNA-RNA interaction, which is highly predictable compared to protein interactions. This allows to generate a library of transmitters and gates ''in silico''. Such a library is essential for the creation of large networks.<br />
*RNA production is fast and energy saving for a cell. Consequently, operating a network that only produces RNA rather than proteins will also be faster and more efficient for the host cell. Since our logic gates are based on transcription, translation and resource consuming protein production will only be required at the very last step. <br />
*As the half-time of RNA can be rather short, transmitter RNA will not accumulate within the cell and it is therefore less likely for the system to become saturated.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Design and functional principle of logic gates==<br />
The concept introduced above provides a framework that can potentially serve as an universal adapter between different BioBricks. However, the [[Team:TU_Munich/Glossary#logic gate | logic gates]] have not been specified more precisely so far. This will be done in the following section.<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, our logic gates are to possess the following characteristics:<br />
*Logic gates, such as AND, OR and NOT, have to be implemented by RNA-interaction based principles (see [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]).<br />
*All logic gates have to recognize their corresponding [[Team:TU_Munich/Glossary#Transmitter (bioLOGICS)| transmitter RNAs]] and, in response, produce an output transmitter molecule.<br />
*Logic gates should follow a basic design rule, in such a way, that their creation can be automated ''in silico''.<br />
*The response efficiency of a logic gate toward a transmitter molecule should be comparable for all logic gates to provide calculable robustness and sensitivity. This will ensure comparable molecular concentrations and functionality of large networks.<br />
*The system has to be designed for ''in vivo'' utilization at the first place. As a reference we always assumed a temperature of 37 °C and an ''E. coli'' environment.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}} <br />
In order to build logic gates for our bioLOGICS system we will first create a simple switch. A switch can be activated by one transmitter RNA and produce an output transmitter RNA. In contrast to a logic gate, a switch does not perform logic operations. However by combining switches, logic gates can be created. The following text will first describe how the developed switch works and secondly, how logic gates such as AND/OR/NOT can be created using these switches.<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Read more{{:Team:TU Munich/Templates/ToggleBoxStart2}}<br />
[[Image:toggle_switch.png|500px|thumb|center|id="hideOnReadMore"|'''A''' The basic structure of a bioLOGICS switch (left) and a transmitter molecule (right).<br>'''B'''The process of switching. See the text in the close-by "Read more" section for details.<br>Rectangles present the composition of our functional units on the level of DNA. Fringed lines represent RNA produced by RNA polymerase. The stem loop structure depicts the switchable terminator. Terminator and target site are illustrated in blue and turquoise, respectively. Recognition sites are indicated in different colors, in this case red for the input transmitter and green for the output transmitter.Each switch and or later logical unit has to be flanked by a promotor and another constitutive terminator, to allow RNA-production by RNA-polymerase in a proper way. ]]<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===Switch===<br />
[[Image:TUM2010_switch-and-transmitter.jpg|550px|right|thumb|The basic strcutrue of a switch (left) and a transmitter RNA (right). See text for details.]]<br />
Roughly speaking, a switch can be regarded as an enhanced switchable transcriptional terminator. The enhancement can be described easier by dividing a switch into its functional components: <br />
*'''Target site'''<br><br />
:The target site is the functional core element of our switches, allowing a shift between an "on" and "off" state. Since we work on the level of RNA-production (transcription), a "switchable" transcriptional terminator is suitable for this purpose. By allowing or preventing formation of a transcriptional terminator, that is by switching between termination and antitermination it is possible to represent an "off" and an "on" state, respectively. Therefore, the target site is the 5' ending of the terminator and is required for a stable terminator formation. It should be noted that this principle was also observed in nature.<br />
:To highlight and illustrate the functional principle of our switches, only the part of the terminator which is involved in interacting with a transmitter molecule and which is responsible for shifting between "on" and "off" state is called target site. The remaining terminator sequence is called terminator in the following, even if both, target site and terminator build up the terminator structure occurring in nature. <br />
:The important aspect of our switches is the fact that all switches will hold the same identical target site. Therefore having found one functional "switchable" terminator, will allow almost unlimited upscaling since this terminator can be used for a large library of switches. This is the main difference to previous works done on this field, which always required developing a new shifting principle for each switch.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup> Beside this scalability, this principle provides a comparable on/off shifting rate (responds function) for all switches, avoiding complex fine tuning of molecular networks.<br />
:To sum it up, the target site, allows to switch between an "on" and "off" state. But so far, the switch is not capable of performing specific interaction with transmitter molecules. This is where the recognition site comes into play.<br />
*'''Recognition site'''<br />
:The recognition site defines which transmitter molecule can actually interact with the switch. Therefore, a unique recognition site is generated for each switch and is positioned right upstream of the target site. In principle the recognition can be any random sequence as long as it remains unique within the molecular network.<br />
Summing up, the recognition site allows a specific interaction between switches and transmitter molecules. Once this interaction is formed, an interaction between the transmitter and the target will actually switch the state of the terminator. This allows the specific arrangement and interconnection of numerous of these switches by transmitter molecules, without changing the target site. Comparable to wires connecting many identical transistors, our target site remains the same.<br />
<br><br />
<br />
===Transmitter RNA´s===<br />
As desccribed above, transmitter RNAs are the input and output of bioLOGICS switches (compare [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]). These transmitters are short ssRNA molecules representing the "trigger" to shift switches between the "on" and "off" state. To fulfill this role, they need to posses the following properties:<br />
*A transmitter may only interact with certain switches. That is, a transmitter has to find the corresponding recognition site of a switch.<br />
*Once an interaction is established between a transmitter and a switch, a transmitter has to be capable of changing the secondary structure of a terminator and thus cause antitermination.<br />
Again, these two properties are fulfilled by two components of the transmitter:<br />
*'''Identity site'''<br />
:This site is capable of forcing an interaction between the transmitter and the switch. Therefore it is complementary to the recognition site of this switch. As the recognition site is unique within a network, so is the identity site. However, the single identity site is not capable of changing the state of the switch. That is were the trigger site comes into play.<br />
*'''Trigger site'''<br />
:Once an interaction is created by the identity site, the trigger site is capable of actually shifting the switch since it is complementary to the target site of the switch. To fulfill this role, it is placed upstream at the 5' end of the identity site. As the target site is the same for all switches, the trigger site is the same for all signals. Therefore it is important, that similar to the identity site, a trigger site cannot function on its own. That is, a single trigger site cannot shift the state of a switch without the help of an identity site.<br />
<br />
Summing up, we applied the principle introduced for the switches to the transmitter molecules. In contrast to previous approaches on this field <sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup>, we introduced the described synthetic trigger site in such a manner that it is not able to change the state of the terminator on its own, but only in combination with the identity site. So the challenge is to arrange and optimize these elementary building blocks thermodynamically, that a trigger site is only able to switch in combination with its respective identity site. This was done by ''in silico'' design using [[TU Munich/Glossary#NUPACK| NUPACK]], presented in section [[TU Munich/Modeling#in silico design based on thermodynamic calculations| in silico design]].<br />
<br />
<br><br />
<br />
===Putting it all together: the switching process===<br />
[[Image:TUM2010_switching-process.jpg|550px|right|thumb|The basic structure of a switch (left) and a transmitter RNA (right). See text for details.]]The functional principle of the designed switches is illustrated in the figure. The switch is positioned on DNA upstream of a desired output transmitter. So in the absence of a triggering transmitter molecule, transcription will be canceled by the formation of a RNA stem loop in the nascent RNA-chain. This will cause the RNA polymerase to stop transcription and fall off the DNA and consequently no output RNA will be produced. This process only relies on [[Team:TU_Munich/Glossary#Termination| rho-independent termination]].<br />
On the other hand, in the presence of a [[Team:TU_Munich/Project#RNA_transmitters | input transmitter]], this small functional RNA inhibits the stem loop formation by complementary base-pairing and hence avoids termination of transcription. In detail, the identity site (red part on transmitter) binds the recognition site (red part on switch) and serves as [[Team:TU_Munich/Glossary#Toehold|toehold]], which will thermodynamically allow the trigger site (turquoise part on transmitter) to perform a [[Team:TU_Munich/Glossary#Strand Displacement| strand displacement]] and open up the stem loop structure. Consequently the polymerase can read all the way through and form the output RNA.<br>Summing up, we use this concept to create a switch that can be toggled by a transmitter RNA molecule and in response, is able to produce another transmitter RNA.<br />
<br><br />
<br><br />
<br><br />
<br />
===From switches towards bioLOGICS logic gates===<br />
As described, each switch can be accessed by a specific RNA-transmitter molecule, illustrating the input. In turn, another RNA-transmitter molecule will be produced if the switch shifts its state. This output transmitter of one switch can serve as input transmitter for the next switch by meaningful selection and design of the respective recognition sites. This easily allows arranging several switches in specific sequences and faulty wiring - the corner stone of a logical network.<br />
<br />
To ease the building of logical networks, applying mathematical logics, e.g. Boolean logics like in computational science would be worthwhile. It is possible to establish general Boolean operators with our switches and thus build "logical modules". <br />
Since AND/OR/NOT are the most simple logic operations which can be implemented with the presented switches, and all remaining operations can be expressed by these three operators according to laws of boolean logics, we exemplary designed them.<br />
<br />
{|<br />
|-<br />
| *AND consists of a parallel circuit of two switches<br />
|-<br />
|[[Image:AND2.png|500px|thumb|center]] <br />
|-<br />
| *OR is implemented by connecting two switches in series<br />
|-<br />
|[[Image:OR2.png|500px|thumb|center]]<br />
|-<br />
| *NOT is more complex to explain. In principle, it consists only of one switch which contains its respective signal molecule intrinsic, so via intramolecular interaction, antitermination is the initial state. The signal is intrinsically of the same components as usual to allow interconnection with other logic gates.<br />
|-<br />
|[[Image:NOT2.png|500px|thumb|center]]<br />
|}<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Network construction==<br />
Designing complex biological networks based on either traditional protein engineering or our new bioLOGICS is still a complex task. We developed a software which allows the fast construction of a bioLOGICS based networks. <br><br />
To read more about this, look at our [https://2010.igem.org/Team:TU_Munich/Software Software page]<br />
<br />
=Our Objective=<br />
Putting the implementation described above into practice, will be a major challenge. For this year's iGEM competition our goal is to do the first step: design and build a switch that can be toggled by a RNA molecule. To be precise, we want to apply the design rules of our switch to modify a transcription terminator in such a way that it interacts with a second RNA molecule and, as a result, is no longer capable of forming a stem loop. This objective will require intensive ''in silico'' designing and modeling of switches based on different terminators and their corresponding transmitters. In connection to this theoretical part, we also have to test and verify the switches. For this step, we establish custom-made assays, ''in vitro'' and ''in vivo''.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Once the objective mentioned above is accomplished, these basic RNA/RNA-interactions have to be modified in such a manner that the described identity/trigger site pattern for the transmitter and the complementary recognition/target site switch composition has to be established. The most important requirement is to is to optimize these modules that the transmitter is only able to switches specifically, meaning only in the presence of both, identity AND trigger site. <br />
<br><br />
Once the objective mentioned above is accomplished, the creation of an OR gate will be rather simple since it only requires two switches. However the creation of an AND or NOT gate and optimizing the logic gates to improve their responds function will remain the goal of future work. Also the creation of small networks and the correct integration of BioBricks as input and output molecules will be future challenges. Furthermore, we wanted to rather focus on the development and the testing of our structural design of the switches, rather than developing a variety of new BioBricks.<br />
<br />
==''In silico'' design==<br />
As described above, our switches are based on certain design rules. However, there still are different structural parameters that need to be tested and optimized (length of recognition site and target site, choice of terminator, etc.).<br />
We used [[Team:TU_Munich/Project#in silico design |''in silico'' design]] and [[Team:TU_Munich/Modeling| modeling]]) to test different parameters. Furthermore we tried to use the [[Team:TU_Munich/Glossary#Antitermination|antitermination principle]] observed in nature, such as [[Team:TU_Munich/Glossary#Attenuation| attenuation]] in ''E. coli'' or [[Team:TU_Munich/Glossary#Tiny Abortive RNA´s| tiny abortive RNA´s]] of T7-phage.<br />
==Evaluation and Measurements==<br />
To evaluate the functionality of our molecular switches, we first had to establish several assays. Therefore, we improved an existing [[Team:TU_Munich/Lab#In vivo Measurements |''in vivo'' assay]] and developed an [[Team:TU_Munich/Lab#In vitro Transcription | ''in vitro'' assay]] for this purpose. For more information please refer to the [[Team:TU_Munich/Lab | lab]] section.<br />
<br><br />
<br><br />
Summarizing, the main challenges are <br />
* to find a suitable terminator construct and design a complementary trigger unit, which is only functional in combination with a specificity site - meaning an optimization of the '''thermodynamically parameters''' (see[[Team:TU_Munich/Project#in silico design| in silico design]])<br />
* to investigate whether the transmitter/switch interaction reaction is on a timescale to be competitive to terminator formation - meaning an comparison of '''kinetic parameters''' (see [[Team:TU_Munich/Modeling|Modeling page]])<br />
* to proof antitermination can be also be caused by synthetically RNA-interaction (see [[Team:TU_Munich/Glossary#Antitermination| Antitermination in nature]] and [[Team:TU_Munich/Project#Results| ''in vivo'' and ''in vitro'' measurements]] )<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
=Results=<br />
Every network starts with a basic unit. While our declared aim is to enable networks allowing fine-tuning of gene expression beyond the regular on/off, exploring such an on/off switch/signal pair is the first step towards a functional network. We constructed several units and tested their efficiency, robustness and reproducibility ''in vivo'', ''in vitro'' and ''in silico''. Furthermore we developed a software which allows easy constructions of networks based on our designed logic gates. Conclusive elaboration of a few first RNA-based logic units is the major contribution of our iGEM team.<br />
<br />
==in silico Design of Switching and Trigger Unit==<br />
===attenuation principle===<br />
<br />
<br />
==Diffusion and RNA Folding Dynamics==<br />
We estimated the diffusion time for our constructs and modeled the folding dynamics of our bioLOGICS switches including the switching process with a stochastic RNA folding program. We were able to provide better insight in their folding dynamics and proved that they are able to interrupt termination. We also optimized the switches and the corresponding signals. Furthermore, we combined the switches what resulted in a logic gate. See our [[Team:TU Munich/Modeling|Modeling page]] for further details.<br />
<br />
==''in vivo'' Functionality Screening==<br />
Since our logic gates are intended to function in living cells, ''in vivo'' measurements were essential. In a set of experiments we concentrated on two different switches based on known [[Team:TU_Munich/Glossary#Attenuation|attenuators]] from nature: the [[Team:TU_Munich/Modeling#Switch|HisTerm]] and [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Focusing on fluorescent proteins for quantifiable input and output we designed a functional and robust screening system. For greater detail see [[Team:TU_Munich/Lab#Experiment_Design|Experimental Design]]. Unfortunately, setting up a working screening system failed twice. Only in redesigning and improving the screening plasmid pSB1A10 we succeeded, but lost precious time.<br />
<br />
Ultimately, the two switches displayed remarkable differences in their terminator efficiency, but neither of them responded to their corresponding signal. However, screening one transmitter signal does not disprove the basic working principle of our system. Limited by time, we hope for future teams to take up our work and to use our improved test system that we submitted to the parts registry, for performing successful in vivo measurement.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Considering the high complexity of ''in vivo'' measurements compared to other experimental challenges, a robust and easy to handle test system for [[Team:TU_Munich/Glossary#PoPS-based devices| PoPS-based devices]] is desirable. As described in [[Team:TU_Munich/Lab#Experiment_Design|Experimental design]], we used fluorescent proteins: RFP or mCherry to measure the amount of produced output and eGFP for normalization. Our first attempt, using the screening plasmid pSB1A10, yielded no interpretable results. Switching the fluorescent protein to mCherry did not work either, but after several experimental setups we determined a transcriptional problem causing no reporter protein expression regardless of the inserted part. Thereby we demonstrated the screening plasmid pSB1A10 to be [[Team:TU_Munich/Biobricks#Falsification| malfunctioning]]. <br />
Finally a new design based on pSB1A10 lead to a functional and robust screening system (compare [[Team:TU_Munich/Parts#Screening system: Backbone BBa_K494001| Screening system: Backbone BBa_K494001]]). A second promoter with identical induction properties inside the BioBrick cloning site enforces transcription of the PoPS-based device and the mCherry output.<br />
<br />
Exemplary, the graph below on the right shows the positive control, induced and uninduced at OD<sub>600</sub>=0.7 followed by 16 h incubation at 25 °C. Clearly visible are eGFP and mCherry fluorescence in the induced samples. The uninduced control showed no fluorescence at all, demonstrating the PBad promoter to be tight and providing very low basal transcription, what is a major advantage for the screening system. This newly designed screening approach renders the characterization of PoPS-based devices in general and switches in particular easy and robust. The low basal transcription furthermore fulfills one of the most important requirements for the designed switches, since output transmitters may only be produced in presence of an input transmitter. This helps to avoid strong "background" noise, which would extremely harden the successful interconnection of several switches. <br />
<br><br />
[[Image:TUM2010_PosControlklein.JPG|200px||thumb|left|Bacteria containing positive control]]<br />
[[Image:TUM2010_graphPosControl1.png|355px|thumb|center|Emission spectra of induced (green/red) and uninduced(black) positive control BBa_K494002 ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
Due to the time limitations of the iGEM completion we had to focus our efforts on few switches after designing the screening system. Relying on the functionality of systems occurring in nature, we choose the [[Team:TU_Munich/Modeling#Switch|HisTerm]] as well as the [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Both switches are based on known natural [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|attenuators]]. Testing synthetic and none-naturally switchable terminators in vivo are goals for future work.<br />
Delorme et al. reported the His-Terminator to be a remarkable effective Terminator with more than 99% termination efficiency.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup> The exemplary measurement below on the right confirms the high terminator efficiency. In fact, we could not detect any mCherry fluorescence in any cells containing the [[Team:TU_Munich/Modeling#Switch|HisTerm]]. Even induction of the corresponding signal transmitter RNA via IPTG did not alter the Terminator efficiency. Again time was the limiting factor and prevented us from testing more than one corresponding transmitter, although the [[Team:TU_Munich/Modeling| Modeling]] highly suggested the necessarily of finding an optimized transmitter length. Thus, the results are insufficient either to prove or to disprove the functionality of the [[Team:TU_Munich/Modeling#Switch|HisTerm]] or our concept in general.<br />
<br><br />
[[Image:TUM2010_HisSwitchklein.JPG|200px|thumb|left|Bacteria containing HisTerm]][[Image:TUM2010_HisSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing HisTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
<br />
Attaining only 90% terminator efficiency, the natural Trp [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|Attenuator]] is known be less effective than the [[Team:TU_Munich/Modeling#Switch|HisTerm]].<sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup> The graph on the right depicts our designed [[Team:TU_Munich/Modeling#Switch|TrpTerm]] characteristic efficiency of about 40 %, notably below the natural standard. Allowing 60% transcription in the “off” state excludes the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] from possible candidates for a scalable network of logic gates, due to the mentioned required "yes or no" function (see [[Team:TU_Munich/Project#Implementation| Implementation and how to connect Biobricks]]). Thus the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] is inoperative as intended, but may still be useful in other contexts. Similar to the [[Team:TU_Munich/Modeling#Switch|HisTerm]], the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] also did not react to the induction of the corresponding signal. Under circumstances, termination efficiencies altered by the transmitter are on a low range and not resolvable within observed 40% basal transcription. <br />
<br><br />
[[Image:TUM2010_TrpSwitchklein.JPG|200px|thumb|left|Bacteria containing TrpTerm]][[Image:TUM2010_TrpSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing TrpTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
<br />
Making use of our improved screening system we also carried out some ''in vivo'' kinetic measurements in addition to the end-point measurements above. In contrast to the ''in vitro'' experiments we did not obtain significant results for the characterization of our switches. As the switching process is many times faster than protein synthesis our ''in vivo'' kinetics include the synthesis of mCherry as well as its maturation. Therefore we centered our attention on end-point experiments. For more information browse the [[Team:TU_Munich/Lab#Lab_Book|lab book]]. <br><br />
Considering our ''in vivo'' measurements, neither of the tested switches showed any effect regarding the signal induction. But due to the small number of tested switches and signals this can hardly be regarded as disprove of concept. In particular in light of the recent findings by Sooncheol proving antitermination in principle using a T7 system.<sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup><br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==''in vitro'' Screening==<br />
To minimize the amount of disturbing factors we decided to countercheck our ''in vivo'' results with a set of ''in vitro'' measurements. While the ''in vitro'' systems are no doubt much less complex than living cells, the work with these set-ups proved to be quite as difficult.<br />
Just as with the ''in vivo'' measurements we could prove our switching system neither right nor wrong, leaving enough work for future iGEM teams.<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
===''in vitro translation''===<br />
<br />
Beside optimization of the reporter proteins in use, the major problem occuring in the experiments was the low capacity of the kit. The signal intensity was very low, which made it difficult to observe any signal intensity alterations, so no conclusion could be drawn from these measurements.<br />
<br />
===''in vitro'' transcription===<br />
We used two completely independent ''in vitro'' systems: Using ''E.coli'' RNA Polymerase we analyzed the His and Trp switches that had already been tested ''in vivo''. In a second set-up, we used the well-established T7 RNA Polymerase and switch based on the T7 terminator as well as several signal sequences.<br />
<br />
====T7 System====<br />
In contradiction to the results of Kang and coworkers and other groups, in our ''in vitro'' set-up the T7 terminator did not seem to terminate at all. The negative control (Promoter_Terminator_malachite binding aptamer) showed a similar increase in fluorescence as the positive control (Promoter_random sequence_malachite binding aptamer). <br />
[[Image:TUM2010_T7Result1.png|360px||thumb|left|''in vitro'' transcription measurement of T7 terminator with no signal(upper left), nonsense signal (upper right) and two different designed signals (below)]]<br />
[[Image:TUM2010_T7Result3.png|360px||thumb|right|''in vitro'' transcription measurement of positive control(upper left and T7 terminator with three different designed signals (remaining traces)]]<br />
Furthermore denaturing Polyacrylamide Gel Electrophoresis (PAGE) confirmed that there was no observeable termination of transcription. The addition of a signaling sequence led to a significantly lower increase in fluorescence, which can be attributed to the fact that both DNA sequences, switch and signal, compete for RNA Polymerases.<br />
However, there is almost no difference between the designed signals and random sequences, which is not a big surprise since there can be no antitermination if the terminator itself does not work.<br><br />
<br />
Possible explanations for the contradiction between our results and those of Kang and coworkers might be the experimental set-up and the RNA Polymerases we used. Different variants of T7 RNA Polymerase might respond in different ways to terminator structures, and the termination might be influenced by the presence or absence of cofactors, depending on the purification methods used in producing the Polymerase.<br><br><br />
<br />
This set-up offers a lot of possible experiments for the future, which we would have loved to conduct with a just a bit more time...<br />
<br />
====''E.coli'' System====<br />
<br />
Compared to the T7 System, the ''E. coli'' RPO system produced poor increases in fluorescence, indicating little RNA synthesis. It was shown that the presence of a terminator decreases, as expected, the production of downstream RNA. This result was also confirmed by denaturing PAGE. However, due to the poor changes in fluorescence we were not able to actually characterize the behaviour of our switches ''in vitro'', and the small RNA concentrations did not allow a quantitative interpretation of our gels. A major problem with this method was the low concentration of the ordered Polymerase resulting in a much weaker overall signal as comparable measurements using the T7 Polymerase. <br><br><br />
In future experiments we might try to work with smaller volumes in order to reach higher concentration of RPO and of the synthesized RNA molecules, so measuring in 96 well plate readers might be a good choice. <br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Software==<br />
Although we could not show the full functionality of bioLOGICS in the lab we still want to demonstrate the potential of our approach. Hence we implemented the idea behind our logic gates in a program which illustrates how bioLOGCIS theoretically would allow the construction of complex information processing networks interconnecting BioBricks. For further details take a look at our [[Team:TU Munich/Software|Software page]].<br />
<br />
<br />
=Outlook=<br />
...<br />
future plans will also work with [[Team:TU_Munich/Glossary#Synthetic Terminator| Synthetic Terminators]], which might retrieve additional informations on what drives the process of Termination<br />
...<br />
<br />
=References=<br />
<html><a name="ref1"></a></html>[1] http://partsregistry.org/cgi/partsdb/Statistics.cgi<br />
<html><a name="ref2"></a></html>[2] https://2009.igem.org/Team:Imperial_College_London/M1 encapsulation<br />
<html><a name="ref3"></a></html>[3] https://2009.igem.org/Team:TUDelft<br />
<html><a name="ref4"></a></html>[4] https://2008.igem.org/Team:Heidelberg<br />
<html><a name="ref5"></a></html>[5] Maung Nyan Win and Christina D. Smolke, Science Oct. 2008 Vol. 322. no. 5900, pp. 456 - 460<br />
<html><a name="ref6"></a></html>[6] http://en.wikipedia.org/wiki/Logic_gate#Symbols<br />
<html><a name="ref6"></a></html>[7] http://en.wikipedia.org/wiki/Moore's_law<br />
<html><a name="ref6"></a></html>[8] http://en.wikipedia.org/wiki/Protein_interaction<br />
<html><a name="ref6"></a></html>[9] http://en.wikipedia.org/wiki/Riboswitch<br />
<html><a name="ref6"></a></html>[10] http://en.wikipedia.org/wiki/Binding_sites + http://en.wikipedia.org/wiki/Recognition_site<br />
<html><a name="ref6"></a></html>[11] irgend ein damn review über directed evolution and so on<br />
<html><a name="ref12"></a></html>[12] Delorme, Ehrlich and Renault, Regulation of Expression of the Lactococcus lactis Histidine Operon. Journal of Bacteriology, Apr. 1999, p. 2026–2037<br />
<html><a name="ref13"></a></html>[13] Trun and Trempy(2003): Fundamental Bacterial Genetics, Wiley-Blackwell, Chapter 12 <br />
<html><a name="ref14"></a></html>[14]Sooncheol Lee, Huong Minh Nguyen and Changwon Kang, Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator. Nucleic Acids Research, 2010, 1–9<br />
<html><a name="ref6"></a></html>[15] <br />
<html><a name="ref6"></a></html>[16]<br />
<br />
<!-- The idea behind our project is to change the way BioBricks have been used up to now. Over the years, many receptors and signals have been constructed as BioBricks during the annual iGEM competition, but still it is not possible to interconnect these Bricks in a complex biological network resuting in a cell, that is able to respond to its environment giving differenciated responses depending on the input signals. (Beispiel: cambridge hat das gemacht, xx dies, aber eine zelle kann nicht beides...<br><br />
We plan to create biological switches, that can function as locial gates inside a cell. Our switches rely on RNA/RNA-interactions, regulating transcriptional termination. This is a major advance of our concept, as regular switches rely on complex regulation including proteins and/or metabolites. Thus, our switches shall offer a greater robustness and their behaviour should be easier to predict. [[switch|Read more]] (hier sollte noch das hochskalieren erwähnt werden...<br><br />
These switches can further be used to build up a logical network inside a bacterial cell, enabling every scientist to connect as many functionalities (in form of BioBricks) as designated. We plan to offer simulation on each specifically designed network.<br />
<br />
<br><br>Over the years, many teams participating in the iGEM competition spent their time on constructing receptors and systems to detect a certain input that a variety of gorgeous oppurtunities is available so far.[[Image:TUM2010 network.png|thumb|300 px|right|Our visioon: A logic network inside the cell]] Nevertheless, until now it is not possible to link all those functionalities and build up a network giving differenciated responses to several of those input signals, where the molecular response depends on the complex composition of the environment a cell faces. We would like to offer this possibility to everyone.<br />
<br><br />
The logic network we want to apply will be based on devices, that can be easily upscaled and therefor offer the chance to build networks of any wanted complexicity. Our devices rely on pure RNA/RNA interactions and thus their behaviour is well predictable.<br />
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The concept we rely on for our design of RNA-switches is based on the principle of [https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation/ '''attenuation'''].<br />
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= Experiments =<br />
We designed several experiments to test our switches, all of them based on fluorescence measurements. We designed experiment setting for measurements ''in vivo'' as well as ''in vitro''. Our ''in vitro'' measurements relied on two different experiment set-ups. While the first was based on a commercial ''E. coli''-lysate, the latter was reporting on a transcriptional level only, eliminating most of the possible side-effects one could expect in the complex behaviour of a living cell or cell-lysate. [[Experiments_main|Read more]]<br />
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= Results =<br />
We ...blablabla<br />
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Text that will present our results...<br />
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= thing to move =<br />
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'''bioLOGICS: Logical RNA-Devices Enabling BioBrick-Network Formation'''<br />
'''Abstract'''<br />
Among the goals of iGEM is the creation of synthetic biological parts and their utilization to achieve novel features and behavior in biological systems. The emphasis of our project is put on this latter, "systems" aspect of iGEM. More precisely, we aim at the development and experimental demonstration of a scalable approach for the realization of logical functions in vivo.<br />
<br />
By developing a computational biological network based on RNA logical devices we will offer everyone the opportunity to 'program' their own cells with individual AND/OR/NOT connections between BioBricks of their choice. Thereby, BioBricks can finally fulfill their original assignment as biological parts that can be connected in many different ways. We will achieve this by engineering simple and easy-to-handle switches based on predictable RNA/RNA-interactions regulating transcriptional termination. These switches represent a complete set of logical functions and are capable of forming arbitrarily complex networks.<br />
<br />
== The Experiments ==<br />
===Fluorescent proteins as reporter===<br />
<div align="justify"><br />
Our initial idea to prove our concept of antitermination was to use flourescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks ''in vivo'' as well as ''in vitro'', the latter using a translation kit based on e.coli lysate. <br>We decided to use the flourescent proteins GFP and RFP, as their spectra do not overlap and we would not measure any FRET. We would use GFP fluorescence as internal control and RFP fluorescence as signal to detect termination/antitermination by our switch we cloned in between the coding sequences of the proteins. Both protein sequences are under the control of one (L-arabinose induced) promoter.<br />
[[Image:TUM2010_gfprfp_schalter_klein.gif|center|our idea]]<br />
<br><br />
When measuring the termination of our BioBricks and the antitermination by their corresponding signal-RNA, we should be able to observe an increasing RFP emission compared to the GFP emission upon induced signal-RNA production in the cells/in the kit:<br><br />
[[Image:TUM2010_Expected_emission_spextra.png|center|our idea]]<br />
Wiith these measurements, it should also be possible to observe differences in efficiency of termination as well as antitermination between our designed switches. <br><br><br />
<br />
===Measurements with the malachite green aptamer as reporter===<br />
A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramaticly (about 3000 times) upon binding of a specific RNA-aptamer.<br><br>#<br />
[[Image:TUM2010_Malachitgruen-2.png|500px|center|our idea]]<br />
---concept to be desribed, as well as literature---<br />
<ref>refs</ref><br />
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To study the switches on the transcriptional level gives the advantage, that we would have less interferences and possible artefacts. Also, we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems.<br><br />
<br />
[[Image:TUM2010_Malachit_emission.png|200px|thumb|left|Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA]]We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.<br>Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level.<br />
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<br><br><br><br><br><br><br><br><br><br><br />
<br />
Results <br />
==Flourescent proteins==<br />
Unfortunatly, we had to change the reporter construct two times during our experiments as several problems occured in our measurements:<br><br><br />
<br />
===First Try: based on the measurement plasmid pSB1A10===<br />
At the beginning, we decided to use the reporter plasmid [http://partsregistry.org/Part:pSB1A10 pSB1A10] from the registry. It consists of the fluorescent proteins eGFP and mRFP1. Each sequence includes a ribosome binding site and a stop-codon; the two genes are divided by a cloning side including the BioBrick cleavage sites.[[Image:ScreeningPlasmid1.0.PNG|300px|thumb|right|pSB1A10]]<br> In front of the eGFP sequence, the plasmid includes an arabinose-inducable promoter. The plasmid also contains an ampicilline resistence.<br><br />
We cloned our switches into the cloning site of the measurement plasmid and used an empty cloning site as control; our signal-RNAs we cloned into the [http://partsregistry.org/Part:pSB1K3 pSB1K3] vector, together with the BioBricks R0011 (Lac promoter) and B0014 (double terminator of transcription). Afterwards, we cut pSB1K3 with Aat2 and Pst1 and pSB1A10 with Nsi1 and Aat2 and ligated those fragments of each plasmid that contained our Bricks to get a Monsterplasmid.<br>We had to do so, as both plasmids contain the same ori mechanism. In addition, having both the switch and the signal RNA transcribed from the same plasmid gives us a high local concentration of the signal, once its transcription is induced.<br><br />
[[Image:TUM2010_Messplasmid_nr1.png|left|the measurement plasmid]]<br />
<br />
We transformed BL21(DE3) cells with the plasmid. We set up cultures, induced the arabinose promoter and measured the GFP and mRFP1 excitation/emission spectra within time.<br><br><br />
Unfortunatly, we were ot able to detect any RFP signal, not even in the positive control with no switch in between the GFP/RFP sequences.<br><br><br />
From these experiments, we concluded, that the mRNA of the RFP variant used was instable and rapidly degraded by RNases, so the RFP was not synthesized in the cells. This was also the conclusion from XXX...<br />
<br><br><br />
As solution to this, we decided to design a measurement plasmid ourselves:<br><br />
<hr width="300"><br />
<br><br />
<br />
===Second Try: A measurement plasmid of our own design===<br />
To design our own plasmid to overcome the problems that occurred in our first try gave us tghe possibility to overcome several other problems:<br><br />
#<br />
#<br />
#<br />
[[Image:TUM2010_Construct_no2.png|400px|our construct|center]]<br />
<br />
===Third Try: One promoter for each protein===<br />
We decided to use the measuremnt plasmid we developed in our second try but to clone another L-arabinose induced promoter into the plasmid, in front of our switch followed by the mCherry sequence.<br><br />
<br>[[Image:TUM2010_Construct_no3.png|left|400px|Construct #3]]In this way, we still can use GFP fluorescence as internal control, because both protein transcription is under the control of a promoter of identical design.<br />
Though we are still not able to tell exactly why our previous measurements did not work, but with this construct we measured the first time fluorescence of the mCherry protein in our positive control.<br />
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{{:Team:TU_Munich/Templates/End}}</div>Hartlmuellerhttp://2010.igem.org/Team:TU_Munich/ProjectTeam:TU Munich/Project2010-10-28T02:59:30Z<p>Hartlmueller: /* How to connect BioBricks */</p>
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<center><font size="5pt" color="#000000">'''bioLOGICS'''</font><font size="4pt" color="#000000">: Logical RNA-Devices Enabling BioBrick-Network Formation</font></center><hr color="black"><br><br />
= Vision=<br />
<br />
Until today, 13.628 biobrick sequences<sup>[[Team:TU_Munich/Project#ref1|&#91;1&#93;]]</sup> have been submitted to partsregistry, thereof 102 reporter units and 12 signaling bricks.<br />
Since then, people are trying to arrange these single biological building blocks in such a manner that allows producing special biotechnological products (metabolic engineering), developing biological sensory circuits (biosensors) and even giving microorganisms the ability to react on multiple environmental factors and serve both as disease indicator and drug. These examples and further promising ideas were implemented on previous iGEM-competitions.<sup>[[Team:TU_Munich/Project#ref2|&#91;2&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref3|&#91;3&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref4|&#91;4&#93;]]</sup> <br><br><br />
The idea of combining the outcome of several iGEM competitions to construct complex synthetic biological systems falls at the last hurdle - the fact, that each team uses a different principle how to access and functionally connect the respectively used biobricks. For example, it is a major challenge to create a system that uses several sensoring BioBricks from different iGEM-teams which in turn regulates reportering BioBricks from various teams. In order to combine and fully take advantage of these promising projects, our vision is to develop an adapter that allows interconnecting arbitrary biobricks on a functional level. Such a system easily allows to setup sensor-reporter circuits and interconnect them to complete biological chips... A further step towards artificial cells.<br><br><br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, the above adapter has to meet the following requirements:<br />
*'''Universality'''<br />
:The adapter has to be compatible to as many BioBricks as possible. This objective will guarantee that a large number of BioBricks can be connected.<br />
*'''Scalability'''<br />
:Once the basic design of the system is established, the construction of the system is supposed to be automated in silico. This way it will be possible to create an adapter connecting a large amount of BioBricks.<br />
*'''Biological orthogonality'''<br />
:Interference with cellular components has to be as low as possible in order to avoid unwanted and perturbing side effects.<br />
*'''Logic'''<br />
:The adapter is supposed to not only associate different BioBricks, but to functionally connect BioBricks in a precisely determined manner (including operations such as AND/OR/NOT).<br />
<br><br />
Several biological logic units, devices and circuits have been developed so far<sup>[[Team:TU_Munich/Project#ref5|&#91;5&#93;]]</sup>, but to our knowledge, none was shown to meet all requirements listed above.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
=Implementation=<br />
To functionally connect BioBricks, there are several possibilities including genetic switches, riboswitches and direct protein-protein interactions. We investigated several hypothetically principles, and decided to focus our practical work on the development of a RNA-RNA interaction-based switch. These switches are capable of changing between two states, a state of antitermination and termination, and make use of highly-specific RNA-RNA interaction. In principle such a switch can fulfill all requirements mentioned previously. The following text clarifies how these switches work in detail.<br />
==How to connect BioBricks==<br />
Our adapter is a system, that activates or disables BioBricks (output BioBricks) in response to the presence of other Biobricks (input Biobricks). Our approach uses a molecular network to put this into practice and consists of four major elements:<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
<br><br />
{|<br />
|-<br />
|[[Image:Networks.png|center|thumb|730px|The general principle how different inputs can be connect to various outputs. For details see text.<br>Inputs (such as proteins or small molecules) are indicated on the left side. blue lines represent transmitter molecules whereas organe lines present logic gates. The type of logic gate is indicated. Green lines indicate transmitter RNA that can function as mRNA and consequently generate any output gene (indicated on the very right).]]<br />
|}<br />
In order to connect different BioBricks, our network requires four major types of components:<br />
*Input elements<br />
*Transmitter molecules<br />
*Logic gates<br />
*Output elements<br />
<br />
{{:Team:TU_Munich/Templates/InfoBoxStart}}'''Computer vs. molecular network - and our approach'''<br><br />
Logic gates in a molecular network are often compared to transistors used in a computer, where billions of transistors are incorporated<sup>[[Team:TU_Munich/Project#ref7|&#91;7&#93;]]</sup>. The main advantage on a computer chip is, all transistors share the same functional principle, and only the way connecting them in a special sequence allows specific addressing of only a subset of other transistors by an input. However, spatially fixed connections of molecular logic gates are not possible in a living cell. The "wiring" within a cell relies on the specific interaction between transmitter molecule and their corresponding logic gates, for example implemented by protein-protein/ligand-protein interactions or specific ligand-riboswitch interactions.<sup>[[Team:TU_Munich/Project#ref8|&#91;8&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref9|&#91;9&#93;]]</sup> As a result, in a cell, each occurring logic gate ("transistor") has to be different, at least in a special recognition site<sup>[[Team:TU_Munich/Project#ref10|&#91;10&#93;]]</sup> - for example like different transcription factors, recognizing different DNA-sites. Thanks to evolution, nature easily can invent a new transistor for each task - science achieves this only on a limited scale, and producing synthetic molecular logic gates artificially by either rational or evolutionary protein or riboswitch engineering, is limited to small circuits so far<sup>[[Team:TU_Munich/Project#ref11|&#91;11&#93;]]</sup>. Our project aims to establish a molecular switch as close as possible to a electronic transistor, thus sharing the same functional principle for all logic gates. At the same time, we want to design a easily exchangeable recognition site, which can individually be designed by everyone! {{:Team:TU_Munich/Templates/InfoBoxEnd}}<br />
<br />
These elements can be combined to build up a molecular network (see illustration). Each input molecule (such as a BioBrick) produces a unique transmitter molecule. All transmitters belong to the same type of molecule and share a common design. However, each transmitter molecule can only interact and activate a certain subset of logic gates. In other words, logic gates have to recognize as well as bind the corresponding transmitter molecules and are capable of producing a new output transmitter molecule. Depending on the type of the logic gate (AND, OR or NOT<sup>[[Team:TU_Munich/Project#ref6|&#91;6&#93;]]</sup>), an output transmitter is only created if both input transmitter molecules are present (AND), at least one of two input transmitters is present (OR) or if no input transmitter is present at all (NOT). Once a logic gate has produced a new output transmitter, these transmitters can in turn address another subset ("layer") of logic gates. In theory many layers of logic gates can be connected this way allowing the creation of large networks. Until this step, various transmitter molecules might have been produced. But in order to create a Biobrick output, the last layer of logic gates finally generates transmitter molecules that will not active logic gates, but will rather interact with the cell metabolism to produce a BioBrick response. In other words, the last layer of transmitter molecules is capable of regulating BioBrick formation.<br />
<br />
<br />
Summarizing, the network establishes a connection between input BioBricks and output BioBricks in a functional manner.<br />
Having addressed the basic layout of the molecular network, the next step is to determine what type of molecules can perform the required functions. We decided to use RNA, both as transmitter molecules and for constructing logic gates. Several advantages result from the utilization of RNA as the central element:<br />
*During the last years, many Biobricks were designed that are sensitive to various chemicals and substances. These BioBricks often function as a transcription factor that binds to a specific DNA sequence and consequently would be capable to produce a specific transmitter RNA molecule. Thus, in principle each BioBrick which involves transcription can be integrated in our network.<br />
*Since all logic gates are capable of producing transmitter RNA, they can also produce functional mRNA encoding any protein. This means, each BioBrick consisting of protein or RNA can be produced as an output of our network.<br />
*If RNA forms both, the transmitter molecule and the logic gates, they can specifically interact by RNA-RNA interaction, which is highly predictable compared to protein interactions. This allows to generate a library of transmitters and gates ''in silico''. Such a library is essential for the creation of large networks.<br />
*RNA production is fast and energy saving for a cell. Consequently, operating a network that only produces RNA rather than proteins will also be faster and more efficient for the host cell. Since our logic gates are based on transcription, translation and resource consuming protein production will only be required at the very last step. <br />
*As the half-time of RNA can be rather short, transmitter RNA will not accumulate within the cell and it is therefore less likely for the system to become saturated.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Design and functional principle of logic gates==<br />
The concept introduced above provides a framework that can potentially serve as an universal adapter between different BioBricks. However, the [[Team:TU_Munich/Glossary#logic gate | logic gates]] have not been specified more precisely so far. This will be done in the following section.<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, our logic gates are to possess the following characteristics:<br />
*Logic gates, such as AND, OR and NOT, have to be implemented by RNA-interaction based principles (see [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]).<br />
*All logic gates have to recognize their corresponding [[Team:TU_Munich/Glossary#Transmitter (bioLOGICS)| transmitter RNAs]] and, in response, produce an output transmitter molecule.<br />
*Logic gates should follow a basic design rule, in such a way, that their creation can be automated ''in silico''.<br />
*The response efficiency of a logic gate toward a transmitter molecule should be comparable for all logic gates to provide calculable robustness and sensitivity. This will ensure comparable molecular concentrations and functionality of large networks.<br />
*The system has to be designed for ''in vivo'' utilization at the first place. As a reference we always assumed a temperature of 37 °C and an ''E. coli'' environment.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}} <br />
In order to build logic gates for our bioLOGICS system we will first create a simple switch. A switch can be activated by one transmitter RNA and produce an output transmitter RNA. In contrast to a logic gate, a switch does not perform logic operations. However by combining switches, logic gates can be created. The following text will first describe how the developed switch works and secondly, how logic gates such as AND/OR/NOT can be created using these switches.<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Read more{{:Team:TU Munich/Templates/ToggleBoxStart2}}<br />
[[Image:toggle_switch.png|500px|thumb|center|id="hideOnReadMore"|'''A''' The basic structure of a bioLOGICS switch (left) and a transmitter molecule (right).<br>'''B'''The process of switching. See the text in the close-by "Read more" section for details.<br>Rectangles present the composition of our functional units on the level of DNA. Fringed lines represent RNA produced by RNA polymerase. The stem loop structure depicts the switchable terminator. Terminator and target site are illustrated in blue and turquoise, respectively. Recognition sites are indicated in different colors, in this case red for the input transmitter and green for the output transmitter.Each switch and or later logical unit has to be flanked by a promotor and another constitutive terminator, to allow RNA-production by RNA-polymerase in a proper way. ]]<br />
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===Switch===<br />
[[Image:TUM2010_switch-and-transmitter.jpg|550px|right|thumb|The basic strcutrue of a switch (left) and a transmitter RNA (right). See text for details.]]<br />
Roughly speaking, a switch can be regarded as an enhanced switchable transcriptional terminator. The enhancement can be described easier by dividing a switch into its functional components: <br />
*'''Target site'''<br><br />
:The target site is the functional core element of our switches, allowing a shift between an "on" and "off" state. Since we work on the level of RNA-production (transcription), a "switchable" transcriptional terminator is suitable for this purpose. By allowing or preventing formation of a transcriptional terminator, that is by switching between termination and antitermination it is possible to represent an "off" and an "on" state, respectively. Therefore, the target site is the 5' ending of the terminator and is required for a stable terminator formation. It should be noted that this principle was also observed in nature.<br />
:To highlight and illustrate the functional principle of our switches, only the part of the terminator which is involved in interacting with a transmitter molecule and which is responsible for shifting between "on" and "off" state is called target site. The remaining terminator sequence is called terminator in the following, even if both, target site and terminator build up the terminator structure occurring in nature. <br />
:The important aspect of our switches is the fact that all switches will hold the same identical target site. Therefore having found one functional "switchable" terminator, will allow almost unlimited upscaling since this terminator can be used for a large library of switches. This is the main difference to previous works done on this field, which always required developing a new shifting principle for each switch.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup> Beside this scalability, this principle provides a comparable on/off shifting rate (responds function) for all switches, avoiding complex fine tuning of molecular networks.<br />
:To sum it up, the target site, allows to switch between an "on" and "off" state. But so far, the switch is not capable of performing specific interaction with transmitter molecules. This is where the recognition site comes into play.<br />
*'''Recognition site'''<br />
:The recognition site defines which transmitter molecule can actually interact with the switch. Therefore, a unique recognition site is generated for each switch and is positioned right upstream of the target site. In principle the recognition can be any random sequence as long as it remains unique within the molecular network.<br />
Summing up, the recognition site allows a specific interaction between switches and transmitter molecules. Once this interaction is formed, an interaction between the transmitter and the target will actually switch the state of the terminator. This allows the specific arrangement and interconnection of numerous of these switches by transmitter molecules, without changing the target site. Comparable to wires connecting many identical transistors, our target site remains the same.<br />
<br><br />
<br />
===Transmitter RNA´s===<br />
As desccribed above, transmitter RNAs are the input and output of bioLOGICS switches (compare [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]). These transmitters are short ssRNA molecules representing the "trigger" to shift switches between the "on" and "off" state. To fulfill this role, they need to posses the following properties:<br />
*A transmitter may only interact with certain switches. That is, a transmitter has to find the corresponding recognition site of a switch.<br />
*Once an interaction is established between a transmitter and a switch, a transmitter has to be capable of changing the secondary structure of a terminator and thus cause antitermination.<br />
Again, these two properties are fulfilled by two components of the transmitter:<br />
*'''Identity site'''<br />
:This site is capable of forcing an interaction between the transmitter and the switch. Therefore it is complementary to the recognition site of this switch. As the recognition site is unique within a network, so is the identity site. However, the single identity site is not capable of changing the state of the switch. That is were the trigger site comes into play.<br />
*'''Trigger site'''<br />
:Once an interaction is created by the identity site, the trigger site is capable of actually shifting the switch since it is complementary to the target site of the switch. To fulfill this role, it is placed upstream at the 5' end of the identity site. As the target site is the same for all switches, the trigger site is the same for all signals. Therefore it is important, that similar to the identity site, a trigger site cannot function on its own. That is, a single trigger site cannot shift the state of a switch without the help of an identity site.<br />
<br />
Summing up, we applied the principle introduced for the switches to the transmitter molecules. In contrast to previous approaches on this field <sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup>, we introduced the described synthetic trigger site in such a manner that it is not able to change the state of the terminator on its own, but only in combination with the identity site. So the challenge is to arrange and optimize these elementary building blocks thermodynamically, that a trigger site is only able to switch in combination with its respective identity site. This was done by ''in silico'' design using [[TU Munich/Glossary#NUPACK| NUPACK]], presented in section [[TU Munich/Modeling#in silico design based on thermodynamic calculations| in silico design]].<br />
<br />
<br><br />
<br />
===Putting it all together: the switching process===<br />
[[Image:TUM2010_switching-process.jpg|620px|right|thumb|The basic structure of a switch (left) and a transmitter RNA (right). See text for details.]]The functional principle of the designed switches is illustrated in the figure. The switch is positioned on DNA upstream of a desired output transmitter. So in the absence of a triggering transmitter molecule, transcription will be canceled by the formation of a RNA stem loop in the nascent RNA-chain. This will cause the RNA polymerase to stop transcription and fall off the DNA and consequently no output RNA will be produced. This process only relies on [[Team:TU_Munich/Glossary#Termination| rho-independent termination]].<br />
On the other hand, in the presence of a [[Team:TU_Munich/Project#RNA_transmitters | input transmitter]], this small functional RNA inhibits the stem loop formation by complementary base-pairing and hence avoids termination of transcription. In detail, the identity site (red part on transmitter) binds the recognition site (red part on switch) and serves as [[Team:TU_Munich/Glossary#Toehold|toehold]], which will thermodynamically allow the trigger site (turquoise part on transmitter) to perform a [[Team:TU_Munich/Glossary#Strand Displacement| strand displacement]] and open up the stem loop structure. Consequently the polymerase can read all the way through and form the output RNA.<br>Summing up, we use this concept to create a switch that can be toggled by a transmitter RNA molecule and in response, is able to produce another transmitter RNA.<br />
<br><br />
<br><br />
<br><br />
<br />
===From switches towards bioLOGICS logic gates===<br />
As described, each switch can be accessed by a specific RNA-transmitter molecule, illustrating the input. In turn, another RNA-transmitter molecule will be produced if the switch shifts its state. This output transmitter of one switch can serve as input transmitter for the next switch by meaningful selection and design of the respective recognition sites. This easily allows arranging several switches in specific sequences and faulty wiring - the corner stone of a logical network.<br />
<br />
To ease the building of logical networks, applying mathematical logics, e.g. Boolean logics like in computational science would be worthwhile. It is possible to establish general Boolean operators with our switches and thus build "logical modules". <br />
Since AND/OR/NOT are the most simple logic operations which can be implemented with the presented switches, and all remaining operations can be expressed by these three operators according to laws of boolean logics, we exemplary designed them.<br />
<br />
{|<br />
|-<br />
| *AND consists of a parallel circuit of two switches<br />
|-<br />
|[[Image:AND2.png|500px|thumb|center]] <br />
|-<br />
| *OR is implemented by connecting two switches in series<br />
|-<br />
|[[Image:OR2.png|500px|thumb|center]]<br />
|-<br />
| *NOT is more complex to explain. In principle, it consists only of one switch which contains its respective signal molecule intrinsic, so via intramolecular interaction, antitermination is the initial state. The signal is intrinsically of the same components as usual to allow interconnection with other logic gates.<br />
|-<br />
|[[Image:NOT2.png|500px|thumb|center]]<br />
|}<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Network construction==<br />
Designing complex biological networks based on either traditional protein engineering or our new bioLOGICS is still a complex task. We developed a software which allows the fast construction of a bioLOGICS based networks. <br><br />
To read more about this, look at our [https://2010.igem.org/Team:TU_Munich/Software Software page]<br />
<br />
=Our Objective=<br />
Putting the implementation described above into practice, will be a major challenge. For this year's iGEM competition our goal is to do the first step: design and build a switch that can be toggled by a RNA molecule. To be precise, we want to apply the design rules of our switch to modify a transcription terminator in such a way that it interacts with a second RNA molecule and, as a result, is no longer capable of forming a stem loop. This objective will require intensive ''in silico'' designing and modeling of switches based on different terminators and their corresponding transmitters. In connection to this theoretical part, we also have to test and verify the switches. For this step, we establish custom-made assays, ''in vitro'' and ''in vivo''.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Once the objective mentioned above is accomplished, these basic RNA/RNA-interactions have to be modified in such a manner that the described identity/trigger site pattern for the transmitter and the complementary recognition/target site switch composition has to be established. The most important requirement is to is to optimize these modules that the transmitter is only able to switches specifically, meaning only in the presence of both, identity AND trigger site. <br />
<br><br />
Once the objective mentioned above is accomplished, the creation of an OR gate will be rather simple since it only requires two switches. However the creation of an AND or NOT gate and optimizing the logic gates to improve their responds function will remain the goal of future work. Also the creation of small networks and the correct integration of BioBricks as input and output molecules will be future challenges. Furthermore, we wanted to rather focus on the development and the testing of our structural design of the switches, rather than developing a variety of new BioBricks.<br />
<br />
==''In silico'' design==<br />
As described above, our switches are based on certain design rules. However, there still are different structural parameters that need to be tested and optimized (length of recognition site and target site, choice of terminator, etc.).<br />
We used [[Team:TU_Munich/Project#in silico design |''in silico'' design]] and [[Team:TU_Munich/Modeling| modeling]]) to test different parameters. Furthermore we tried to use the [[Team:TU_Munich/Glossary#Antitermination|antitermination principle]] observed in nature, such as [[Team:TU_Munich/Glossary#Attenuation| attenuation]] in ''E. coli'' or [[Team:TU_Munich/Glossary#Tiny Abortive RNA´s| tiny abortive RNA´s]] of T7-phage.<br />
==Evaluation and Measurements==<br />
To evaluate the functionality of our molecular switches, we first had to establish several assays. Therefore, we improved an existing [[Team:TU_Munich/Lab#In vivo Measurements |''in vivo'' assay]] and developed an [[Team:TU_Munich/Lab#In vitro Transcription | ''in vitro'' assay]] for this purpose. For more information please refer to the [[Team:TU_Munich/Lab | lab]] section.<br />
<br><br />
<br><br />
Summarizing, the main challenges are <br />
* to find a suitable terminator construct and design a complementary trigger unit, which is only functional in combination with a specificity site - meaning an optimization of the '''thermodynamically parameters''' (see[[Team:TU_Munich/Project#in silico design| in silico design]])<br />
* to investigate whether the transmitter/switch interaction reaction is on a timescale to be competitive to terminator formation - meaning an comparison of '''kinetic parameters''' (see [[Team:TU_Munich/Modeling|Modeling page]])<br />
* to proof antitermination can be also be caused by synthetically RNA-interaction (see [[Team:TU_Munich/Glossary#Antitermination| Antitermination in nature]] and [[Team:TU_Munich/Project#Results| ''in vivo'' and ''in vitro'' measurements]] )<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
=Results=<br />
Every network starts with a basic unit. While our declared aim is to enable networks allowing fine-tuning of gene expression beyond the regular on/off, exploring such an on/off switch/signal pair is the first step towards a functional network. We constructed several units and tested their efficiency, robustness and reproducibility ''in vivo'', ''in vitro'' and ''in silico''. Furthermore we developed a software which allows easy constructions of networks based on our designed logic gates. Conclusive elaboration of a few first RNA-based logic units is the major contribution of our iGEM team.<br />
<br />
==in silico Design of Switching and Trigger Unit==<br />
===attenuation principle===<br />
<br />
<br />
==Diffusion and RNA Folding Dynamics==<br />
We estimated the diffusion time for our constructs and modeled the folding dynamics of our bioLOGICS switches including the switching process with a stochastic RNA folding program. We were able to provide better insight in their folding dynamics and proved that they are able to interrupt termination. We also optimized the switches and the corresponding signals. Furthermore, we combined the switches what resulted in a logic gate. See our [[Team:TU Munich/Modeling|Modeling page]] for further details.<br />
<br />
==''in vivo'' Functionality Screening==<br />
Since our logic gates are intended to function in living cells, ''in vivo'' measurements were essential. In a set of experiments we concentrated on two different switches based on known [[Team:TU_Munich/Glossary#Attenuation|attenuators]] from nature: the [[Team:TU_Munich/Modeling#Switch|HisTerm]] and [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Focusing on fluorescent proteins for quantifiable input and output we designed a functional and robust screening system. For greater detail see [[Team:TU_Munich/Lab#Experiment_Design|Experimental Design]]. Unfortunately, setting up a working screening system failed twice. Only in redesigning and improving the screening plasmid pSB1A10 we succeeded, but lost precious time.<br />
<br />
Ultimately, the two switches displayed remarkable differences in their terminator efficiency, but neither of them responded to their corresponding signal. However, screening one transmitter signal does not disprove the basic working principle of our system. Limited by time, we hope for future teams to take up our work and to use our improved test system that we submitted to the parts registry, for performing successful in vivo measurement.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Considering the high complexity of ''in vivo'' measurements compared to other experimental challenges, a robust and easy to handle test system for [[Team:TU_Munich/Glossary#PoPS-based devices| PoPS-based devices]] is desirable. As described in [[Team:TU_Munich/Lab#Experiment_Design|Experimental design]], we used fluorescent proteins: RFP or mCherry to measure the amount of produced output and eGFP for normalization. Our first attempt, using the screening plasmid pSB1A10, yielded no interpretable results. Switching the fluorescent protein to mCherry did not work either, but after several experimental setups we determined a transcriptional problem causing no reporter protein expression regardless of the inserted part. Thereby we demonstrated the screening plasmid pSB1A10 to be [[Team:TU_Munich/Biobricks#Falsification| malfunctioning]]. <br />
Finally a new design based on pSB1A10 lead to a functional and robust screening system (compare [[Team:TU_Munich/Parts#Screening system: Backbone BBa_K494001| Screening system: Backbone BBa_K494001]]). A second promoter with identical induction properties inside the BioBrick cloning site enforces transcription of the PoPS-based device and the mCherry output.<br />
<br />
Exemplary, the graph below on the right shows the positive control, induced and uninduced at OD<sub>600</sub>=0.7 followed by 16 h incubation at 25 °C. Clearly visible are eGFP and mCherry fluorescence in the induced samples. The uninduced control showed no fluorescence at all, demonstrating the PBad promoter to be tight and providing very low basal transcription, what is a major advantage for the screening system. This newly designed screening approach renders the characterization of PoPS-based devices in general and switches in particular easy and robust. The low basal transcription furthermore fulfills one of the most important requirements for the designed switches, since output transmitters may only be produced in presence of an input transmitter. This helps to avoid strong "background" noise, which would extremely harden the successful interconnection of several switches. <br />
<br><br />
[[Image:TUM2010_PosControlklein.JPG|200px||thumb|left|Bacteria containing positive control]]<br />
[[Image:TUM2010_graphPosControl1.png|355px|thumb|center|Emission spectra of induced (green/red) and uninduced(black) positive control BBa_K494002 ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
Due to the time limitations of the iGEM completion we had to focus our efforts on few switches after designing the screening system. Relying on the functionality of systems occurring in nature, we choose the [[Team:TU_Munich/Modeling#Switch|HisTerm]] as well as the [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Both switches are based on known natural [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|attenuators]]. Testing synthetic and none-naturally switchable terminators in vivo are goals for future work.<br />
Delorme et al. reported the His-Terminator to be a remarkable effective Terminator with more than 99% termination efficiency.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup> The exemplary measurement below on the right confirms the high terminator efficiency. In fact, we could not detect any mCherry fluorescence in any cells containing the [[Team:TU_Munich/Modeling#Switch|HisTerm]]. Even induction of the corresponding signal transmitter RNA via IPTG did not alter the Terminator efficiency. Again time was the limiting factor and prevented us from testing more than one corresponding transmitter, although the [[Team:TU_Munich/Modeling| Modeling]] highly suggested the necessarily of finding an optimized transmitter length. Thus, the results are insufficient either to prove or to disprove the functionality of the [[Team:TU_Munich/Modeling#Switch|HisTerm]] or our concept in general.<br />
<br><br />
[[Image:TUM2010_HisSwitchklein.JPG|200px|thumb|left|Bacteria containing HisTerm]][[Image:TUM2010_HisSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing HisTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
<br />
Attaining only 90% terminator efficiency, the natural Trp [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|Attenuator]] is known be less effective than the [[Team:TU_Munich/Modeling#Switch|HisTerm]].<sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup> The graph on the right depicts our designed [[Team:TU_Munich/Modeling#Switch|TrpTerm]] characteristic efficiency of about 40 %, notably below the natural standard. Allowing 60% transcription in the “off” state excludes the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] from possible candidates for a scalable network of logic gates, due to the mentioned required "yes or no" function (see [[Team:TU_Munich/Project#Implementation| Implementation and how to connect Biobricks]]). Thus the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] is inoperative as intended, but may still be useful in other contexts. Similar to the [[Team:TU_Munich/Modeling#Switch|HisTerm]], the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] also did not react to the induction of the corresponding signal. Under circumstances, termination efficiencies altered by the transmitter are on a low range and not resolvable within observed 40% basal transcription. <br />
<br><br />
[[Image:TUM2010_TrpSwitchklein.JPG|200px|thumb|left|Bacteria containing TrpTerm]][[Image:TUM2010_TrpSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing TrpTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
<br />
Making use of our improved screening system we also carried out some ''in vivo'' kinetic measurements in addition to the end-point measurements above. In contrast to the ''in vitro'' experiments we did not obtain significant results for the characterization of our switches. As the switching process is many times faster than protein synthesis our ''in vivo'' kinetics include the synthesis of mCherry as well as its maturation. Therefore we centered our attention on end-point experiments. For more information browse the [[Team:TU_Munich/Lab#Lab_Book|lab book]]. <br><br />
Considering our ''in vivo'' measurements, neither of the tested switches showed any effect regarding the signal induction. But due to the small number of tested switches and signals this can hardly be regarded as disprove of concept. In particular in light of the recent findings by Sooncheol proving antitermination in principle using a T7 system.<sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup><br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==''in vitro'' Screening==<br />
To minimize the amount of disturbing factors we decided to countercheck our ''in vivo'' results with a set of ''in vitro'' measurements. While the ''in vitro'' systems are no doubt much less complex than living cells, the work with these set-ups proved to be quite as difficult.<br />
Just as with the ''in vivo'' measurements we could prove our switching system neither right nor wrong, leaving enough work for future iGEM teams.<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
===''in vitro translation''===<br />
<br />
Beside optimization of the reporter proteins in use, the major problem occuring in the experiments was the low capacity of the kit. The signal intensity was very low, which made it difficult to observe any signal intensity alterations, so no conclusion could be drawn from these measurements.<br />
<br />
===''in vitro'' transcription===<br />
We used two completely independent ''in vitro'' systems: Using ''E.coli'' RNA Polymerase we analyzed the His and Trp switches that had already been tested ''in vivo''. In a second set-up, we used the well-established T7 RNA Polymerase and switch based on the T7 terminator as well as several signal sequences.<br />
<br />
====T7 System====<br />
In contradiction to the results of Kang and coworkers and other groups, in our ''in vitro'' set-up the T7 terminator did not seem to terminate at all. The negative control (Promoter_Terminator_malachite binding aptamer) showed a similar increase in fluorescence as the positive control (Promoter_random sequence_malachite binding aptamer). <br />
[[Image:TUM2010_T7Result1.png|360px||thumb|left|''in vitro'' transcription measurement of T7 terminator with no signal(upper left), nonsense signal (upper right) and two different designed signals (below)]]<br />
[[Image:TUM2010_T7Result3.png|360px||thumb|right|''in vitro'' transcription measurement of positive control(upper left and T7 terminator with three different designed signals (remaining traces)]]<br />
Furthermore denaturing Polyacrylamide Gel Electrophoresis (PAGE) confirmed that there was no observeable termination of transcription. The addition of a signaling sequence led to a significantly lower increase in fluorescence, which can be attributed to the fact that both DNA sequences, switch and signal, compete for RNA Polymerases.<br />
However, there is almost no difference between the designed signals and random sequences, which is not a big surprise since there can be no antitermination if the terminator itself does not work.<br><br />
<br />
Possible explanations for the contradiction between our results and those of Kang and coworkers might be the experimental set-up and the RNA Polymerases we used. Different variants of T7 RNA Polymerase might respond in different ways to terminator structures, and the termination might be influenced by the presence or absence of cofactors, depending on the purification methods used in producing the Polymerase.<br><br><br />
<br />
This set-up offers a lot of possible experiments for the future, which we would have loved to conduct with a just a bit more time...<br />
<br />
====''E.coli'' System====<br />
<br />
Compared to the T7 System, the ''E. coli'' RPO system produced poor increases in fluorescence, indicating little RNA synthesis. It was shown that the presence of a terminator decreases, as expected, the production of downstream RNA. This result was also confirmed by denaturing PAGE. However, due to the poor changes in fluorescence we were not able to actually characterize the behaviour of our switches ''in vitro'', and the small RNA concentrations did not allow a quantitative interpretation of our gels. A major problem with this method was the low concentration of the ordered Polymerase resulting in a much weaker overall signal as comparable measurements using the T7 Polymerase. <br><br><br />
In future experiments we might try to work with smaller volumes in order to reach higher concentration of RPO and of the synthesized RNA molecules, so measuring in 96 well plate readers might be a good choice. <br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Software==<br />
Although we could not show the full functionality of bioLOGICS in the lab we still want to demonstrate the potential of our approach. Hence we implemented the idea behind our logic gates in a program which illustrates how bioLOGCIS theoretically would allow the construction of complex information processing networks interconnecting BioBricks. For further details take a look at our [[Team:TU Munich/Software|Software page]].<br />
<br />
<br />
=Outlook=<br />
...<br />
future plans will also work with [[Team:TU_Munich/Glossary#Synthetic Terminator| Synthetic Terminators]], which might retrieve additional informations on what drives the process of Termination<br />
...<br />
<br />
=References=<br />
<html><a name="ref1"></a></html>[1] http://partsregistry.org/cgi/partsdb/Statistics.cgi<br />
<html><a name="ref2"></a></html>[2] https://2009.igem.org/Team:Imperial_College_London/M1 encapsulation<br />
<html><a name="ref3"></a></html>[3] https://2009.igem.org/Team:TUDelft<br />
<html><a name="ref4"></a></html>[4] https://2008.igem.org/Team:Heidelberg<br />
<html><a name="ref5"></a></html>[5] Maung Nyan Win and Christina D. Smolke, Science Oct. 2008 Vol. 322. no. 5900, pp. 456 - 460<br />
<html><a name="ref6"></a></html>[6] http://en.wikipedia.org/wiki/Logic_gate#Symbols<br />
<html><a name="ref6"></a></html>[7] http://en.wikipedia.org/wiki/Moore's_law<br />
<html><a name="ref6"></a></html>[8] http://en.wikipedia.org/wiki/Protein_interaction<br />
<html><a name="ref6"></a></html>[9] http://en.wikipedia.org/wiki/Riboswitch<br />
<html><a name="ref6"></a></html>[10] http://en.wikipedia.org/wiki/Binding_sites + http://en.wikipedia.org/wiki/Recognition_site<br />
<html><a name="ref6"></a></html>[11] irgend ein damn review über directed evolution and so on<br />
<html><a name="ref12"></a></html>[12] Delorme, Ehrlich and Renault, Regulation of Expression of the Lactococcus lactis Histidine Operon. Journal of Bacteriology, Apr. 1999, p. 2026–2037<br />
<html><a name="ref13"></a></html>[13] Trun and Trempy(2003): Fundamental Bacterial Genetics, Wiley-Blackwell, Chapter 12 <br />
<html><a name="ref14"></a></html>[14]Sooncheol Lee, Huong Minh Nguyen and Changwon Kang, Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator. Nucleic Acids Research, 2010, 1–9<br />
<html><a name="ref6"></a></html>[15] <br />
<html><a name="ref6"></a></html>[16]<br />
<br />
<!-- The idea behind our project is to change the way BioBricks have been used up to now. Over the years, many receptors and signals have been constructed as BioBricks during the annual iGEM competition, but still it is not possible to interconnect these Bricks in a complex biological network resuting in a cell, that is able to respond to its environment giving differenciated responses depending on the input signals. (Beispiel: cambridge hat das gemacht, xx dies, aber eine zelle kann nicht beides...<br><br />
We plan to create biological switches, that can function as locial gates inside a cell. Our switches rely on RNA/RNA-interactions, regulating transcriptional termination. This is a major advance of our concept, as regular switches rely on complex regulation including proteins and/or metabolites. Thus, our switches shall offer a greater robustness and their behaviour should be easier to predict. [[switch|Read more]] (hier sollte noch das hochskalieren erwähnt werden...<br><br />
These switches can further be used to build up a logical network inside a bacterial cell, enabling every scientist to connect as many functionalities (in form of BioBricks) as designated. We plan to offer simulation on each specifically designed network.<br />
<br />
<br><br>Over the years, many teams participating in the iGEM competition spent their time on constructing receptors and systems to detect a certain input that a variety of gorgeous oppurtunities is available so far.[[Image:TUM2010 network.png|thumb|300 px|right|Our visioon: A logic network inside the cell]] Nevertheless, until now it is not possible to link all those functionalities and build up a network giving differenciated responses to several of those input signals, where the molecular response depends on the complex composition of the environment a cell faces. We would like to offer this possibility to everyone.<br />
<br><br />
The logic network we want to apply will be based on devices, that can be easily upscaled and therefor offer the chance to build networks of any wanted complexicity. Our devices rely on pure RNA/RNA interactions and thus their behaviour is well predictable.<br />
<br><br />
<br />
The concept we rely on for our design of RNA-switches is based on the principle of [https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation/ '''attenuation'''].<br />
<br />
= Experiments =<br />
We designed several experiments to test our switches, all of them based on fluorescence measurements. We designed experiment setting for measurements ''in vivo'' as well as ''in vitro''. Our ''in vitro'' measurements relied on two different experiment set-ups. While the first was based on a commercial ''E. coli''-lysate, the latter was reporting on a transcriptional level only, eliminating most of the possible side-effects one could expect in the complex behaviour of a living cell or cell-lysate. [[Experiments_main|Read more]]<br />
<br />
= Results =<br />
We ...blablabla<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Text that will present our results...<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
= thing to move =<br />
<br />
'''bioLOGICS: Logical RNA-Devices Enabling BioBrick-Network Formation'''<br />
'''Abstract'''<br />
Among the goals of iGEM is the creation of synthetic biological parts and their utilization to achieve novel features and behavior in biological systems. The emphasis of our project is put on this latter, "systems" aspect of iGEM. More precisely, we aim at the development and experimental demonstration of a scalable approach for the realization of logical functions in vivo.<br />
<br />
By developing a computational biological network based on RNA logical devices we will offer everyone the opportunity to 'program' their own cells with individual AND/OR/NOT connections between BioBricks of their choice. Thereby, BioBricks can finally fulfill their original assignment as biological parts that can be connected in many different ways. We will achieve this by engineering simple and easy-to-handle switches based on predictable RNA/RNA-interactions regulating transcriptional termination. These switches represent a complete set of logical functions and are capable of forming arbitrarily complex networks.<br />
<br />
== The Experiments ==<br />
===Fluorescent proteins as reporter===<br />
<div align="justify"><br />
Our initial idea to prove our concept of antitermination was to use flourescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks ''in vivo'' as well as ''in vitro'', the latter using a translation kit based on e.coli lysate. <br>We decided to use the flourescent proteins GFP and RFP, as their spectra do not overlap and we would not measure any FRET. We would use GFP fluorescence as internal control and RFP fluorescence as signal to detect termination/antitermination by our switch we cloned in between the coding sequences of the proteins. Both protein sequences are under the control of one (L-arabinose induced) promoter.<br />
[[Image:TUM2010_gfprfp_schalter_klein.gif|center|our idea]]<br />
<br><br />
When measuring the termination of our BioBricks and the antitermination by their corresponding signal-RNA, we should be able to observe an increasing RFP emission compared to the GFP emission upon induced signal-RNA production in the cells/in the kit:<br><br />
[[Image:TUM2010_Expected_emission_spextra.png|center|our idea]]<br />
Wiith these measurements, it should also be possible to observe differences in efficiency of termination as well as antitermination between our designed switches. <br><br><br />
<br />
===Measurements with the malachite green aptamer as reporter===<br />
A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramaticly (about 3000 times) upon binding of a specific RNA-aptamer.<br><br>#<br />
[[Image:TUM2010_Malachitgruen-2.png|500px|center|our idea]]<br />
---concept to be desribed, as well as literature---<br />
<ref>refs</ref><br />
<br><br><br />
To study the switches on the transcriptional level gives the advantage, that we would have less interferences and possible artefacts. Also, we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems.<br><br />
<br />
[[Image:TUM2010_Malachit_emission.png|200px|thumb|left|Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA]]We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.<br>Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level.<br />
<br />
<br><br><br><br><br><br><br><br><br><br><br />
<br />
Results <br />
==Flourescent proteins==<br />
Unfortunatly, we had to change the reporter construct two times during our experiments as several problems occured in our measurements:<br><br><br />
<br />
===First Try: based on the measurement plasmid pSB1A10===<br />
At the beginning, we decided to use the reporter plasmid [http://partsregistry.org/Part:pSB1A10 pSB1A10] from the registry. It consists of the fluorescent proteins eGFP and mRFP1. Each sequence includes a ribosome binding site and a stop-codon; the two genes are divided by a cloning side including the BioBrick cleavage sites.[[Image:ScreeningPlasmid1.0.PNG|300px|thumb|right|pSB1A10]]<br> In front of the eGFP sequence, the plasmid includes an arabinose-inducable promoter. The plasmid also contains an ampicilline resistence.<br><br />
We cloned our switches into the cloning site of the measurement plasmid and used an empty cloning site as control; our signal-RNAs we cloned into the [http://partsregistry.org/Part:pSB1K3 pSB1K3] vector, together with the BioBricks R0011 (Lac promoter) and B0014 (double terminator of transcription). Afterwards, we cut pSB1K3 with Aat2 and Pst1 and pSB1A10 with Nsi1 and Aat2 and ligated those fragments of each plasmid that contained our Bricks to get a Monsterplasmid.<br>We had to do so, as both plasmids contain the same ori mechanism. In addition, having both the switch and the signal RNA transcribed from the same plasmid gives us a high local concentration of the signal, once its transcription is induced.<br><br />
[[Image:TUM2010_Messplasmid_nr1.png|left|the measurement plasmid]]<br />
<br />
We transformed BL21(DE3) cells with the plasmid. We set up cultures, induced the arabinose promoter and measured the GFP and mRFP1 excitation/emission spectra within time.<br><br><br />
Unfortunatly, we were ot able to detect any RFP signal, not even in the positive control with no switch in between the GFP/RFP sequences.<br><br><br />
From these experiments, we concluded, that the mRNA of the RFP variant used was instable and rapidly degraded by RNases, so the RFP was not synthesized in the cells. This was also the conclusion from XXX...<br />
<br><br><br />
As solution to this, we decided to design a measurement plasmid ourselves:<br><br />
<hr width="300"><br />
<br><br />
<br />
===Second Try: A measurement plasmid of our own design===<br />
To design our own plasmid to overcome the problems that occurred in our first try gave us tghe possibility to overcome several other problems:<br><br />
#<br />
#<br />
#<br />
[[Image:TUM2010_Construct_no2.png|400px|our construct|center]]<br />
<br />
===Third Try: One promoter for each protein===<br />
We decided to use the measuremnt plasmid we developed in our second try but to clone another L-arabinose induced promoter into the plasmid, in front of our switch followed by the mCherry sequence.<br><br />
<br>[[Image:TUM2010_Construct_no3.png|left|400px|Construct #3]]In this way, we still can use GFP fluorescence as internal control, because both protein transcription is under the control of a promoter of identical design.<br />
Though we are still not able to tell exactly why our previous measurements did not work, but with this construct we measured the first time fluorescence of the mCherry protein in our positive control.<br />
<br><br><br><br><br />
<br />
FORCE_TOC<br />
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{{:Team:TU_Munich/Templates/End}}</div>Hartlmuellerhttp://2010.igem.org/File:Networks.pngFile:Networks.png2010-10-28T02:57:09Z<p>Hartlmueller: uploaded a new version of "Image:Networks.png"</p>
<hr />
<div></div>Hartlmuellerhttp://2010.igem.org/Team:TU_Munich/BeyondTheLab/WikiTutorialTeam:TU Munich/BeyondTheLab/WikiTutorial2010-10-28T02:51:52Z<p>Hartlmueller: </p>
<hr />
<div>{{:Team:TU_Munich/Templates/Beginn}}<br />
<!-- Title of this page here--><br />
Social Project<br />
{{:Team:TU_Munich/Templates/Middle}}<br />
<br />
<!-- ############## WIKI-PAGE STARTS HERE ############## --><br />
<br />
One major task for every iGEM team is the creation of a wiki describing their project. For this reason the MIT is hosting a MediaWiki and every iGEM participant is allowed to create wiki pages on this server. The great advantage of this setup is that no team has to run their own server for hosting their wiki. On the other hand, team members are not allowed to change any MediaWiki settings, such as changing the skin of the wiki or installing extensions.<br />
As a part of your "Beyond the Lab" project we want to help other team getting started on their wiki. Therefore we will show how to create a team wiki that is easy to use and can still be customized as desired. Furthermore, we will also explain how to use this wiki even if somebody else of your team created the wiki. After running through this tutorial, you will have a basic framework that can be extended and enhanced later on.<br><br />
The tutorial will be divided into two parts:<br />
*'''Part I''' describes how to create and setup a team wiki at the first place. This part should be read by members in charge of the layout and design of the wiki and will require basic computer skills. An understanding of HTML and CSS will also be very helpful.<br />
*'''Part II''' focuses on the everyday usage of the wiki create in part I and gibes some general advice how to use the iGEM MediaWiki. All team member that want to contribute to their wiki should read this part.<br />
<br />
So the goal of your social project is to give every teams an easier access to the iGEM wiki: Because a wiki should simplify your life and not make you struggle!<br />
<br />
=Part I: Setup your Wiki=<br />
Before creating your own wiki, you should be aware of some aspects that you have to deal with when you create your team wiki.<br />
The intention of a wiki is to provide an easy-to-use platform where team member can enter information without having to know any HTML, CSS, etc. Secondly, most teams want to add their own style and design to their wiki. The normal way to customize MediaWiki is by using skins and extensions. As all iGEM teams share you common wiki, this is not possible. Changes done by one team would screw up the wiki of some another team. Summing up, the following text has to goals:<br />
Create a wiki that every team member can easily edit.<br />
Demonstrate how to customize the look of your wiki.<br />
==Create an easy-to-use Wiki==<br />
Although team wikis tend to vary, most of them contain two areas. A navigation area is always found as well as a content section. These two sections are the most relevant parts of your wiki since every team member should be capable of adding information or arranging your wiki pages. In this tutorial we will use a straight forward, table-based layout that will be put into practice using an invisible HTML table (see figure). Please note that HTML code within a wiki page has to be marked as such, by surrounding it with an opening "<nowiki><html></nowiki>" and a closing "<nowiki></html></nowiki>". For a very short introduction on HTML open the up the following "Read more" section.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
HTML (Hypertext Markup Language) is a computer language that tells a browser (e.g. Firefox, Internet Explorer, …) what a webpage is supposed to looks like. In fact, a HTML file comprises plain text and holds instructions, so-called tags, for the browser. Every tag is marked with brackets and has a name that specifies the tag type. The beginning of a tag always contains an opening an a closing bracket (<...>) and the ending of a tag additionally has a forward slash / (</...>). For example, <table> and </table> tell the browser that there will be a table, whereas all text between <p> and </p> will be in one paragraph. Consequently, a complete webpage is build by just stringing together several tags.<br />
It should be noted that every webpage is to be placed between a <html> and a </html> tag. Within this html-tag, there has to be one <head> … </head> that holds some invisible information about the webpage (e.g. the title of the page, the author, fonts and font-sizes, information for search robots such as Google, …). After the head-section, the body-section is declared (<body> … </body>). This is where all visible content of the page is specified (e.g. tables, paragraphs and text, images, flash content, …).<br />
Another important aspect concerning tags are so-called attributes. Attributes represent options that can be applied to certain tags. Attributes are places inside the opening brackets of a tag. For example, <table border=1> ...</table> set the width of the border of this table to 1 pixel.<br />
In order to learn more about HTML or to just look up some tags or attributes, many tutorials exist on the web. A small collection is available in the [http://en.wikipedia.org/wiki/HTML#External_links Wikipedia reference list].<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
[[Image:TUM2010_wikiHowTo_table.jpg | thumb | 765px | '''A''' The top of the page will contain a header showing a banner representing your team. Underneath will be a horizontal navigation area. Below this area on the left, the large content area will be positioned. To the right of the content will be a narrow vertical range, where you can add optional extras, such as a countdown to the jamboree, a visitor counter or some art work etc.<br />
<br>'''B''' HTML code for this table]]<br />
Another important aspect is how to apply this table layout to the wiki page. In general, a wiki software allows the user to create different pages and to add content to these pages. To view such a wiki page using a browser, the MediaWiki software has to generates the corresponding HTML page every time a page is requested. For this purpose, the MediaWiki software is supplied with a HTML framework holding the typical wiki interface and places the content of the requested page into a container within the HTML framework. So when a wiki page is edited, the only part that is actually changed lies within the container of this HTML framework. It is important to note, that in the case of the iGEM wiki this HTML framework cannot be modified. In other words, the layout and design has to be entered into the textbox on every edit page.<br><br />
But as the layout and navigation bar is identical for every page, it is useful to have these elements at one central place, rather than copying the same information on every single wiki page. For this purpose MediaWiki can handle so-called templates. These templates are wiki pages themselves and can be included into other wiki page. The import of a template into another wiki page is accomplished by placing the name of the template between and opening <nowiki>"{{:" and a closing "}}"</nowiki>, for example <nowiki>{{:Team:xyz/Templates/Layout}}</nowiki> will import the wiki page "Team:xyz/Templates/Layout". When this page is requested by a browser, the MediaWiki software will replace <nowiki>{{:Team:xyz/Templates/Layout}}</nowiki> with the content of the page Team:xyz/Templates/Layout (Please note, that MediaWiki always treats names in a case-sensitive manner). Another handy aspect about templates are so-called parameters that can serve as a placeholder for content. The great advantage araises from the fact, that you can import one template several times but each time you tell MediaWiki to replace the placeholder with another content. A placeholder or parameter is inserted by using "{{{" and "}}}" with the name of the placeholder inbetween. For example, the template page "Team:xyz/Templates/Layout" containing the placeholder <nowiki>"{{{text}}}"</nowiki> can be imported using <nowiki>{{:Team:xyz/Templates/Layout | text=This will be put into the template}}</nowiki>, where the <nowiki>{{{text}}}</nowiki> will be replaced by "This will be put into the template".<br><br />
<br />
By now we already have enough knowledge to implement the basic layout of the wiki. We will use the table layout described above and, to provide an easy-to-use wiki, we will split this table into different template page. The figure summaries the basic structure of the wiki:<br />
[[Image:TUM2010_wikiHowTo_structure.jpg | thumb | 765px | Figure illustrating the basic structure of the wiki.<br>Every box represents a wiki page, whereas the top left page is the main wiki page that holds the content. All other boxes represent template pages, that will imported into each other as indicated. For details see the following text.]]<br />
Every wiki page first includes the template ".../Templates/Header" which holds the first part of the HTML table. The bottom part of the table is include at the very last line in every wiki page (".../Templates/Footer"). Furthermore the template ".../Templates/Header" includes another template ".../Templates/Navigation" containing the navigation. The navigation itself is build by include multiple times the same template called ".../Templates/Button" where a two parameters are set each time. Looking at the template ".../Templates/Button", it can be seen that the text-parameter will eventually be the text of the link, where as the link-parameter will complete the URL "<nowiki>https://2010.igem.org/Team:xyz</nowiki>" with the page name (e.g. "<nowiki>https://2010.igem.org/Team:xyz</nowiki>" and "/Project" will be merged to "<nowiki>https://2010.igem.org/Team:xyz/Project</nowiki>").<br />
<br><br><br />
To see what the wiki actually looks like, take a look at this Team:TU_Munich/Social_Project/Demonstration. Please note, that for better visualization the table cells were colored using the bgcolor attribute in the td-tags. Also, the height was adjusted using the height-attribute. <br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}More details about the demonstration{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
This a list of all page used for the demonstration:<br />
*[[Team:TU_Munich/Social Project/Demonstration]]<br />
*[[Team:TU_Munich/Social Project/Templates/Header]]<br />
*[[Team:TU_Munich/Social Project/Templates/Footer]]<br />
*[[Team:TU Munich/Social Project/Templates/Navigation]]<br />
*[[Team:TU Munich/Social Project/Templates/Button]]<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br><br><br />
The great advantage that comes with these template pages is that it is much easier to maintain the wiki afterwards. Any member of your team can edit the wiki page without bothering about HTML and the layout of the page. Just let your team members know, that they always have to leave the first and the last line of code on every wiki page since these lines import the templates. Furthermore, with very little effort, the navigation can be changed by editing the page ".../Templates/Navigation".<br>The extensive use of templates is not only an advangtage to the every day usage, but also in the process of building the wiki. For example, if you want to add a new banner, you just have insert an <nowiki><img src="..."></nowiki> tag in the corresponding template and after saving the templates all your wiki pages will contain this new banner.<br />
<br />
<br />
<br><br><br />
<br />
Another application for templates is the easy integration of special elements such as a "Read more" section. On our wiki we use this technique to give the user a better overview of the page and to find the desired content faster. These toggle boxes use javascript and make use of the external javascript library [http://jquery.com "jQuerry"].<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}More information about "Read more" sections{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
The "Read more" boxes are based on displaying and hiding a <nowiki><div></nowiki> … <nowiki></div></nowiki>. Therefore overtime a box is used, you have to import a template page before and after the text that is supposed to be inside the box.<br><br />
To setup "Read more" boxes you have to do following steps:<br />
*Include the following javascript into a template page (e.g. .../Templates/Header):<br />
<nowiki><script type="text/javascript" src="http://code.jquery.com/jquery-latest.js"></script></nowiki><br />
<nowiki><script type="text/javascript"></nowiki><br />
<nowiki>$(document).ready(function(){</nowiki><br />
<nowiki> //Hide (Collapse) the toggle containers on load</nowiki><br />
<nowiki> $(".toggle_container").hide(); </nowiki><br />
<nowiki> //Switch the "Open" and "Close" state per click then slide up/down (depending on open/close state)</nowiki><br />
<nowiki> $("p.trigger").click(function(){</nowiki><br />
<nowiki> $(this).toggleClass("activeToggle");</nowiki><br />
<nowiki> var nextElem = $(this).next();</nowiki><br />
<nowiki> while(nextElem!= null) {</nowiki><br />
<nowiki> if(!nextElem.is(".toggle_container")) {</nowiki><br />
<nowiki> nextElem = nextElem.next();</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> else {</nowiki><br />
<nowiki> break;</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> if(nextElem.is(".toggle_container")) {</nowiki><br />
<nowiki> nextElem.slideToggle("slow");</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> return false; //Prevent the browser jump to the link anchor</nowiki><br />
<nowiki> });</nowiki><br />
<nowiki> $("a.toggle_close").click(function(){</nowiki><br />
<nowiki> var nextParent = $(this).parent();</nowiki><br />
<nowiki> while(nextParent!= null) {</nowiki><br />
<nowiki> if(!nextParent.is(".toggle_container")) {</nowiki><br />
<nowiki> nextParent = nextParent.parent();</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> else {</nowiki><br />
<nowiki> break;</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> if(nextParent.is(".toggle_container")) {</nowiki><br />
<nowiki> nextParent.slideToggle("slow");</nowiki><br />
<nowiki> var prevElem = nextParent.prev();</nowiki><br />
<nowiki> while(prevElem!= null) {</nowiki><br />
<nowiki> if(!prevElem.is("p.trigger")) {</nowiki><br />
<nowiki> prevElem = prevElem.prev();</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> else {</nowiki><br />
<nowiki> break;</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> if(prevElem.is("p.trigger")) {</nowiki><br />
<nowiki> prevElem.toggleClass("activeToggle");</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> return false; //Prevent the browser jump to the link anchor</nowiki><br />
<nowiki> });</nowiki><br />
<nowiki> });</nowiki><br />
<nowiki></script></nowiki><br />
*Create a template page (e.g. Team:xyz/Templates/ToggleBoxStart) and insert the following code:<br />
<nowiki><html></nowiki><br />
<nowiki><p class="trigger"><a class="toggle_text" href="#" style="font-size: 12px; color: #4e9d20">{{{text}}}</a></p></nowiki><br />
<nowiki><div></div></nowiki><br />
<nowiki><div class="toggle_container"></nowiki><br />
<nowiki> <div class="block"> <p align="justified"></nowiki><br />
<nowiki></html></nowiki><br />
*Create a template page (e.g. Team:xyz/Templates/ToggleBoxEnd) and insert the following code:<br />
<nowiki><html></p><a class="toggle_close" href="#" style="font-size: 12px; color: #4e9d20" align="left">Close</a></nowiki><br />
<nowiki> </div></nowiki><br />
<nowiki></div></nowiki><br />
<nowiki><br></nowiki><br />
<nowiki></html></nowiki><br />
*To style your "Read more" sections you can use optional CSS code, that you can include in the CSS section in (.../Templates/Header). To give you an example, this is the CSS code we used to style your toggle boxes:<br />
p.activeToggle<br />
{<br />
padding-left: 17px;<br />
background-image: url('https://static.igem.org/mediawiki/2010/5/56/Team_TU_Munich2010_Images_Arrow_close_small.jpg');<br />
background-repeat: no-repeat;<br />
background-position: center left;<br />
}<br />
a.toggle_close<br />
{<br />
padding-left: 17px;<br />
background-image: url('https://static.igem.org/mediawiki/2010/5/5d/Team_TU_Munich2010_Images_Cross_small.jpg');<br />
background-repeat: no-repeat;<br />
background-position: center left;<br />
float: left;<br />
text-align: left;<br />
}<br />
div.toggle_container<br />
{<br />
border-left: 1px solid #4E9D20;<br />
padding-left: 7px;<br />
margin-left: 5px;<br />
margin-bottom: 25px;<br />
}<br />
:Please note, that "a.toggle_close" refers to the close-Button displayed at the bottom of every "Read more" section, "div.toggle_container" refers to the box surrounding the content and "p.activeToggle" contains style instruction that only apply to the text of an open "Read more" section.<br><br />
The only information that your team mates will need to create a toggle box are the names of the two templates that have to be put before and after the text. A simple toggle could look like this:<br />
<nowiki>{{:Team:xyz/Templates/ToggleBoxStart | text=Read more}}</nowiki><br />
<nowiki>This is text will be hidden at the beginning, but can be read if the box is open.</nowiki><br />
<nowiki>{{:Team:xyz/Templates/ToggleBoxEnd}}</nowiki><br />
Your wiki is now ready and offers say-so-use "Read more" sections.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br><br />
==Customize and style your wiki==<br />
So far be we created the backbone of your wiki by using templates and a table layout. Besides, every teams wants to have their only style. However, in the case of an iGEM team wiki, the normal way of change the look and feel of a wiki does not work. This is due to the fact, that all teams share one big wiki and changes done to the layout will change all other team wikis as well.<br><br />
The best way of styling your wiki anyway is by using CSS (Cascading Style Sheets). To read more about CSS read the following section.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
CSS ([http://en.wikipedia.org/wiki/Css Cascading Style Sheets]) is a language that is used to describe what elements on a web page are supposed to look like. So a webpage is created using HTML code and can be styled using CSS. The CSS instruction are to be placed between an opening <nowiki>"<style>"</nowiki> and a closing <nowiki>"</style>"</nowiki> in the header of a HTML page or can be directly included in HTML tags using the "style" attribute:<br />
<br />
<nowiki><html></nowiki><br />
<nowiki><head></nowiki><br />
<nowiki><title>CSS example</title></nowiki><br />
...<br />
<nowiki><style type="text/css"></nowiki><br />
a {<br />
color: green;<br />
}<br />
a.external {<br />
color: red;<br />
}<br />
#bestImage {<br />
border: 1px solid green;<br />
width: 500px;<br />
}<br />
.red {<br />
color: red;<br />
}<br />
...<br />
<nowiki></style></nowiki><br />
<nowiki></head></nowiki><br />
<nowiki><body></nowiki><br />
<nowiki><p '''style="color: red;">Just some text</p></nowiki><br />
<nowiki></body></nowiki><br />
<nowiki></html></nowiki><br />
<br />
If the style-attribute is used, the CSS instruction will only affect this tag and all its child-tags. In case of the <nowiki>"<style>"</nowiki> block in the header section, two types of information have to be provided: What is to be styled and how is it to be styled.<br><br />
The first information is provided by so-called selectors. A selector can select an element of the page in many different ways. A selector can generally select all tags of a certain kind (e.g. all <nowiki><p></nowiki>-tags). Secondly, a selecotr can select an element that has a certain identifier. An identifier is a unique ID for an element within the webpage and is specified by the attibute "id" (e.g. <nowiki><img id="bestImage"><nowiki>). Thirdly, a selector can select a subgroup of elements, called class. HTML elements can be grouped into a classes by using the attribute "class" (e.g. <nowiki><img class="goodImages"></nowiki>).<br>A selector is always placed before the opening bracket "{". In the example above all three types have been used: "a" is a selector that selects all link elements (<nowiki><a></nowiki>-tags). "a.external" addresses all links that have been assigned to the group "external" (<nowiki><a class="external"></nowiki>-tags). "#bestImage" refers to the element that has the ID "bestImage" (e.g. <nowiki><img id="bestImage"></nowiki>-tag). And finally ".red" selects all tags within the class "red" (e.g. <nowiki><a class="red">, <p class="red">, <table class="red"></nowiki>).<br><br />
All style instruction are place inside brakets "{ ... }". They all share a common syntax. and the left side of a colon is a keyword that specifies what property is to be changed, and on the right side is the new value that will be set. An instruction is terminated by a semicolon. There are many different keywords that can set all kinds of properties (colors, borders, dimensions, visibility, text-decoration, etc.). Once you learn more about CSS, you will get to know the most important ones.<br>Again, there many tutorials about CSS on the web that go far beyond this short introduction. If you want to create a sophisticated wiki, you will definitely have to deal with CSS.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
Once you are familiar with CSS, changing the appearance of the wiki is very straight forward. The nice thing about MediaWiki is that nearly every element in the wiki framework (the big banner on the top of the page, the table of content, the title of the page, etc.) has a unique identifier or belongs to a certain class. An identifier is specified using the HTML tag id, whereas an element can be assigned to a class called "title" by using class="title". <br />
Consequently, to change the look of a certain element, you have to first get to know the name of the class or the identifier and then use this name to change the appearance using CSS.<br><br />
To find out the name of the identifier or the class, you can either take a direct look at the source of the page, or you can use a very powerful Firefox extension to easily extract this information. Take a look at the following "read more" section for details about this.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
To use Firebug you have to install the plug-in first. Open up FireFox, go to [https://addons.mozilla.org/de/firefox/addon/1843/ the Mozilla plug-in webpage], install the plug-in and restart FireFox. As soon as you start FireFox you will notice an icon with a small bug on the very bottom of the FireFox window. Clicking on it will open and close the FireBug screen.<br><br />
To find out the id or the class of a specific element on the webpage, just right-click on it and in the menu you can choose to inspect this element. The firebug screen will now show the HTML code of this element. Now, you can either look for "id=" or "name=" which will give you a name you can address this element using CSS selectors (see also the following figure).<br />
[[Image:TUM2010_FireBug.jpg| thumb | 720px | A typical FireBig screen<br>After right-clicking on the heading, it can be seen that this heading is assigned to the class "firstHeading" and can thus be styled using an appropriate selector]]<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
Once you found out the name, you have to get into CSS. Since the appearance of your wiki is very likely to be the same for all pages, we can make use of our template structure that we created before. As the template ".../Templates/Header" is imported into every wiki page, putting your CSS styling in this template will affect all pages. Just edit the .../Templates/Header page and insert your CSS code:<br />
<nowiki><style type="text/css"></nowiki><br />
body {<br />
background-color: white; /* defines the default background color of the document*/<br />
color: black; /* defines the default font-color of the document*/<br />
}<br />
a {<br />
color: black; /* defines the default color of links */<br />
}<br />
a:hover {<br />
color: gray; /* defines the default color of links hovered by the cursor */<br />
}<br />
a {<br />
color: black; /* defines the default color of links */<br />
}<br />
.firstHeading<br />
{<br />
display: none; /* hides the default heading */<br />
}<br />
...<br />
<nowiki></style></nowiki><br />
<br />
You can not only modify the framework of MediaWiki, but you can also define classes in your templates and modify them in your global css section. For example, in the button template the link is assigned to the class "button", allowing the modification of all assigned links in your global CSS section. Now, you can also change all the default elements of the iGEM wiki. But please note that changing to much might can also confuse the user. For example, you should leave default buttons such as "Login, search, …". It is also appreciated to provide a link to the iGEM mainpage (http://20xx.igem.org/) in your header section.<br />
CSS is a very powerful technique that cannot be explained in this tutorial completely. Learning more about CSS can save a lot of time when customizing your wiki. You might also want to take a look at [https://2010.igem.org/Team:TU_Delft#page=Modeling/wiki-tips-tricks this page from this year's TU Delft team], where some more iGEM-specific CSS styling is listed. You will also find many tutorials or look-up pages about CSS on the web. And don't forget to take a look at previous teams, they might inspire you or have that piece of code you where looking for...<br />
<br><br><br />
Congratulations! You made it through the tutorial. Now you are ready to play around with CSS and add some more eye candy in your templates to produce a nice looking wiki. Have fun and don't hesitate to try new things, you can always go back using the MediaWiki history!<br />
<br />
=Part II: How to use the iGEM MediaWiki=<br />
<br />
This part will give you a short introduction, how to use the iGEM wiki and will gibe you some advice that will save your time and your nervs. Furthermore it will give some hints, specific to a wiki created according to the above tutorial. <br />
==Login in==<br />
First of all, you always have to be login into the iGEM wiki before you can change your page. Just click the log in button on the top of this page or any other iGEM page and make sure you have your account set up.<br />
==Creating a new wiki page==<br />
There are several ways of creating a new page. The easiest way is to type the name of the new page into your browser, for example <nowiki>http://20xx.igem.org/Team:xxx/bane of the page you want to create.</nowiki>. Please note, that all names are treated in a case sensitive manner! After typing in the name, just click "Create" or "Edit" and you can add content to your newly created page. Hit "Save" to save the new page.<br><br />
Since all iGEM teams share one common wiki, it is important that every page you create is in your namespace. In other words, all pages you create have to have your team name as a prefix. For instance, all page of our wiki are in the namespace "Team:TU_Munich".<br><br />
One other important aspect is, that at the beginning and at the end of every wiki page, your have to link to so-called template pages that contain all your styling and layout of your wiki. Please ask your team member in charge of the wiki to add these lines to your new page, or take a look at an existing page.<br />
==Adding content to a wiki page==<br />
Before you start adding a lot of text into the wiki page please make yourself familiar with the Markup language of MediaWiki. MediaWiki offers a very intuitive way to mark headings, pictures, etc. As the Wikipedia is also based on MediaWiki, might already be familiar with these commands. However, you still want to take a look at the [http://en.wikipedia.org/wiki/Help:Wiki_markup Wikipedia help page] or the [http://www.mediawiki.org/wiki/Help:Contents MediaWiki documentation].<br><br />
MediaWiki features a powerful history. You can undo any changed to a page or any replacement of a file. Make sure you are familiar with this feature.<br />
<br />
==Uploading files==<br />
In case you have to upload a picture or any other file you should be aware of some aspects. To upload a file you can scroll down on any wiki page and click on "upload file". Alternatively you can link to a picture on your page before you even uploaded it. MediaWiki will no show a red link at the corresponding page. Clicking on it will also get you to the upload page.<br><br />
For security reasons, the iGEM wiki does not accept any type of file. Refer to the upload page to see a list of allowed file extensions. Make sure whether you can upload a file in advance, before you spend time generating files that you cannot send to the iGEM server.<br><br />
Another iGEM-specific aspect, is that all teams share one common pool to upload pictures and other files. Your team should agree on a prefix that you add to all your filenames. In case you upload a picture named "team.jpg", chances a high that by accident another team will replace your file. Use "OUR_COLLEGE_20xx_team.jpg" instead!<br />
==Editing your navigation bar==<br />
As the navigation section is the same for all wiki pages, it can be stored at one central page. In case your wiki was created according to this tutorial, the computer expert in your team can give the name of the corresponding page where you can edit the navigation. In this page you will find entries similar to "<nowiki>{{:Team:xxx/Templates/Button | text=Home | link=/}}</nowiki>". Each entry resembles one button or link to a page. Please note, that your team members might have changed the exact spelling. In the above example, you can just add a new button to your navigation by copying a new entry "<nowiki>{{:Team:xxx/Templates/Button | text=Home | link=/}}</nowiki>". Now replace "Home" by the text you want to appear in the button. The target of your button will be specified by "link=". Change "/" to "/Modeling" to link to the page "Modeling" in our namespace.<br />
==Using "Read more" sections==<br />
With the help of "Read more" sections you can give the user a better overview of the page. Your team member in charge of the wiki can tell you more details. In general, you only have to add two lines to your page to create a "Read more" box:<br />
*Place "<nowiki>{{:Team:xyz/Templates/ToggleBoxStart | text=Read more}}</nowiki> before the content you want to be inside the box. Note that you can change the text "Read more" to some custom words by change the text after text=<br />
*Place "<nowiki>{{:Team:xyz/Templates/ToggleBoxEnd}}</nowiki> behind the text that you want to be within the box.<br />
To give you an example, a simple toggle could look something like this:<br />
<nowiki>{{:Team:xyz/Templates/ToggleBoxStart | text=Read more}}</nowiki><br />
<nowiki>This is text will be hidden at the beginning, but can be read if the box is open.</nowiki><br />
<nowiki>{{:Team:xyz/Templates/ToggleBoxEnd}}</nowiki><br />
Your wiki is now ready and offers say-so-use "Read more" sections.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
<br />
<!-- ############## WIKI-PAGE STOPS HERE ############## --><br />
{{:Team:TU_Munich/Templates/End}}</div>Hartlmuellerhttp://2010.igem.org/Team:TU_Munich/BeyondTheLab/WikiTutorialTeam:TU Munich/BeyondTheLab/WikiTutorial2010-10-28T02:49:46Z<p>Hartlmueller: </p>
<hr />
<div>{{:Team:TU_Munich/Templates/Beginn}}<br />
<!-- Title of this page here--><br />
Social Project<br />
{{:Team:TU_Munich/Templates/Middle}}<br />
<br />
<!-- ############## WIKI-PAGE STARTS HERE ############## --><br />
<br />
One major task for every iGEM team is the creation of a wiki describing their project. For this reason the MIT is hosting a MediaWiki and every iGEM participant is allowed to create wiki pages on this server. The great advantage of this setup is that no team has to run their own server for hosting their wiki. On the other hand, team members are not allowed to change any MediaWiki settings, such as changing the skin of the wiki or installing extensions.<br />
As a part of your "Beyond the Lab" project we want to help other team getting started on their wiki. Therefore we will show how to create a team wiki that is easy to use and can still be customized as desired. Furthermore, we will also explain how to use this wiki even if somebody else of your team created the wiki. After running through this tutorial, you will have a basic framework that can be extended and enhanced later on.<br><br />
The tutorial will be divided into two parts:<br />
*'''Part I''' describes how to create and setup a team wiki at the first place. This part should be read by members in charge of the layout and design of the wiki and will require basic computer skills. An understanding of HTML and CSS will also be very helpful.<br />
*'''Part II''' focuses on the everyday usage of the wiki create in part I and gibes some general advice how to use the iGEM MediaWiki. All team member that want to contribute to their wiki should read this part.<br />
<br />
So the goal of your social project is to give every teams an easier access to the iGEM wiki: Because a wiki should simplify your life and not make you struggle!<br />
<br />
=Part I: Setup your Wiki=<br />
Before creating your own wiki, you should be aware of some aspects that you have to deal with when you create your team wiki.<br />
The intention of a wiki is to provide an easy-to-use platform where team member can enter information without having to know any HTML, CSS, etc. Secondly, most teams want to add their own style and design to their wiki. The normal way to customize MediaWiki is by using skins and extensions. As all iGEM teams share you common wiki, this is not possible. Changes done by one team would screw up the wiki of some another team. Summing up, the following text has to goals:<br />
Create a wiki that every team member can easily edit.<br />
Demonstrate how to customize the look of your wiki.<br />
==Create an easy-to-use Wiki==<br />
Although team wikis tend to vary, most of them contain two areas. A navigation area is always found as well as a content section. These two sections are the most relevant parts of your wiki since every team member should be capable of adding information or arranging your wiki pages. In this tutorial we will use a straight forward, table-based layout that will be put into practice using an invisible HTML table (see figure). Please note that HTML code within a wiki page has to be marked as such, by surrounding it with an opening "<nowiki><html></nowiki>" and a closing "<nowiki></html></nowiki>". For a very short introduction on HTML open the up the following "Read more" section.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
HTML (Hypertext Markup Language) is a computer language that tells a browser (e.g. Firefox, Internet Explorer, …) what a webpage is supposed to looks like. In fact, a HTML file comprises plain text and holds instructions, so-called tags, for the browser. Every tag is marked with brackets and has a name that specifies the tag type. The beginning of a tag always contains an opening an a closing bracket (<...>) and the ending of a tag additionally has a forward slash / (</...>). For example, <table> and </table> tell the browser that there will be a table, whereas all text between <p> and </p> will be in one paragraph. Consequently, a complete webpage is build by just stringing together several tags.<br />
It should be noted that every webpage is to be placed between a <html> and a </html> tag. Within this html-tag, there has to be one <head> … </head> that holds some invisible information about the webpage (e.g. the title of the page, the author, fonts and font-sizes, information for search robots such as Google, …). After the head-section, the body-section is declared (<body> … </body>). This is where all visible content of the page is specified (e.g. tables, paragraphs and text, images, flash content, …).<br />
Another important aspect concerning tags are so-called attributes. Attributes represent options that can be applied to certain tags. Attributes are places inside the opening brackets of a tag. For example, <table border=1> ...</table> set the width of the border of this table to 1 pixel.<br />
In order to learn more about HTML or to just look up some tags or attributes, many tutorials exist on the web. A small collection is available in the [http://en.wikipedia.org/wiki/HTML#External_links Wikipedia reference list].<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
[[Image:TUM2010_wikiHowTo_table.jpg | thumb | 765px | '''A''' The top of the page will contain a header showing a banner representing your team. Underneath will be a horizontal navigation area. Below this area on the left, the large content area will be positioned. To the right of the content will be a narrow vertical range, where you can add optional extras, such as a countdown to the jamboree, a visitor counter or some art work etc.<br />
<br>'''B''' HTML code for this table]]<br />
Another important aspect is how to apply this table layout to the wiki page. In general, a wiki software allows the user to create different pages and to add content to these pages. To view such a wiki page using a browser, the MediaWiki software has to generates the corresponding HTML page every time a page is requested. For this purpose, the MediaWiki software is supplied with a HTML framework holding the typical wiki interface and places the content of the requested page into a container within the HTML framework. So when a wiki page is edited, the only part that is actually changed lies within the container of this HTML framework. It is important to note, that in the case of the iGEM wiki this HTML framework cannot be modified. In other words, the layout and design has to be entered into the textbox on every edit page.<br><br />
But as the layout and navigation bar is identical for every page, it is useful to have these elements at one central place, rather than copying the same information on every single wiki page. For this purpose MediaWiki can handle so-called templates. These templates are wiki pages themselves and can be included into other wiki page. The import of a template into another wiki page is accomplished by placing the name of the template between and opening <nowiki>"{{:" and a closing "}}"</nowiki>, for example <nowiki>{{:Team:xyz/Templates/Layout}}</nowiki> will import the wiki page "Team:xyz/Templates/Layout". When this page is requested by a browser, the MediaWiki software will replace <nowiki>{{:Team:xyz/Templates/Layout}}</nowiki> with the content of the page Team:xyz/Templates/Layout (Please note, that MediaWiki always treats names in a case-sensitive manner). Another handy aspect about templates are so-called parameters that can serve as a placeholder for content. The great advantage araises from the fact, that you can import one template several times but each time you tell MediaWiki to replace the placeholder with another content. A placeholder or parameter is inserted by using "{{{" and "}}}" with the name of the placeholder inbetween. For example, the template page "Team:xyz/Templates/Layout" containing the placeholder <nowiki>"{{{text}}}"</nowiki> can be imported using <nowiki>{{:Team:xyz/Templates/Layout | text=This will be put into the template}}</nowiki>, where the <nowiki>{{{text}}}</nowiki> will be replaced by "This will be put into the template".<br><br />
<br />
By now we already have enough knowledge to implement the basic layout of the wiki. We will use the table layout described above and, to provide an easy-to-use wiki, we will split this table into different template page. The figure summaries the basic structure of the wiki:<br />
[[Image:TUM2010_wikiHowTo_structure.jpg | thumb | 765px | Figure illustrating the basic structure of the wiki.<br>Every box represents a wiki page, whereas the top left page is the main wiki page that holds the content. All other boxes represent template pages, that will imported into each other as indicated. For details see the following text.]]<br />
Every wiki page first includes the template ".../Templates/Header" which holds the first part of the HTML table. The bottom part of the table is include at the very last line in every wiki page (".../Templates/Footer"). Furthermore the template ".../Templates/Header" includes another template ".../Templates/Navigation" containing the navigation. The navigation itself is build by include multiple times the same template called ".../Templates/Button" where a two parameters are set each time. Looking at the template ".../Templates/Button", it can be seen that the text-parameter will eventually be the text of the link, where as the link-parameter will complete the URL "<nowiki>https://2010.igem.org/Team:xyz</nowiki>" with the page name (e.g. "<nowiki>https://2010.igem.org/Team:xyz</nowiki>" and "/Project" will be merged to "<nowiki>https://2010.igem.org/Team:xyz/Project</nowiki>").<br />
<br><br><br />
To see what the wiki actually looks like, take a look at this Team:TU_Munich/Social_Project/Demonstration. Please note, that for better visualization the table cells were colored using the bgcolor attribute in the td-tags. Also, the height was adjusted using the height-attribute. <br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}More details about the demonstration{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
This a list of all page used for the demonstration:<br />
*[[Team:TU_Munich/Social Project/Demonstration]]<br />
*[[Team:TU_Munich/Social Project/Templates/Header]]<br />
*[[Team:TU_Munich/Social Project/Templates/Footer]]<br />
*[[Team:TU Munich/Social Project/Templates/Navigation]]<br />
*[[Team:TU Munich/Social Project/Templates/Button]]<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br><br><br />
The great advantage that comes with these template pages is that it is much easier to maintain the wiki afterwards. Any member of your team can edit the wiki page without bothering about HTML and the layout of the page. Just let your team members know, that they always have to leave the first and the last line of code on every wiki page since these lines import the templates. Furthermore, with very little effort, the navigation can be changed by editing the page ".../Templates/Navigation".<br>The extensive use of templates is not only an advangtage to the every day usage, but also in the process of building the wiki. For example, if you want to add a new banner, you just have insert an <nowiki><img src="..."></nowiki> tag in the corresponding template and after saving the templates all your wiki pages will contain this new banner.<br />
<br />
<br />
<br><br><br />
<br />
Another application for templates is the easy integration of special elements such as a "Read more" section. On our wiki we use this technique to give the user a better overview of the page and to find the desired content faster. These toggle boxes use javascript and make use of the external javascript library [http://jquery.com "jQuerry"].<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}More information about "Read more" sections{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
The "Read more" boxes are based on displaying and hiding a <nowiki><div></nowiki> … <nowiki></div></nowiki>. Therefore overtime a box is used, you have to import a template page before and after the text that is supposed to be inside the box.<br><br />
To setup "Read more" boxes you have to do following steps:<br />
*Include the following javascript into a template page (e.g. .../Templates/Header):<br />
<nowiki><script type="text/javascript" src="http://code.jquery.com/jquery-latest.js"></script></nowiki><br />
<nowiki><script type="text/javascript"></nowiki><br />
<nowiki>$(document).ready(function(){</nowiki><br />
<nowiki> //Hide (Collapse) the toggle containers on load</nowiki><br />
<nowiki> $(".toggle_container").hide(); </nowiki><br />
<nowiki> //Switch the "Open" and "Close" state per click then slide up/down (depending on open/close state)</nowiki><br />
<nowiki> $("p.trigger").click(function(){</nowiki><br />
<nowiki> $(this).toggleClass("activeToggle");</nowiki><br />
<nowiki> var nextElem = $(this).next();</nowiki><br />
<nowiki> while(nextElem!= null) {</nowiki><br />
<nowiki> if(!nextElem.is(".toggle_container")) {</nowiki><br />
<nowiki> nextElem = nextElem.next();</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> else {</nowiki><br />
<nowiki> break;</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> if(nextElem.is(".toggle_container")) {</nowiki><br />
<nowiki> nextElem.slideToggle("slow");</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> return false; //Prevent the browser jump to the link anchor</nowiki><br />
<nowiki> });</nowiki><br />
<nowiki> $("a.toggle_close").click(function(){</nowiki><br />
<nowiki> var nextParent = $(this).parent();</nowiki><br />
<nowiki> while(nextParent!= null) {</nowiki><br />
<nowiki> if(!nextParent.is(".toggle_container")) {</nowiki><br />
<nowiki> nextParent = nextParent.parent();</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> else {</nowiki><br />
<nowiki> break;</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> if(nextParent.is(".toggle_container")) {</nowiki><br />
<nowiki> nextParent.slideToggle("slow");</nowiki><br />
<nowiki> var prevElem = nextParent.prev();</nowiki><br />
<nowiki> while(prevElem!= null) {</nowiki><br />
<nowiki> if(!prevElem.is("p.trigger")) {</nowiki><br />
<nowiki> prevElem = prevElem.prev();</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> else {</nowiki><br />
<nowiki> break;</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> if(prevElem.is("p.trigger")) {</nowiki><br />
<nowiki> prevElem.toggleClass("activeToggle");</nowiki><br />
<nowiki> }</nowiki><br />
<nowiki> }<br />
<nowiki> return false; //Prevent the browser jump to the link anchor</nowiki><br />
<nowiki> });</nowiki><br />
<nowiki> });</nowiki><br />
<nowiki></script></nowiki><br />
*Create a template page (e.g. Team:xyz/Templates/ToggleBoxStart) and insert the following code:<br />
<nowiki><html></nowiki><br />
<nowiki><p class="trigger"><a class="toggle_text" href="#" style="font-size: 12px; color: #4e9d20">{{{text}}}</a></p></nowiki><br />
<nowiki><div></div></nowiki><br />
<nowiki><div class="toggle_container"></nowiki><br />
<nowiki> <div class="block"> <p align="justified"></nowiki><br />
<nowiki></html></nowiki><br />
*Create a template page (e.g. Team:xyz/Templates/ToggleBoxEnd) and insert the following code:<br />
<nowiki><html></p><a class="toggle_close" href="#" style="font-size: 12px; color: #4e9d20" align="left">Close</a></nowiki><br />
<nowiki> </div></nowiki><br />
<nowiki></div></nowiki><br />
<nowiki><br></nowiki><br />
<nowiki></html></nowiki><br />
*To style your "Read more" sections you can use optional CSS code, that you can include in the CSS section in (.../Templates/Header). To give you an example, this is the CSS code we used to style your toggle boxes:<br />
p.activeToggle<br />
{<br />
padding-left: 17px;<br />
background-image: url('https://static.igem.org/mediawiki/2010/5/56/Team_TU_Munich2010_Images_Arrow_close_small.jpg');<br />
background-repeat: no-repeat;<br />
background-position: center left;<br />
}<br />
a.toggle_close<br />
{<br />
padding-left: 17px;<br />
background-image: url('https://static.igem.org/mediawiki/2010/5/5d/Team_TU_Munich2010_Images_Cross_small.jpg');<br />
background-repeat: no-repeat;<br />
background-position: center left;<br />
float: left;<br />
text-align: left;<br />
}<br />
div.toggle_container<br />
{<br />
border-left: 1px solid #4E9D20;<br />
padding-left: 7px;<br />
margin-left: 5px;<br />
margin-bottom: 25px;<br />
}<br />
:Please note, that "a.toggle_close" refers to the close-Button displayed at the bottom of every "Read more" section, "div.toggle_container" refers to the box surrounding the content and "p.activeToggle" contains style instruction that only apply to the text of an open "Read more" section.<br><br />
The only information that your team mates will need to create a toggle box are the names of the two templates that have to be put before and after the text. A simple toggle could look like this:<br />
<nowiki>{{:Team:xyz/Templates/ToggleBoxStart | text=Read more}}</nowiki><br />
<nowiki>This is text will be hidden at the beginning, but can be read if the box is open.</nowiki><br />
<nowiki>{{:Team:xyz/Templates/ToggleBoxEnd}}</nowiki><br />
Your wiki is now ready and offers say-so-use "Read more" sections.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br><br />
==Customize and style your wiki==<br />
So far be we created the backbone of your wiki by using templates and a table layout. Besides, every teams wants to have their only style. However, in the case of an iGEM team wiki, the normal way of change the look and feel of a wiki does not work. This is due to the fact, that all teams share one big wiki and changes done to the layout will change all other team wikis as well.<br><br />
The best way of styling your wiki anyway is by using CSS (Cascading Style Sheets). To read more about CSS read the following section.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
CSS ([http://en.wikipedia.org/wiki/Css Cascading Style Sheets]) is a language that is used to describe what elements on a web page are supposed to look like. So a webpage is created using HTML code and can be styled using CSS. The CSS instruction are to be placed between an opening <nowiki>"<style>"</nowiki> and a closing <nowiki>"</style>"</nowiki> in the header of a HTML page or can be directly included in HTML tags using the "style" attribute:<br />
<br />
<nowiki><html></nowiki><br />
<nowiki><head></nowiki><br />
<nowiki><title>CSS example</title></nowiki><br />
...<br />
<nowiki><style type="text/css"></nowiki><br />
a {<br />
color: green;<br />
}<br />
a.external {<br />
color: red;<br />
}<br />
#bestImage {<br />
border: 1px solid green;<br />
width: 500px;<br />
}<br />
.red {<br />
color: red;<br />
}<br />
...<br />
<nowiki></style></nowiki><br />
<nowiki></head></nowiki><br />
<nowiki><body></nowiki><br />
<nowiki><p '''style="color: red;">Just some text</p></nowiki><br />
<nowiki></body></nowiki><br />
<nowiki></html></nowiki><br />
<br />
If the style-attribute is used, the CSS instruction will only affect this tag and all its child-tags. In case of the <nowiki>"<style>"</nowiki> block in the header section, two types of information have to be provided: What is to be styled and how is it to be styled.<br><br />
The first information is provided by so-called selectors. A selector can select an element of the page in many different ways. A selector can generally select all tags of a certain kind (e.g. all <nowiki><p></nowiki>-tags). Secondly, a selecotr can select an element that has a certain identifier. An identifier is a unique ID for an element within the webpage and is specified by the attibute "id" (e.g. <nowiki><img id="bestImage"><nowiki>). Thirdly, a selector can select a subgroup of elements, called class. HTML elements can be grouped into a classes by using the attribute "class" (e.g. <nowiki><img class="goodImages"></nowiki>).<br>A selector is always placed before the opening bracket "{". In the example above all three types have been used: "a" is a selector that selects all link elements (<nowiki><a></nowiki>-tags). "a.external" addresses all links that have been assigned to the group "external" (<nowiki><a class="external"></nowiki>-tags). "#bestImage" refers to the element that has the ID "bestImage" (e.g. <nowiki><img id="bestImage"></nowiki>-tag). And finally ".red" selects all tags within the class "red" (e.g. <nowiki><a class="red">, <p class="red">, <table class="red"></nowiki>).<br><br />
All style instruction are place inside brakets "{ ... }". They all share a common syntax. and the left side of a colon is a keyword that specifies what property is to be changed, and on the right side is the new value that will be set. An instruction is terminated by a semicolon. There are many different keywords that can set all kinds of properties (colors, borders, dimensions, visibility, text-decoration, etc.). Once you learn more about CSS, you will get to know the most important ones.<br>Again, there many tutorials about CSS on the web that go far beyond this short introduction. If you want to create a sophisticated wiki, you will definitely have to deal with CSS.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
Once you are familiar with CSS, changing the appearance of the wiki is very straight forward. The nice thing about MediaWiki is that nearly every element in the wiki framework (the big banner on the top of the page, the table of content, the title of the page, etc.) has a unique identifier or belongs to a certain class. An identifier is specified using the HTML tag id, whereas an element can be assigned to a class called "title" by using class="title". <br />
Consequently, to change the look of a certain element, you have to first get to know the name of the class or the identifier and then use this name to change the appearance using CSS.<br><br />
To find out the name of the identifier or the class, you can either take a direct look at the source of the page, or you can use a very powerful Firefox extension to easily extract this information. Take a look at the following "read more" section for details about this.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
To use Firebug you have to install the plug-in first. Open up FireFox, go to [https://addons.mozilla.org/de/firefox/addon/1843/ the Mozilla plug-in webpage], install the plug-in and restart FireFox. As soon as you start FireFox you will notice an icon with a small bug on the very bottom of the FireFox window. Clicking on it will open and close the FireBug screen.<br><br />
To find out the id or the class of a specific element on the webpage, just right-click on it and in the menu you can choose to inspect this element. The firebug screen will now show the HTML code of this element. Now, you can either look for "id=" or "name=" which will give you a name you can address this element using CSS selectors (see also the following figure).<br />
[[Image:TUM2010_FireBug.jpg| thumb | 720px | A typical FireBig screen<br>After right-clicking on the heading, it can be seen that this heading is assigned to the class "firstHeading" and can thus be styled using an appropriate selector]]<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
Once you found out the name, you have to get into CSS. Since the appearance of your wiki is very likely to be the same for all pages, we can make use of our template structure that we created before. As the template ".../Templates/Header" is imported into every wiki page, putting your CSS styling in this template will affect all pages. Just edit the .../Templates/Header page and insert your CSS code:<br />
<nowiki><style type="text/css"></nowiki><br />
body {<br />
background-color: white; /* defines the default background color of the document*/<br />
color: black; /* defines the default font-color of the document*/<br />
}<br />
a {<br />
color: black; /* defines the default color of links */<br />
}<br />
a:hover {<br />
color: gray; /* defines the default color of links hovered by the cursor */<br />
}<br />
a {<br />
color: black; /* defines the default color of links */<br />
}<br />
.firstHeading<br />
{<br />
display: none; /* hides the default heading */<br />
}<br />
...<br />
<nowiki></style></nowiki><br />
<br />
You can not only modify the framework of MediaWiki, but you can also define classes in your templates and modify them in your global css section. For example, in the button template the link is assigned to the class "button", allowing the modification of all assigned links in your global CSS section. Now, you can also change all the default elements of the iGEM wiki. But please note that changing to much might can also confuse the user. For example, you should leave default buttons such as "Login, search, …". It is also appreciated to provide a link to the iGEM mainpage (http://20xx.igem.org/) in your header section.<br />
CSS is a very powerful technique that cannot be explained in this tutorial completely. Learning more about CSS can save a lot of time when customizing your wiki. You might also want to take a look at [https://2010.igem.org/Team:TU_Delft#page=Modeling/wiki-tips-tricks this page from this year's TU Delft team], where some more iGEM-specific CSS styling is listed. You will also find many tutorials or look-up pages about CSS on the web. And don't forget to take a look at previous teams, they might inspire you or have that piece of code you where looking for...<br />
<br><br><br />
Congratulations! You made it through the tutorial. Now you are ready to play around with CSS and add some more eye candy in your templates to produce a nice looking wiki. Have fun and don't hesitate to try new things, you can always go back using the MediaWiki history!<br />
<br />
=Part II: How to use the iGEM MediaWiki=<br />
<br />
This part will give you a short introduction, how to use the iGEM wiki and will gibe you some advice that will save your time and your nervs. Furthermore it will give some hints, specific to a wiki created according to the above tutorial. <br />
==Login in==<br />
First of all, you always have to be login into the iGEM wiki before you can change your page. Just click the log in button on the top of this page or any other iGEM page and make sure you have your account set up.<br />
==Creating a new wiki page==<br />
There are several ways of creating a new page. The easiest way is to type the name of the new page into your browser, for example <nowiki>http://20xx.igem.org/Team:xxx/bane of the page you want to create.</nowiki>. Please note, that all names are treated in a case sensitive manner! After typing in the name, just click "Create" or "Edit" and you can add content to your newly created page. Hit "Save" to save the new page.<br><br />
Since all iGEM teams share one common wiki, it is important that every page you create is in your namespace. In other words, all pages you create have to have your team name as a prefix. For instance, all page of our wiki are in the namespace "Team:TU_Munich".<br><br />
One other important aspect is, that at the beginning and at the end of every wiki page, your have to link to so-called template pages that contain all your styling and layout of your wiki. Please ask your team member in charge of the wiki to add these lines to your new page, or take a look at an existing page.<br />
==Adding content to a wiki page==<br />
Before you start adding a lot of text into the wiki page please make yourself familiar with the Markup language of MediaWiki. MediaWiki offers a very intuitive way to mark headings, pictures, etc. As the Wikipedia is also based on MediaWiki, might already be familiar with these commands. However, you still want to take a look at the [http://en.wikipedia.org/wiki/Help:Wiki_markup Wikipedia help page] or the [http://www.mediawiki.org/wiki/Help:Contents MediaWiki documentation].<br><br />
MediaWiki features a powerful history. You can undo any changed to a page or any replacement of a file. Make sure you are familiar with this feature.<br />
<br />
==Uploading files==<br />
In case you have to upload a picture or any other file you should be aware of some aspects. To upload a file you can scroll down on any wiki page and click on "upload file". Alternatively you can link to a picture on your page before you even uploaded it. MediaWiki will no show a red link at the corresponding page. Clicking on it will also get you to the upload page.<br><br />
For security reasons, the iGEM wiki does not accept any type of file. Refer to the upload page to see a list of allowed file extensions. Make sure whether you can upload a file in advance, before you spend time generating files that you cannot send to the iGEM server.<br><br />
Another iGEM-specific aspect, is that all teams share one common pool to upload pictures and other files. Your team should agree on a prefix that you add to all your filenames. In case you upload a picture named "team.jpg", chances a high that by accident another team will replace your file. Use "OUR_COLLEGE_20xx_team.jpg" instead!<br />
==Editing your navigation bar==<br />
As the navigation section is the same for all wiki pages, it can be stored at one central page. In case your wiki was created according to this tutorial, the computer expert in your team can give the name of the corresponding page where you can edit the navigation. In this page you will find entries similar to "<nowiki>{{:Team:xxx/Templates/Button | text=Home | link=/}}</nowiki>". Each entry resembles one button or link to a page. Please note, that your team members might have changed the exact spelling. In the above example, you can just add a new button to your navigation by copying a new entry "<nowiki>{{:Team:xxx/Templates/Button | text=Home | link=/}}</nowiki>". Now replace "Home" by the text you want to appear in the button. The target of your button will be specified by "link=". Change "/" to "/Modeling" to link to the page "Modeling" in our namespace.<br />
==Using "Read more" sections==<br />
With the help of "Read more" sections you can give the user a better overview of the page. Your team member in charge of the wiki can tell you more details. In general, you only have to add two lines to your page to create a "Read more" box:<br />
*Place "<nowiki>{{:Team:xyz/Templates/ToggleBoxStart | text=Read more}}</nowiki> before the content you want to be inside the box. Note that you can change the text "Read more" to some custom words by change the text after text=<br />
*Place "<nowiki>{{:Team:xyz/Templates/ToggleBoxEnd}}</nowiki> behind the text that you want to be within the box.<br />
To give you an example, a simple toggle could look something like this:<br />
<nowiki>{{:Team:xyz/Templates/ToggleBoxStart | text=Read more}}</nowiki><br />
<nowiki>This is text will be hidden at the beginning, but can be read if the box is open.</nowiki><br />
<nowiki>{{:Team:xyz/Templates/ToggleBoxEnd}}</nowiki><br />
Your wiki is now ready and offers say-so-use "Read more" sections.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
<br />
<!-- ############## WIKI-PAGE STOPS HERE ############## --><br />
{{:Team:TU_Munich/Templates/End}}</div>Hartlmuellerhttp://2010.igem.org/Team:TU_Munich/ProjectTeam:TU Munich/Project2010-10-28T02:45:41Z<p>Hartlmueller: /* Design and functional principle of logic gates */</p>
<hr />
<div>{{:Team:TU_Munich/Templates/Beginn}}<br />
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Project<br />
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<center><font size="5pt" color="#000000">'''bioLOGICS'''</font><font size="4pt" color="#000000">: Logical RNA-Devices Enabling BioBrick-Network Formation</font></center><hr color="black"><br><br />
= Vision=<br />
<br />
Until today, 13.628 biobrick sequences<sup>[[Team:TU_Munich/Project#ref1|&#91;1&#93;]]</sup> have been submitted to partsregistry, thereof 102 reporter units and 12 signaling bricks.<br />
Since then, people are trying to arrange these single biological building blocks in such a manner that allows producing special biotechnological products (metabolic engineering), developing biological sensory circuits (biosensors) and even giving microorganisms the ability to react on multiple environmental factors and serve both as disease indicator and drug. These examples and further promising ideas were implemented on previous iGEM-competitions.<sup>[[Team:TU_Munich/Project#ref2|&#91;2&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref3|&#91;3&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref4|&#91;4&#93;]]</sup> <br><br><br />
The idea of combining the outcome of several iGEM competitions to construct complex synthetic biological systems falls at the last hurdle - the fact, that each team uses a different principle how to access and functionally connect the respectively used biobricks. For example, it is a major challenge to create a system that uses several sensoring BioBricks from different iGEM-teams which in turn regulates reportering BioBricks from various teams. In order to combine and fully take advantage of these promising projects, our vision is to develop an adapter that allows interconnecting arbitrary biobricks on a functional level. Such a system easily allows to setup sensor-reporter circuits and interconnect them to complete biological chips... A further step towards artificial cells.<br><br><br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, the above adapter has to meet the following requirements:<br />
*'''Universality'''<br />
:The adapter has to be compatible to as many BioBricks as possible. This objective will guarantee that a large number of BioBricks can be connected.<br />
*'''Scalability'''<br />
:Once the basic design of the system is established, the construction of the system is supposed to be automated in silico. This way it will be possible to create an adapter connecting a large amount of BioBricks.<br />
*'''Biological orthogonality'''<br />
:Interference with cellular components has to be as low as possible in order to avoid unwanted and perturbing side effects.<br />
*'''Logic'''<br />
:The adapter is supposed to not only associate different BioBricks, but to functionally connect BioBricks in a precisely determined manner (including operations such as AND/OR/NOT).<br />
<br><br />
Several biological logic units, devices and circuits have been developed so far<sup>[[Team:TU_Munich/Project#ref5|&#91;5&#93;]]</sup>, but to our knowledge, none was shown to meet all requirements listed above.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
=Implementation=<br />
To functionally connect BioBricks, there are several possibilities including genetic switches, riboswitches and direct protein-protein interactions. We investigated several hypothetically principles, and decided to focus our practical work on the development of a RNA-RNA interaction-based switch. These switches are capable of changing between two states, a state of antitermination and termination, and make use of highly-specific RNA-RNA interaction. In principle such a switch can fulfill all requirements mentioned previously. The following text clarifies how these switches work in detail.<br />
==How to connect BioBricks==<br />
Our adapter is a system, that activates or disables BioBricks (output BioBricks) in response to the presence of other Biobricks (input Biobricks). Our approach uses a molecular network to put this into practice and consists of four major elements:<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
<br><br />
{|<br />
|-<br />
|[[Image:Networks.png|center|thumb|730px]]<br />
|}<br />
In order to connect different BioBricks, our network requires four major types of components:<br />
*Input elements<br />
*Transmitter molecules<br />
*Logic gates<br />
*Output elements<br />
<br />
{{:Team:TU_Munich/Templates/InfoBoxStart}}'''Computer vs. molecular network - and our approach'''<br><br />
Logic gates in a molecular network are often compared to transistors used in a computer, where billions of transistors are incorporated<sup>[[Team:TU_Munich/Project#ref7|&#91;7&#93;]]</sup>. The main advantage on a computer chip is, all transistors share the same functional principle, and only the way connecting them in a special sequence allows specific addressing of only a subset of other transistors by an input. However, spatially fixed connections of molecular logic gates are not possible in a living cell. The "wiring" within a cell relies on the specific interaction between transmitter molecule and their corresponding logic gates, for example implemented by protein-protein/ligand-protein interactions or specific ligand-riboswitch interactions.<sup>[[Team:TU_Munich/Project#ref8|&#91;8&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref9|&#91;9&#93;]]</sup> As a result, in a cell, each occurring logic gate ("transistor") has to be different, at least in a special recognition site<sup>[[Team:TU_Munich/Project#ref10|&#91;10&#93;]]</sup> - for example like different transcription factors, recognizing different DNA-sites. Thanks to evolution, nature easily can invent a new transistor for each task - science achieves this only on a limited scale, and producing synthetic molecular logic gates artificially by either rational or evolutionary protein or riboswitch engineering, is limited to small circuits so far<sup>[[Team:TU_Munich/Project#ref11|&#91;11&#93;]]</sup>. Our project aims to establish a molecular switch as close as possible to a electronic transistor, thus sharing the same functional principle for all logic gates. At the same time, we want to design a easily exchangeable recognition site, which can individually be designed by everyone! {{:Team:TU_Munich/Templates/InfoBoxEnd}}<br />
<br />
These elements can be combined to build up a molecular network (see illustration). Each input molecule (such as a BioBrick) produces a unique transmitter molecule. All transmitters belong to the same type of molecule and share a common design. However, each transmitter molecule can only interact and activate a certain subset of logic gates. In other words, logic gates have to recognize as well as bind the corresponding transmitter molecules and are capable of producing a new output transmitter molecule. Depending on the type of the logic gate (AND, OR or NOT<sup>[[Team:TU_Munich/Project#ref6|&#91;6&#93;]]</sup>), an output transmitter is only created if both input transmitter molecules are present (AND), at least one of two input transmitters is present (OR) or if no input transmitter is present at all (NOT). Once a logic gate has produced a new output transmitter, these transmitters can in turn address another subset ("layer") of logic gates. In theory many layers of logic gates can be connected this way allowing the creation of large networks. Until this step, various transmitter molecules might have been produced. But in order to create a Biobrick output, the last layer of logic gates finally generates transmitter molecules that will not active logic gates, but will rather interact with the cell metabolism to produce a BioBrick response. In other words, the last layer of transmitter molecules is capable of regulating BioBrick formation.<br />
<br />
<br />
Summarizing, the network establishes a connection between input BioBricks and output BioBricks in a functional manner.<br />
Having addressed the basic layout of the molecular network, the next step is to determine what type of molecules can perform the required functions. We decided to use RNA, both as transmitter molecules and for constructing logic gates. Several advantages result from the utilization of RNA as the central element:<br />
*During the last years, many Biobricks were designed that are sensitive to various chemicals and substances. These BioBricks often function as a transcription factor that binds to a specific DNA sequence and consequently would be capable to produce a specific transmitter RNA molecule. Thus, in principle each BioBrick which involves transcription can be integrated in our network.<br />
*Since all logic gates are capable of producing transmitter RNA, they can also produce functional mRNA encoding any protein. This means, each BioBrick consisting of protein or RNA can be produced as an output of our network.<br />
*If RNA forms both, the transmitter molecule and the logic gates, they can specifically interact by RNA-RNA interaction, which is highly predictable compared to protein interactions. This allows to generate a library of transmitters and gates ''in silico''. Such a library is essential for the creation of large networks.<br />
*RNA production is fast and energy saving for a cell. Consequently, operating a network that only produces RNA rather than proteins will also be faster and more efficient for the host cell. Since our logic gates are based on transcription, translation and resource consuming protein production will only be required at the very last step. <br />
*As the half-time of RNA can be rather short, transmitter RNA will not accumulate within the cell and it is therefore less likely for the system to become saturated.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Design and functional principle of logic gates==<br />
The concept introduced above provides a framework that can potentially serve as an universal adapter between different BioBricks. However, the [[Team:TU_Munich/Glossary#logic gate | logic gates]] have not been specified more precisely so far. This will be done in the following section.<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Generally speaking, our logic gates are to possess the following characteristics:<br />
*Logic gates, such as AND, OR and NOT, have to be implemented by RNA-interaction based principles (see [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]).<br />
*All logic gates have to recognize their corresponding [[Team:TU_Munich/Glossary#Transmitter (bioLOGICS)| transmitter RNAs]] and, in response, produce an output transmitter molecule.<br />
*Logic gates should follow a basic design rule, in such a way, that their creation can be automated ''in silico''.<br />
*The response efficiency of a logic gate toward a transmitter molecule should be comparable for all logic gates to provide calculable robustness and sensitivity. This will ensure comparable molecular concentrations and functionality of large networks.<br />
*The system has to be designed for ''in vivo'' utilization at the first place. As a reference we always assumed a temperature of 37 °C and an ''E. coli'' environment.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}} <br />
In order to build logic gates for our bioLOGICS system we will first create a simple switch. A switch can be activated by one transmitter RNA and produce an output transmitter RNA. In contrast to a logic gate, a switch does not perform logic operations. However by combining switches, logic gates can be created. The following text will first describe how the developed switch works and secondly, how logic gates such as AND/OR/NOT can be created using these switches.<br />
<html><br />
<script type="text/javascript"><br />
$(document).ready(function(){<br />
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$("p.trigger").click(function(){<br />
$(this).next("div").find(".thumbinner").slideToggle("slow");<br />
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return false; //Prevent the browser jump to the link anchor<br />
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$("a.toggle_close").click(function(){<br />
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while(nextParent!= null) {<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Read more{{:Team:TU Munich/Templates/ToggleBoxStart2}}<br />
[[Image:toggle_switch.png|500px|thumb|center|id="hideOnReadMore"|'''A''' The basic structure of a bioLOGICS switch (left) and a transmitter molecule (right).<br>'''B'''The process of switching. See the text in the close-by "Read more" section for details.<br>Rectangles present the composition of our functional units on the level of DNA. Fringed lines represent RNA produced by RNA polymerase. The stem loop structure depicts the switchable terminator. Terminator and target site are illustrated in blue and turquoise, respectively. Recognition sites are indicated in different colors, in this case red for the input transmitter and green for the output transmitter.Each switch and or later logical unit has to be flanked by a promotor and another constitutive terminator, to allow RNA-production by RNA-polymerase in a proper way. ]]<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===Switch===<br />
[[Image:TUM2010_switch-and-transmitter.jpg|550px|right|thumb|The basic strcutrue of a switch (left) and a transmitter RNA (right). See text for details.]]<br />
Roughly speaking, a switch can be regarded as an enhanced switchable transcriptional terminator. The enhancement can be described easier by dividing a switch into its functional components: <br />
*'''Target site'''<br><br />
:The target site is the functional core element of our switches, allowing a shift between an "on" and "off" state. Since we work on the level of RNA-production (transcription), a "switchable" transcriptional terminator is suitable for this purpose. By allowing or preventing formation of a transcriptional terminator, that is by switching between termination and antitermination it is possible to represent an "off" and an "on" state, respectively. Therefore, the target site is the 5' ending of the terminator and is required for a stable terminator formation. It should be noted that this principle was also observed in nature.<br />
:To highlight and illustrate the functional principle of our switches, only the part of the terminator which is involved in interacting with a transmitter molecule and which is responsible for shifting between "on" and "off" state is called target site. The remaining terminator sequence is called terminator in the following, even if both, target site and terminator build up the terminator structure occurring in nature. <br />
:The important aspect of our switches is the fact that all switches will hold the same identical target site. Therefore having found one functional "switchable" terminator, will allow almost unlimited upscaling since this terminator can be used for a large library of switches. This is the main difference to previous works done on this field, which always required developing a new shifting principle for each switch.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup> Beside this scalability, this principle provides a comparable on/off shifting rate (responds function) for all switches, avoiding complex fine tuning of molecular networks.<br />
:To sum it up, the target site, allows to switch between an "on" and "off" state. But so far, the switch is not capable of performing specific interaction with transmitter molecules. This is where the recognition site comes into play.<br />
*'''Recognition site'''<br />
:The recognition site defines which transmitter molecule can actually interact with the switch. Therefore, a unique recognition site is generated for each switch and is positioned right upstream of the target site. In principle the recognition can be any random sequence as long as it remains unique within the molecular network.<br />
Summing up, the recognition site allows a specific interaction between switches and transmitter molecules. Once this interaction is formed, an interaction between the transmitter and the target will actually switch the state of the terminator. This allows the specific arrangement and interconnection of numerous of these switches by transmitter molecules, without changing the target site. Comparable to wires connecting many identical transistors, our target site remains the same.<br />
<br><br />
<br />
===Transmitter RNA´s===<br />
As desccribed above, transmitter RNAs are the input and output of bioLOGICS switches (compare [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]). These transmitters are short ssRNA molecules representing the "trigger" to shift switches between the "on" and "off" state. To fulfill this role, they need to posses the following properties:<br />
*A transmitter may only interact with certain switches. That is, a transmitter has to find the corresponding recognition site of a switch.<br />
*Once an interaction is established between a transmitter and a switch, a transmitter has to be capable of changing the secondary structure of a terminator and thus cause antitermination.<br />
Again, these two properties are fulfilled by two components of the transmitter:<br />
*'''Identity site'''<br />
:This site is capable of forcing an interaction between the transmitter and the switch. Therefore it is complementary to the recognition site of this switch. As the recognition site is unique within a network, so is the identity site. However, the single identity site is not capable of changing the state of the switch. That is were the trigger site comes into play.<br />
*'''Trigger site'''<br />
:Once an interaction is created by the identity site, the trigger site is capable of actually shifting the switch since it is complementary to the target site of the switch. To fulfill this role, it is placed upstream at the 5' end of the identity site. As the target site is the same for all switches, the trigger site is the same for all signals. Therefore it is important, that similar to the identity site, a trigger site cannot function on its own. That is, a single trigger site cannot shift the state of a switch without the help of an identity site.<br />
<br />
Summing up, we applied the principle introduced for the switches to the transmitter molecules. In contrast to previous approaches on this field <sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup>, we introduced the described synthetic trigger site in such a manner that it is not able to change the state of the terminator on its own, but only in combination with the identity site. So the challenge is to arrange and optimize these elementary building blocks thermodynamically, that a trigger site is only able to switch in combination with its respective identity site. This was done by ''in silico'' design using [[TU Munich/Glossary#NUPACK| NUPACK]], presented in section [[TU Munich/Modeling#in silico design based on thermodynamic calculations| in silico design]].<br />
<br />
<br><br />
<br />
===Putting it all together: the switching process===<br />
[[Image:TUM2010_switching-process.jpg|620px|right|thumb|The basic structure of a switch (left) and a transmitter RNA (right). See text for details.]]The functional principle of the designed switches is illustrated in the figure. The switch is positioned on DNA upstream of a desired output transmitter. So in the absence of a triggering transmitter molecule, transcription will be canceled by the formation of a RNA stem loop in the nascent RNA-chain. This will cause the RNA polymerase to stop transcription and fall off the DNA and consequently no output RNA will be produced. This process only relies on [[Team:TU_Munich/Glossary#Termination| rho-independent termination]].<br />
On the other hand, in the presence of a [[Team:TU_Munich/Project#RNA_transmitters | input transmitter]], this small functional RNA inhibits the stem loop formation by complementary base-pairing and hence avoids termination of transcription. In detail, the identity site (red part on transmitter) binds the recognition site (red part on switch) and serves as [[Team:TU_Munich/Glossary#Toehold|toehold]], which will thermodynamically allow the trigger site (turquoise part on transmitter) to perform a [[Team:TU_Munich/Glossary#Strand Displacement| strand displacement]] and open up the stem loop structure. Consequently the polymerase can read all the way through and form the output RNA.<br>Summing up, we use this concept to create a switch that can be toggled by a transmitter RNA molecule and in response, is able to produce another transmitter RNA.<br />
<br><br />
<br><br />
<br><br />
<br />
===From switches towards bioLOGICS logic gates===<br />
As described, each switch can be accessed by a specific RNA-transmitter molecule, illustrating the input. In turn, another RNA-transmitter molecule will be produced if the switch shifts its state. This output transmitter of one switch can serve as input transmitter for the next switch by meaningful selection and design of the respective recognition sites. This easily allows arranging several switches in specific sequences and faulty wiring - the corner stone of a logical network.<br />
<br />
To ease the building of logical networks, applying mathematical logics, e.g. Boolean logics like in computational science would be worthwhile. It is possible to establish general Boolean operators with our switches and thus build "logical modules". <br />
Since AND/OR/NOT are the most simple logic operations which can be implemented with the presented switches, and all remaining operations can be expressed by these three operators according to laws of boolean logics, we exemplary designed them.<br />
<br />
{|<br />
|-<br />
| *AND consists of a parallel circuit of two switches<br />
|-<br />
|[[Image:AND2.png|500px|thumb|center]] <br />
|-<br />
| *OR is implemented by connecting two switches in series<br />
|-<br />
|[[Image:OR2.png|500px|thumb|center]]<br />
|-<br />
| *NOT is more complex to explain. In principle, it consists only of one switch which contains its respective signal molecule intrinsic, so via intramolecular interaction, antitermination is the initial state. The signal is intrinsically of the same components as usual to allow interconnection with other logic gates.<br />
|-<br />
|[[Image:NOT2.png|500px|thumb|center]]<br />
|}<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Network construction==<br />
Designing complex biological networks based on either traditional protein engineering or our new bioLOGICS is still a complex task. We developed a software which allows the fast construction of a bioLOGICS based networks. <br><br />
To read more about this, look at our [https://2010.igem.org/Team:TU_Munich/Software Software page]<br />
<br />
=Our Objective=<br />
Putting the implementation described above into practice, will be a major challenge. For this year's iGEM competition our goal is to do the first step: design and build a switch that can be toggled by a RNA molecule. To be precise, we want to apply the design rules of our switch to modify a transcription terminator in such a way that it interacts with a second RNA molecule and, as a result, is no longer capable of forming a stem loop. This objective will require intensive ''in silico'' designing and modeling of switches based on different terminators and their corresponding transmitters. In connection to this theoretical part, we also have to test and verify the switches. For this step, we establish custom-made assays, ''in vitro'' and ''in vivo''.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Once the objective mentioned above is accomplished, these basic RNA/RNA-interactions have to be modified in such a manner that the described identity/trigger site pattern for the transmitter and the complementary recognition/target site switch composition has to be established. The most important requirement is to is to optimize these modules that the transmitter is only able to switches specifically, meaning only in the presence of both, identity AND trigger site. <br />
<br><br />
Once the objective mentioned above is accomplished, the creation of an OR gate will be rather simple since it only requires two switches. However the creation of an AND or NOT gate and optimizing the logic gates to improve their responds function will remain the goal of future work. Also the creation of small networks and the correct integration of BioBricks as input and output molecules will be future challenges. Furthermore, we wanted to rather focus on the development and the testing of our structural design of the switches, rather than developing a variety of new BioBricks.<br />
<br />
==''In silico'' design==<br />
As described above, our switches are based on certain design rules. However, there still are different structural parameters that need to be tested and optimized (length of recognition site and target site, choice of terminator, etc.).<br />
We used [[Team:TU_Munich/Project#in silico design |''in silico'' design]] and [[Team:TU_Munich/Modeling| modeling]]) to test different parameters. Furthermore we tried to use the [[Team:TU_Munich/Glossary#Antitermination|antitermination principle]] observed in nature, such as [[Team:TU_Munich/Glossary#Attenuation| attenuation]] in ''E. coli'' or [[Team:TU_Munich/Glossary#Tiny Abortive RNA´s| tiny abortive RNA´s]] of T7-phage.<br />
==Evaluation and Measurements==<br />
To evaluate the functionality of our molecular switches, we first had to establish several assays. Therefore, we improved an existing [[Team:TU_Munich/Lab#In vivo Measurements |''in vivo'' assay]] and developed an [[Team:TU_Munich/Lab#In vitro Transcription | ''in vitro'' assay]] for this purpose. For more information please refer to the [[Team:TU_Munich/Lab | lab]] section.<br />
<br><br />
<br><br />
Summarizing, the main challenges are <br />
* to find a suitable terminator construct and design a complementary trigger unit, which is only functional in combination with a specificity site - meaning an optimization of the '''thermodynamically parameters''' (see[[Team:TU_Munich/Project#in silico design| in silico design]])<br />
* to investigate whether the transmitter/switch interaction reaction is on a timescale to be competitive to terminator formation - meaning an comparison of '''kinetic parameters''' (see [[Team:TU_Munich/Modeling|Modeling page]])<br />
* to proof antitermination can be also be caused by synthetically RNA-interaction (see [[Team:TU_Munich/Glossary#Antitermination| Antitermination in nature]] and [[Team:TU_Munich/Project#Results| ''in vivo'' and ''in vitro'' measurements]] )<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
=Results=<br />
Every network starts with a basic unit. While our declared aim is to enable networks allowing fine-tuning of gene expression beyond the regular on/off, exploring such an on/off switch/signal pair is the first step towards a functional network. We constructed several units and tested their efficiency, robustness and reproducibility ''in vivo'', ''in vitro'' and ''in silico''. Furthermore we developed a software which allows easy constructions of networks based on our designed logic gates. Conclusive elaboration of a few first RNA-based logic units is the major contribution of our iGEM team.<br />
<br />
==in silico Design of Switching and Trigger Unit==<br />
===attenuation principle===<br />
<br />
<br />
==Diffusion and RNA Folding Dynamics==<br />
We estimated the diffusion time for our constructs and modeled the folding dynamics of our bioLOGICS switches including the switching process with a stochastic RNA folding program. We were able to provide better insight in their folding dynamics and proved that they are able to interrupt termination. We also optimized the switches and the corresponding signals. Furthermore, we combined the switches what resulted in a logic gate. See our [[Team:TU Munich/Modeling|Modeling page]] for further details.<br />
<br />
==''in vivo'' Functionality Screening==<br />
Since our logic gates are intended to function in living cells, ''in vivo'' measurements were essential. In a set of experiments we concentrated on two different switches based on known [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|attenuators]] from nature: the [[Team:TU_Munich/Modeling#Switch|HisTerm]] and [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Focusing on fluorescent proteins for quantifiable input and output we designed a functional and robust screening system. For greater detail see [[Team:TU_Munich/Lab#Experiment_Design|Experimental Design]]. Unfortunately, setting up a working screening system failed twice. Only in redesigning and improving the screening plasmid pSB1A10 we succeeded, but lost precious time.<br />
<br />
Ultimately, the two switches displayed remarkable differences in their terminator efficiency, but neither of them responded to their corresponding signal. However, screening one transmitter signal does not disprove the basic working principle of our system. Limited by time, we hope for future teams to take up our work and to use our improved test system that we submitted to the parts registry, for performing successful in vivo measurement.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Considering the high complexity of ''in vivo'' measurements compared to other experimental challenges, a robust and easy to handle test system for [[Team:TU_Munich/Glossary#PoPS-based devices| PoPS-based devices]] is desirable. As described in [[Team:TU_Munich/Lab#Experiment_Design|Experimental design]], we used fluorescent proteins: RFP or mCherry to measure the amount of produced output and eGFP for normalization. Our first attempt, using the screening plasmid pSB1A10, yielded no interpretable results. Switching the fluorescent protein to mCherry did not work either, but after several experimental setups we determined a transcriptional problem causing no reporter protein expression regardless of the inserted part. Thereby we demonstrated the screening plasmid pSB1A10 to be [[Team:TU_Munich/Biobricks#Falsification| malfunctioning]]. <br />
Finally a new design based on pSB1A10 lead to a functional and robust screening system (compare [[Team:TU_Munich/Parts#Screening system: Backbone BBa_K494001| Screening system: Backbone BBa_K494001]]). A second promoter with identical induction properties inside the BioBrick cloning site enforces transcription of the PoPS-based device and the mCherry output.<br />
<br />
Exemplary, the graph below on the right shows the positive control, induced and uninduced at OD<sub>600</sub>=0.7 followed by 16 h incubation at 25 °C. Clearly visible are eGFP and mCherry fluorescence in the induced samples. The uninduced control showed no fluorescence at all, demonstrating the PBad promoter to be tight and providing very low basal transcription, what is a major advantage for the screening system. This newly designed screening approach renders the characterization of PoPS-based devices in general and switches in particular easy and robust. The low basal transcription furthermore fulfills one of the most important requirements for the designed switches, since output transmitters may only be produced in presence of an input transmitter. This helps to avoid strong "background" noise, which would extremely harden the successful interconnection of several switches. <br />
<br><br />
[[Image:TUM2010_PosControlklein.JPG|200px||thumb|left|Bacteria containing positive control]]<br />
[[Image:TUM2010_graphPosControl1.png|355px|thumb|center|Emission spectra of induced (green/red) and uninduced(black) positive control BBa_K494002 ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
Due to the time limitations of the iGEM completion we had to focus our efforts on few switches after designing the screening system. Relying on the functionality of systems occurring in nature, we choose the [[Team:TU_Munich/Modeling#Switch|HisTerm]] as well as the [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Both switches are based on known natural [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|attenuators]]. Testing synthetic and none-naturally switchable terminators in vivo are goals for future work.<br />
Delorme et al. reported the His-Terminator to be a remarkable effective Terminator with more than 99% termination efficiency.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup> The exemplary measurement below on the right confirms the high terminator efficiency. In fact, we could not detect any mCherry fluorescence in any cells containing the [[Team:TU_Munich/Modeling#Switch|HisTerm]]. Even induction of the corresponding signal transmitter RNA via IPTG did not alter the Terminator efficiency. Again time was the limiting factor and prevented us from testing more than one corresponding transmitter, although the [[Team:TU_Munich/Modeling| Modeling]] highly suggested the necessarily of finding an optimized transmitter length. Thus, the results are insufficient either to prove or to disprove the functionality of the [[Team:TU_Munich/Modeling#Switch|HisTerm]] or our concept in general.<br />
<br><br />
[[Image:TUM2010_HisSwitchklein.JPG|200px|thumb|left|Bacteria containing HisTerm]][[Image:TUM2010_HisSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing HisTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
<br />
Attaining only 90% terminator efficiency, the natural Trp [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|Attenuator]] is known be less effective than the [[Team:TU_Munich/Modeling#Switch|HisTerm]].<sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup> The graph on the right depicts our designed [[Team:TU_Munich/Modeling#Switch|TrpTerm]] characteristic efficiency of about 40 %, notably below the natural standard. Allowing 60% transcription in the “off” state excludes the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] from possible candidates for a scalable network of logic gates, due to the mentioned required "yes or no" function (see [[Team:TU_Munich/Project#Implementation| Implementation and how to connect Biobricks]]). Thus the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] is inoperative as intended, but may still be useful in other contexts. Similar to the [[Team:TU_Munich/Modeling#Switch|HisTerm]], the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] also did not react to the induction of the corresponding signal. Under circumstances, termination efficiencies altered by the transmitter are on a low range and not resolvable within observed 40% basal transcription. <br />
<br><br />
[[Image:TUM2010_TrpSwitchklein.JPG|200px|thumb|left|Bacteria containing TrpTerm]][[Image:TUM2010_TrpSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing TrpTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
<br><br />
<br />
Making use of our improved screening system we also carried out some ''in vivo'' kinetic measurements in addition to the end-point measurements above. In contrast to the ''in vitro'' experiments we did not obtain significant results for the characterization of our switches. As the switching process is many times faster than protein synthesis our ''in vivo'' kinetics include the synthesis of mCherry as well as its maturation. Therefore we centered our attention on end-point experiments. For more information browse the [[Team:TU_Munich/Lab#Lab_Book|lab book]]. <br><br />
Considering our ''in vivo'' measurements, neither of the tested switches showed any effect regarding the signal induction. But due to the small number of tested switches and signals this can hardly be regarded as disprove of concept. In particular in light of the recent findings by Sooncheol proving antitermination in principle using a T7 system.<sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup><br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==''in vitro'' Screening==<br />
To minimize the amount of disturbing factors we decided to countercheck our ''in vivo'' results with a set of ''in vitro'' measurements. While the ''in vitro'' systems are no doubt much less complex than living cells, the work with these set-ups proved to be quite as difficult.<br />
Just as with the ''in vivo'' measurements we could prove our switching system neither right nor wrong, leaving enough work for future iGEM teams.<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
===''in vitro translation''===<br />
<br />
Beside optimization of the reporter proteins in use, the major problem occuring in the experiments was the low capacity of the kit. The signal intensity was very low, which made it difficult to observe any signal intensity alterations, so no conclusion could be drawn from these measurements.<br />
<br />
===''in vitro'' transcription===<br />
We used two completely independent ''in vitro'' systems: Using ''E.coli'' RNA Polymerase we analyzed the His and Trp switches that had already been tested ''in vivo''. In a second set-up, we used the well-established T7 RNA Polymerase and switch based on the T7 terminator as well as several signal sequences.<br />
<br />
====T7 System====<br />
In contradiction to the results of Kang and coworkers and other groups, in our ''in vitro'' set-up the T7 terminator did not seem to terminate at all. The negative control (Promoter_Terminator_malachite binding aptamer) showed a similar increase in fluorescence as the positive control (Promoter_random sequence_malachite binding aptamer). <br />
[[Image:TUM2010_T7Result1.png|360px||thumb|left|''in vitro'' transcription measurement of T7 terminator with no signal(upper left), nonsense signal (upper right) and two different designed signals (below)]]<br />
[[Image:TUM2010_T7Result3.png|360px||thumb|right|''in vitro'' transcription measurement of positive control(upper left and T7 terminator with three different designed signals (remaining traces)]]<br />
Furthermore denaturing Polyacrylamide Gel Electrophoresis (PAGE) confirmed that there was no observeable termination of transcription. The addition of a signaling sequence led to a significantly lower increase in fluorescence, which can be attributed to the fact that both DNA sequences, switch and signal, compete for RNA Polymerases.<br />
However, there is almost no difference between the designed signals and random sequences, which is not a big surprise since there can be no antitermination if the terminator itself does not work.<br><br />
<br />
Possible explanations for the contradiction between our results and those of Kang and coworkers might be the experimental set-up and the RNA Polymerases we used. Different variants of T7 RNA Polymerase might respond in different ways to terminator structures, and the termination might be influenced by the presence or absence of cofactors, depending on the purification methods used in producing the Polymerase.<br><br><br />
<br />
This set-up offers a lot of possible experiments for the future, which we would have loved to conduct with a just a bit more time...<br />
<br />
====''E.coli'' System====<br />
<br />
Compared to the T7 System, the ''E. coli'' RPO system produced poor increases in fluorescence, indicating little RNA synthesis. It was shown that the presence of a terminator decreases, as expected, the production of downstream RNA. This result was also confirmed by denaturing PAGE. However, due to the poor changes in fluorescence we were not able to actually characterize the behaviour of our switches ''in vitro'', and the small RNA concentrations did not allow a quantitative interpretation of our gels. A major problem with this method was the low concentration of the ordered Polymerase resulting in a much weaker overall signal as comparable measurements using the T7 Polymerase. <br><br><br />
In future experiments we might try to work with smaller volumes in order to reach higher concentration of RPO and of the synthesized RNA molecules, so measuring in 96 well plate readers might be a good choice. <br />
<br />
<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Software==<br />
Although we could not show the full functionality of bioLOGICS in the lab we still want to demonstrate the potential of our approach. Hence we implemented the idea behind our logic gates in a program which illustrates how bioLOGCIS theoretically would allow the construction of complex information processing networks interconnecting BioBricks. For further details take a look at our [[Team:TU Munich/Software|Software page]].<br />
<br />
<br />
=Outlook=<br />
...<br />
future plans will also work with [[Team:TU_Munich/Glossary#Synthetic Terminator| Synthetic Terminators]], which might retrieve additional informations on what drives the process of Termination<br />
...<br />
<br />
=References=<br />
<html><a name="ref1"></a></html>[1] http://partsregistry.org/cgi/partsdb/Statistics.cgi<br />
<html><a name="ref2"></a></html>[2] https://2009.igem.org/Team:Imperial_College_London/M1 encapsulation<br />
<html><a name="ref3"></a></html>[3] https://2009.igem.org/Team:TUDelft<br />
<html><a name="ref4"></a></html>[4] https://2008.igem.org/Team:Heidelberg<br />
<html><a name="ref5"></a></html>[5] Maung Nyan Win and Christina D. Smolke, Science Oct. 2008 Vol. 322. no. 5900, pp. 456 - 460<br />
<html><a name="ref6"></a></html>[6] http://en.wikipedia.org/wiki/Logic_gate#Symbols<br />
<html><a name="ref6"></a></html>[7] http://en.wikipedia.org/wiki/Moore's_law<br />
<html><a name="ref6"></a></html>[8] http://en.wikipedia.org/wiki/Protein_interaction<br />
<html><a name="ref6"></a></html>[9] http://en.wikipedia.org/wiki/Riboswitch<br />
<html><a name="ref6"></a></html>[10] http://en.wikipedia.org/wiki/Binding_sites + http://en.wikipedia.org/wiki/Recognition_site<br />
<html><a name="ref6"></a></html>[11] irgend ein damn review über directed evolution and so on<br />
<html><a name="ref12"></a></html>[12] Delorme, Ehrlich and Renault, Regulation of Expression of the Lactococcus lactis Histidine Operon. Journal of Bacteriology, Apr. 1999, p. 2026–2037<br />
<html><a name="ref13"></a></html>[13] Trun and Trempy(2003): Fundamental Bacterial Genetics, Wiley-Blackwell, Chapter 12 <br />
<html><a name="ref14"></a></html>[14]Sooncheol Lee, Huong Minh Nguyen and Changwon Kang, Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator. Nucleic Acids Research, 2010, 1–9<br />
<html><a name="ref6"></a></html>[15] <br />
<html><a name="ref6"></a></html>[16]<br />
<br />
<!-- The idea behind our project is to change the way BioBricks have been used up to now. Over the years, many receptors and signals have been constructed as BioBricks during the annual iGEM competition, but still it is not possible to interconnect these Bricks in a complex biological network resuting in a cell, that is able to respond to its environment giving differenciated responses depending on the input signals. (Beispiel: cambridge hat das gemacht, xx dies, aber eine zelle kann nicht beides...<br><br />
We plan to create biological switches, that can function as locial gates inside a cell. Our switches rely on RNA/RNA-interactions, regulating transcriptional termination. This is a major advance of our concept, as regular switches rely on complex regulation including proteins and/or metabolites. Thus, our switches shall offer a greater robustness and their behaviour should be easier to predict. [[switch|Read more]] (hier sollte noch das hochskalieren erwähnt werden...<br><br />
These switches can further be used to build up a logical network inside a bacterial cell, enabling every scientist to connect as many functionalities (in form of BioBricks) as designated. We plan to offer simulation on each specifically designed network.<br />
<br />
<br><br>Over the years, many teams participating in the iGEM competition spent their time on constructing receptors and systems to detect a certain input that a variety of gorgeous oppurtunities is available so far.[[Image:TUM2010 network.png|thumb|300 px|right|Our visioon: A logic network inside the cell]] Nevertheless, until now it is not possible to link all those functionalities and build up a network giving differenciated responses to several of those input signals, where the molecular response depends on the complex composition of the environment a cell faces. We would like to offer this possibility to everyone.<br />
<br><br />
The logic network we want to apply will be based on devices, that can be easily upscaled and therefor offer the chance to build networks of any wanted complexicity. Our devices rely on pure RNA/RNA interactions and thus their behaviour is well predictable.<br />
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The concept we rely on for our design of RNA-switches is based on the principle of [https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation/ '''attenuation'''].<br />
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= Experiments =<br />
We designed several experiments to test our switches, all of them based on fluorescence measurements. We designed experiment setting for measurements ''in vivo'' as well as ''in vitro''. Our ''in vitro'' measurements relied on two different experiment set-ups. While the first was based on a commercial ''E. coli''-lysate, the latter was reporting on a transcriptional level only, eliminating most of the possible side-effects one could expect in the complex behaviour of a living cell or cell-lysate. [[Experiments_main|Read more]]<br />
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= Results =<br />
We ...blablabla<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
Text that will present our results...<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
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= thing to move =<br />
<br />
'''bioLOGICS: Logical RNA-Devices Enabling BioBrick-Network Formation'''<br />
'''Abstract'''<br />
Among the goals of iGEM is the creation of synthetic biological parts and their utilization to achieve novel features and behavior in biological systems. The emphasis of our project is put on this latter, "systems" aspect of iGEM. More precisely, we aim at the development and experimental demonstration of a scalable approach for the realization of logical functions in vivo.<br />
<br />
By developing a computational biological network based on RNA logical devices we will offer everyone the opportunity to 'program' their own cells with individual AND/OR/NOT connections between BioBricks of their choice. Thereby, BioBricks can finally fulfill their original assignment as biological parts that can be connected in many different ways. We will achieve this by engineering simple and easy-to-handle switches based on predictable RNA/RNA-interactions regulating transcriptional termination. These switches represent a complete set of logical functions and are capable of forming arbitrarily complex networks.<br />
<br />
== The Experiments ==<br />
===Fluorescent proteins as reporter===<br />
<div align="justify"><br />
Our initial idea to prove our concept of antitermination was to use flourescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks ''in vivo'' as well as ''in vitro'', the latter using a translation kit based on e.coli lysate. <br>We decided to use the flourescent proteins GFP and RFP, as their spectra do not overlap and we would not measure any FRET. We would use GFP fluorescence as internal control and RFP fluorescence as signal to detect termination/antitermination by our switch we cloned in between the coding sequences of the proteins. Both protein sequences are under the control of one (L-arabinose induced) promoter.<br />
[[Image:TUM2010_gfprfp_schalter_klein.gif|center|our idea]]<br />
<br><br />
When measuring the termination of our BioBricks and the antitermination by their corresponding signal-RNA, we should be able to observe an increasing RFP emission compared to the GFP emission upon induced signal-RNA production in the cells/in the kit:<br><br />
[[Image:TUM2010_Expected_emission_spextra.png|center|our idea]]<br />
Wiith these measurements, it should also be possible to observe differences in efficiency of termination as well as antitermination between our designed switches. <br><br><br />
<br />
===Measurements with the malachite green aptamer as reporter===<br />
A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramaticly (about 3000 times) upon binding of a specific RNA-aptamer.<br><br>#<br />
[[Image:TUM2010_Malachitgruen-2.png|500px|center|our idea]]<br />
---concept to be desribed, as well as literature---<br />
<ref>refs</ref><br />
<br><br><br />
To study the switches on the transcriptional level gives the advantage, that we would have less interferences and possible artefacts. Also, we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems.<br><br />
<br />
[[Image:TUM2010_Malachit_emission.png|200px|thumb|left|Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA]]We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.<br>Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level.<br />
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<br><br><br><br><br><br><br><br><br><br><br />
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Results <br />
==Flourescent proteins==<br />
Unfortunatly, we had to change the reporter construct two times during our experiments as several problems occured in our measurements:<br><br><br />
<br />
===First Try: based on the measurement plasmid pSB1A10===<br />
At the beginning, we decided to use the reporter plasmid [http://partsregistry.org/Part:pSB1A10 pSB1A10] from the registry. It consists of the fluorescent proteins eGFP and mRFP1. Each sequence includes a ribosome binding site and a stop-codon; the two genes are divided by a cloning side including the BioBrick cleavage sites.[[Image:ScreeningPlasmid1.0.PNG|300px|thumb|right|pSB1A10]]<br> In front of the eGFP sequence, the plasmid includes an arabinose-inducable promoter. The plasmid also contains an ampicilline resistence.<br><br />
We cloned our switches into the cloning site of the measurement plasmid and used an empty cloning site as control; our signal-RNAs we cloned into the [http://partsregistry.org/Part:pSB1K3 pSB1K3] vector, together with the BioBricks R0011 (Lac promoter) and B0014 (double terminator of transcription). Afterwards, we cut pSB1K3 with Aat2 and Pst1 and pSB1A10 with Nsi1 and Aat2 and ligated those fragments of each plasmid that contained our Bricks to get a Monsterplasmid.<br>We had to do so, as both plasmids contain the same ori mechanism. In addition, having both the switch and the signal RNA transcribed from the same plasmid gives us a high local concentration of the signal, once its transcription is induced.<br><br />
[[Image:TUM2010_Messplasmid_nr1.png|left|the measurement plasmid]]<br />
<br />
We transformed BL21(DE3) cells with the plasmid. We set up cultures, induced the arabinose promoter and measured the GFP and mRFP1 excitation/emission spectra within time.<br><br><br />
Unfortunatly, we were ot able to detect any RFP signal, not even in the positive control with no switch in between the GFP/RFP sequences.<br><br><br />
From these experiments, we concluded, that the mRNA of the RFP variant used was instable and rapidly degraded by RNases, so the RFP was not synthesized in the cells. This was also the conclusion from XXX...<br />
<br><br><br />
As solution to this, we decided to design a measurement plasmid ourselves:<br><br />
<hr width="300"><br />
<br><br />
<br />
===Second Try: A measurement plasmid of our own design===<br />
To design our own plasmid to overcome the problems that occurred in our first try gave us tghe possibility to overcome several other problems:<br><br />
#<br />
#<br />
#<br />
[[Image:TUM2010_Construct_no2.png|400px|our construct|center]]<br />
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===Third Try: One promoter for each protein===<br />
We decided to use the measuremnt plasmid we developed in our second try but to clone another L-arabinose induced promoter into the plasmid, in front of our switch followed by the mCherry sequence.<br><br />
<br>[[Image:TUM2010_Construct_no3.png|left|400px|Construct #3]]In this way, we still can use GFP fluorescence as internal control, because both protein transcription is under the control of a promoter of identical design.<br />
Though we are still not able to tell exactly why our previous measurements did not work, but with this construct we measured the first time fluorescence of the mCherry protein in our positive control.<br />
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One major task for every iGEM team is the creation of a wiki describing their project. For this reason the MIT is hosting a MediaWiki and every iGEM participant is allowed to create wiki pages on this server. The great advantage of this setup is that no team has to run their own server for hosting their wiki. On the other hand, team members are not allowed to change any MediaWiki settings, such as changing the skin of the wiki or installing extensions.<br />
As a part of your "Beyond the Lab" project we want to help other team getting started on their wiki. Therefore we will show how to create a team wiki that is easy to use and can still be customized as desired. Furthermore, we will also explain how to use this wiki even if somebody else of your team created the wiki. After running through this tutorial, you will have a basic framework that can be extended and enhanced later on.<br><br />
The tutorial will be divided into two parts:<br />
*'''Part I''' describes how to create and setup a team wiki at the first place. This part should be read by members in charge of the layout and design of the wiki and will require basic computer skills. An understanding of HTML and CSS will also be very helpful.<br />
*'''Part II''' focuses on the everyday usage of the wiki create in part I and gibes some general advice how to use the iGEM MediaWiki. All team member that want to contribute to their wiki should read this part.<br />
<br />
So the goal of your social project is to give every teams an easier access to the iGEM wiki: Because a wiki should simplify your life and not make you struggle!<br />
<br />
=Part I: Setup your Wiki=<br />
Before creating your own wiki, you should be aware of some aspects that you have to deal with when you create your team wiki.<br />
The intention of a wiki is to provide an easy-to-use platform where team member can enter information without having to know any HTML, CSS, etc. Secondly, most teams want to add their own style and design to their wiki. The normal way to customize MediaWiki is by using skins and extensions. As all iGEM teams share you common wiki, this is not possible. Changes done by one team would screw up the wiki of some another team. Summing up, the following text has to goals:<br />
Create a wiki that every team member can easily edit.<br />
Demonstrate how to customize the look of your wiki.<br />
==Create an easy-to-use Wiki==<br />
Although team wikis tend to vary, most of them contain two areas. A navigation area is always found as well as a content section. These two sections are the most relevant parts of your wiki since every team member should be capable of adding information or arranging your wiki pages. In this tutorial we will use a straight forward, table-based layout that will be put into practice using an invisible HTML table (see figure). Please note that HTML code within a wiki page has to be marked as such, by surrounding it with an opening "<nowiki><html></nowiki>" and a closing "<nowiki></html></nowiki>". For a very short introduction on HTML open the up the following "Read more" section.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
HTML (Hypertext Markup Language) is a computer language that tells a browser (e.g. Firefox, Internet Explorer, …) what a webpage is supposed to looks like. In fact, a HTML file comprises plain text and holds instructions, so-called tags, for the browser. Every tag is marked with brackets and has a name that specifies the tag type. The beginning of a tag always contains an opening an a closing bracket (<...>) and the ending of a tag additionally has a forward slash / (</...>). For example, <table> and </table> tell the browser that there will be a table, whereas all text between <p> and </p> will be in one paragraph. Consequently, a complete webpage is build by just stringing together several tags.<br />
It should be noted that every webpage is to be placed between a <html> and a </html> tag. Within this html-tag, there has to be one <head> … </head> that holds some invisible information about the webpage (e.g. the title of the page, the author, fonts and font-sizes, information for search robots such as Google, …). After the head-section, the body-section is declared (<body> … </body>). This is where all visible content of the page is specified (e.g. tables, paragraphs and text, images, flash content, …).<br />
Another important aspect concerning tags are so-called attributes. Attributes represent options that can be applied to certain tags. Attributes are places inside the opening brackets of a tag. For example, <table border=1> ...</table> set the width of the border of this table to 1 pixel.<br />
In order to learn more about HTML or to just look up some tags or attributes, many tutorials exist on the web. A small collection is available in the [http://en.wikipedia.org/wiki/HTML#External_links Wikipedia reference list].<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
[[Image:TUM2010_wikiHowTo_table.jpg | thumb | 765px | '''A''' The top of the page will contain a header showing a banner representing your team. Underneath will be a horizontal navigation area. Below this area on the left, the large content area will be positioned. To the right of the content will be a narrow vertical range, where you can add optional extras, such as a countdown to the jamboree, a visitor counter or some art work etc.<br />
<br>'''B''' HTML code for this table]]<br />
Another important aspect is how to apply this table layout to the wiki page. In general, a wiki software allows the user to create different pages and to add content to these pages. To view such a wiki page using a browser, the MediaWiki software has to generates the corresponding HTML page every time a page is requested. For this purpose, the MediaWiki software is supplied with a HTML framework holding the typical wiki interface and places the content of the requested page into a container within the HTML framework. So when a wiki page is edited, the only part that is actually changed lies within the container of this HTML framework. It is important to note, that in the case of the iGEM wiki this HTML framework cannot be modified. In other words, the layout and design has to be entered into the textbox on every edit page.<br><br />
But as the layout and navigation bar is identical for every page, it is useful to have these elements at one central place, rather than copying the same information on every single wiki page. For this purpose MediaWiki can handle so-called templates. These templates are wiki pages themselves and can be included into other wiki page. The import of a template into another wiki page is accomplished by placing the name of the template between and opening <nowiki>"{{:" and a closing "}}"</nowiki>, for example <nowiki>{{:Team:xyz/Templates/Layout}}</nowiki> will import the wiki page "Team:xyz/Templates/Layout". When this page is requested by a browser, the MediaWiki software will replace <nowiki>{{:Team:xyz/Templates/Layout}}</nowiki> with the content of the page Team:xyz/Templates/Layout (Please note, that MediaWiki always treats names in a case-sensitive manner). Another handy aspect about templates are so-called parameters that can serve as a placeholder for content. The great advantage araises from the fact, that you can import one template several times but each time you tell MediaWiki to replace the placeholder with another content. A placeholder or parameter is inserted by using "{{{" and "}}}" with the name of the placeholder inbetween. For example, the template page "Team:xyz/Templates/Layout" containing the placeholder <nowiki>"{{{text}}}"</nowiki> can be imported using <nowiki>{{:Team:xyz/Templates/Layout | text=This will be put into the template}}</nowiki>, where the <nowiki>{{{text}}}</nowiki> will be replaced by "This will be put into the template".<br><br />
<br />
By now we already have enough knowledge to implement the basic layout of the wiki. We will use the table layout described above and, to provide an easy-to-use wiki, we will split this table into different template page. The figure summaries the basic structure of the wiki:<br />
[[Image:TUM2010_wikiHowTo_structure.jpg | thumb | 765px | Figure illustrating the basic structure of the wiki.<br>Every box represents a wiki page, whereas the top left page is the main wiki page that holds the content. All other boxes represent template pages, that will imported into each other as indicated. For details see the following text.]]<br />
Every wiki page first includes the template ".../Templates/Header" which holds the first part of the HTML table. The bottom part of the table is include at the very last line in every wiki page (".../Templates/Footer"). Furthermore the template ".../Templates/Header" includes another template ".../Templates/Navigation" containing the navigation. The navigation itself is build by include multiple times the same template called ".../Templates/Button" where a two parameters are set each time. Looking at the template ".../Templates/Button", it can be seen that the text-parameter will eventually be the text of the link, where as the link-parameter will complete the URL "<nowiki>https://2010.igem.org/Team:xyz</nowiki>" with the page name (e.g. "<nowiki>https://2010.igem.org/Team:xyz</nowiki>" and "/Project" will be merged to "<nowiki>https://2010.igem.org/Team:xyz/Project</nowiki>").<br />
<br><br><br />
To see what the wiki actually looks like, take a look at this Team:TU_Munich/Social_Project/Demonstration. Please note, that for better visualization the table cells were colored using the bgcolor attribute in the td-tags. Also, the height was adjusted using the height-attribute. <br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}More details about the demonstration{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
This a list of all page used for the demonstration:<br />
*[[Team:TU_Munich/Social Project/Demonstration]]<br />
*[[Team:TU_Munich/Social Project/Templates/Header]]<br />
*[[Team:TU_Munich/Social Project/Templates/Footer]]<br />
*[[Team:TU Munich/Social Project/Templates/Navigation]]<br />
*[[Team:TU Munich/Social Project/Templates/Button]]<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br><br><br />
The great advantage that comes with these template pages is that it is much easier to maintain the wiki afterwards. Any member of your team can edit the wiki page without bothering about HTML and the layout of the page. Just let your team members know, that they always have to leave the first and the last line of code on every wiki page since these lines import the templates. Furthermore, with very little effort, the navigation can be changed by editing the page ".../Templates/Navigation".<br>The extensive use of templates is not only an advangtage to the every day usage, but also in the process of building the wiki. For example, if you want to add a new banner, you just have insert an <nowiki><img src="..."></nowiki> tag in the corresponding template and after saving the templates all your wiki pages will contain this new banner.<br />
<br />
<br />
<br><br><br />
<br />
Another application for templates is the easy integration of special elements such as a "Read more" section. On our wiki we use this technique to give the user a better overview of the page and to find the desired content faster. These toggle boxes use javascript and make use of the external javascript library [http://jquery.com "jQuerry"].<br />
{{:Team:TU Munich/Templates/ToggleBoxStart1}}More information about "Read more" sections{{:Team:TU Munich/Templates/ToggleBoxStart2}} {{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
The "Read more" boxes are based on displaying and hiding a <nowiki><div></nowiki> … <nowiki></div></nowiki>. Therefore overtime a box is used, you have to import a template page before and after the text that is supposed to be inside the box.<br><br />
To setup "Read more" boxes you have to do following steps:<br />
*Include the following javascript into a template page (e.g. .../Templates/Header):<br />
<nowiki><script type="text/javascript" src="http://code.jquery.com/jquery-latest.js"></script></nowiki><br />
<nowiki><script type="text/javascript"></nowiki><br />
$(document).ready(function(){<br />
//Hide (Collapse) the toggle containers on load<br />
$(".toggle_container").hide(); <br />
//Switch the "Open" and "Close" state per click then slide up/down (depending on open/close state)<br />
$("p.trigger").click(function(){<br />
$(this).toggleClass("activeToggle");<br />
var nextElem = $(this).next();<br />
while(nextElem!= null) {<br />
if(!nextElem.is(".toggle_container")) {<br />
nextElem = nextElem.next();<br />
}<br />
else {<br />
break;<br />
}<br />
}<br />
if(nextElem.is(".toggle_container")) {<br />
nextElem.slideToggle("slow");<br />
}<br />
return false; //Prevent the browser jump to the link anchor<br />
});<br />
$("a.toggle_close").click(function(){<br />
var nextParent = $(this).parent();<br />
while(nextParent!= null) {<br />
if(!nextParent.is(".toggle_container")) {<br />
nextParent = nextParent.parent();<br />
}<br />
else {<br />
break;<br />
}<br />
}<br />
if(nextParent.is(".toggle_container")) {<br />
nextParent.slideToggle("slow");<br />
var prevElem = nextParent.prev();<br />
while(prevElem!= null) {<br />
if(!prevElem.is("p.trigger")) {<br />
prevElem = prevElem.prev();<br />
}<br />
else {<br />
break;<br />
}<br />
}<br />
if(prevElem.is("p.trigger")) {<br />
prevElem.toggleClass("activeToggle");<br />
}<br />
}<br />
return false; //Prevent the browser jump to the link anchor<br />
});<br />
});<br />
<nowiki></script></nowiki><br />
*Create a template page (e.g. Team:xyz/Templates/ToggleBoxStart) and insert the following code:<br />
<nowiki><html></nowiki><br />
<nowiki><p class="trigger"><a class="toggle_text" href="#" style="font-size: 12px; color: #4e9d20">{{{text}}}</a></p></nowiki><br />
<nowiki><div></div></nowiki><br />
<nowiki><div class="toggle_container"></nowiki><br />
<nowiki> <div class="block"> <p align="justified"></nowiki><br />
<nowiki></html></nowiki><br />
*Create a template page (e.g. Team:xyz/Templates/ToggleBoxEnd) and insert the following code:<br />
<nowiki><html></p><a class="toggle_close" href="#" style="font-size: 12px; color: #4e9d20" align="left">Close</a></nowiki><br />
<nowiki> </div></nowiki><br />
<nowiki></div></nowiki><br />
<nowiki><br></nowiki><br />
<nowiki></html></nowiki><br />
*To style your "Read more" sections you can use optional CSS code, that you can include in the CSS section in (.../Templates/Header). To give you an example, this is the CSS code we used to style your toggle boxes:<br />
p.activeToggle<br />
{<br />
padding-left: 17px;<br />
background-image: url('https://static.igem.org/mediawiki/2010/5/56/Team_TU_Munich2010_Images_Arrow_close_small.jpg');<br />
background-repeat: no-repeat;<br />
background-position: center left;<br />
}<br />
a.toggle_close<br />
{<br />
padding-left: 17px;<br />
background-image: url('https://static.igem.org/mediawiki/2010/5/5d/Team_TU_Munich2010_Images_Cross_small.jpg');<br />
background-repeat: no-repeat;<br />
background-position: center left;<br />
float: left;<br />
text-align: left;<br />
}<br />
div.toggle_container<br />
{<br />
border-left: 1px solid #4E9D20;<br />
padding-left: 7px;<br />
margin-left: 5px;<br />
margin-bottom: 25px;<br />
}<br />
:Please note, that "a.toggle_close" refers to the close-Button displayed at the bottom of every "Read more" section, "div.toggle_container" refers to the box surrounding the content and "p.activeToggle" contains style instruction that only apply to the text of an open "Read more" section.<br><br />
The only information that your team mates will need to create a toggle box are the names of the two templates that have to be put before and after the text. A simple toggle could look like this:<br />
<nowiki>{{:Team:xyz/Templates/ToggleBoxStart | text=Read more}}</nowiki><br />
<nowiki>This is text will be hidden at the beginning, but can be read if the box is open.</nowiki><br />
<nowiki>{{:Team:xyz/Templates/ToggleBoxEnd}}</nowiki><br />
Your wiki is now ready and offers say-so-use "Read more" sections.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
<br />
==Customize and style your wiki==<br />
So far be we created the backbone of your wiki by using templates and a table layout. Besides, every teams wants to have their only style. However, in the case of an iGEM team wiki, the normal way of change the look and feel of a wiki does not work. This is due to the fact, that all teams share one big wiki and changes done to the layout will change all other team wikis as well.<br><br />
The best way of styling your wiki anyway is by using CSS (Cascading Style Sheets). To read more about CSS read the following section.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
CSS ([http://en.wikipedia.org/wiki/Css Cascading Style Sheets]) is a language that is used to describe what elements on a web page are supposed to look like. So a webpage is created using HTML code and can be styled using CSS. The CSS instruction are to be placed between an opening <nowiki>"<style>"</nowiki> and a closing <nowiki>"</style>"</nowiki> in the header of a HTML page or can be directly included in HTML tags using the "style" attribute:<br />
<br />
<nowiki><html></nowiki><br />
<nowiki><head></nowiki><br />
<nowiki><title>CSS example</title></nowiki><br />
...<br />
<nowiki><style type="text/css"></nowiki><br />
a {<br />
color: green;<br />
}<br />
a.external {<br />
color: red;<br />
}<br />
#bestImage {<br />
border: 1px solid green;<br />
width: 500px;<br />
}<br />
.red {<br />
color: red;<br />
}<br />
...<br />
<nowiki></style></nowiki><br />
<nowiki></head></nowiki><br />
<nowiki><body></nowiki><br />
<nowiki><p '''style="color: red;">Just some text</p></nowiki><br />
<nowiki></body></nowiki><br />
<nowiki></html></nowiki><br />
<br />
If the style-attribute is used, the CSS instruction will only affect this tag and all its child-tags. In case of the <nowiki>"<style>"</nowiki> block in the header section, two types of information have to be provided: What is to be styled and how is it to be styled.<br><br />
The first information is provided by so-called selectors. A selector can select an element of the page in many different ways. A selector can generally select all tags of a certain kind (e.g. all <nowiki><p></nowiki>-tags). Secondly, a selecotr can select an element that has a certain identifier. An identifier is a unique ID for an element within the webpage and is specified by the attibute "id" (e.g. <nowiki><img id="bestImage"><nowiki>). Thirdly, a selector can select a subgroup of elements, called class. HTML elements can be grouped into a classes by using the attribute "class" (e.g. <nowiki><img class="goodImages"></nowiki>).<br>A selector is always placed before the opening bracket "{". In the example above all three types have been used: "a" is a selector that selects all link elements (<nowiki><a></nowiki>-tags). "a.external" addresses all links that have been assigned to the group "external" (<nowiki><a class="external"></nowiki>-tags). "#bestImage" refers to the element that has the ID "bestImage" (e.g. <nowiki><img id="bestImage"></nowiki>-tag). And finally ".red" selects all tags within the class "red" (e.g. <nowiki><a class="red">, <p class="red">, <table class="red"></nowiki>).<br><br />
All style instruction are place inside brakets "{ ... }". They all share a common syntax. and the left side of a colon is a keyword that specifies what property is to be changed, and on the right side is the new value that will be set. An instruction is terminated by a semicolon. There are many different keywords that can set all kinds of properties (colors, borders, dimensions, visibility, text-decoration, etc.). Once you learn more about CSS, you will get to know the most important ones.<br>Again, there many tutorials about CSS on the web that go far beyond this short introduction. If you want to create a sophisticated wiki, you will definitely have to deal with CSS.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
Once you are familiar with CSS, changing the appearance of the wiki is very straight forward. The nice thing about MediaWiki is that nearly every element in the wiki framework (the big banner on the top of the page, the table of content, the title of the page, etc.) has a unique identifier or belongs to a certain class. An identifier is specified using the HTML tag id, whereas an element can be assigned to a class called "title" by using class="title". <br />
Consequently, to change the look of a certain element, you have to first get to know the name of the class or the identifier and then use this name to change the appearance using CSS.<br><br />
To find out the name of the identifier or the class, you can either take a direct look at the source of the page, or you can use a very powerful Firefox extension to easily extract this information. Take a look at the following "read more" section for details about this.<br />
{{:Team:TU Munich/Templates/ToggleBoxStart}}<br />
To use Firebug you have to install the plug-in first. Open up FireFox, go to [https://addons.mozilla.org/de/firefox/addon/1843/ the Mozilla plug-in webpage], install the plug-in and restart FireFox. As soon as you start FireFox you will notice an icon with a small bug on the very bottom of the FireFox window. Clicking on it will open and close the FireBug screen.<br><br />
To find out the id or the class of a specific element on the webpage, just right-click on it and in the menu you can choose to inspect this element. The firebug screen will now show the HTML code of this element. Now, you can either look for "id=" or "name=" which will give you a name you can address this element using CSS selectors (see also the following figure).<br />
[[Image:TUM2010_FireBug.jpg| thumb | 720px | A typical FireBig screen<br>After right-clicking on the heading, it can be seen that this heading is assigned to the class "firstHeading" and can thus be styled using an appropriate selector]]<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
Once you found out the name, you have to get into CSS. Since the appearance of your wiki is very likely to be the same for all pages, we can make use of our template structure that we created before. As the template ".../Templates/Header" is imported into every wiki page, putting your CSS styling in this template will affect all pages. Just edit the .../Templates/Header page and insert your CSS code:<br />
<nowiki><style type="text/css"></nowiki><br />
body {<br />
background-color: white; /* defines the default background color of the document*/<br />
color: black; /* defines the default font-color of the document*/<br />
}<br />
a {<br />
color: black; /* defines the default color of links */<br />
}<br />
a:hover {<br />
color: gray; /* defines the default color of links hovered by the cursor */<br />
}<br />
a {<br />
color: black; /* defines the default color of links */<br />
}<br />
.firstHeading<br />
{<br />
display: none; /* hides the default heading */<br />
}<br />
...<br />
<nowiki></style></nowiki><br />
<br />
You can not only modify the framework of MediaWiki, but you can also define classes in your templates and modify them in your global css section. For example, in the button template the link is assigned to the class "button", allowing the modification of all assigned links in your global CSS section. Now, you can also change all the default elements of the iGEM wiki. But please note that changing to much might can also confuse the user. For example, you should leave default buttons such as "Login, search, …". It is also appreciated to provide a link to the iGEM mainpage (http://20xx.igem.org/) in your header section.<br />
CSS is a very powerful technique that cannot be explained in this tutorial completely. Learning more about CSS can save a lot of time when customizing your wiki. You might also want to take a look at [https://2010.igem.org/Team:TU_Delft#page=Modeling/wiki-tips-tricks this page from this year's TU Delft team], where some more iGEM-specific CSS styling is listed. You will also find many tutorials or look-up pages about CSS on the web. And don't forget to take a look at previous teams, they might inspire you or have that piece of code you where looking for...<br />
<br><br><br />
Congratulations! You made it through the tutorial. Now you are ready to play around with CSS and add some more eye candy in your templates to produce a nice looking wiki. Have fun and don't hesitate to try new things, you can always go back using the MediaWiki history!<br />
<br />
=Part II: How to use the iGEM MediaWiki=<br />
<br />
This part will give you a short introduction, how to use the iGEM wiki and will gibe you some advice that will save your time and your nervs. Furthermore it will give some hints, specific to a wiki created according to the above tutorial. <br />
==Login in==<br />
First of all, you always have to be login into the iGEM wiki before you can change your page. Just click the log in button on the top of this page or any other iGEM page and make sure you have your account set up.<br />
==Creating a new wiki page==<br />
There are several ways of creating a new page. The easiest way is to type the name of the new page into your browser, for example <nowiki>http://20xx.igem.org/Team:xxx/bane of the page you want to create.</nowiki>. Please note, that all names are treated in a case sensitive manner! After typing in the name, just click "Create" or "Edit" and you can add content to your newly created page. Hit "Save" to save the new page.<br><br />
Since all iGEM teams share one common wiki, it is important that every page you create is in your namespace. In other words, all pages you create have to have your team name as a prefix. For instance, all page of our wiki are in the namespace "Team:TU_Munich".<br><br />
One other important aspect is, that at the beginning and at the end of every wiki page, your have to link to so-called template pages that contain all your styling and layout of your wiki. Please ask your team member in charge of the wiki to add these lines to your new page, or take a look at an existing page.<br />
==Adding content to a wiki page==<br />
Before you start adding a lot of text into the wiki page please make yourself familiar with the Markup language of MediaWiki. MediaWiki offers a very intuitive way to mark headings, pictures, etc. As the Wikipedia is also based on MediaWiki, might already be familiar with these commands. However, you still want to take a look at the [http://en.wikipedia.org/wiki/Help:Wiki_markup Wikipedia help page] or the [http://www.mediawiki.org/wiki/Help:Contents MediaWiki documentation].<br><br />
MediaWiki features a powerful history. You can undo any changed to a page or any replacement of a file. Make sure you are familiar with this feature.<br />
<br />
==Uploading files==<br />
In case you have to upload a picture or any other file you should be aware of some aspects. To upload a file you can scroll down on any wiki page and click on "upload file". Alternatively you can link to a picture on your page before you even uploaded it. MediaWiki will no show a red link at the corresponding page. Clicking on it will also get you to the upload page.<br><br />
For security reasons, the iGEM wiki does not accept any type of file. Refer to the upload page to see a list of allowed file extensions. Make sure whether you can upload a file in advance, before you spend time generating files that you cannot send to the iGEM server.<br><br />
Another iGEM-specific aspect, is that all teams share one common pool to upload pictures and other files. Your team should agree on a prefix that you add to all your filenames. In case you upload a picture named "team.jpg", chances a high that by accident another team will replace your file. Use "OUR_COLLEGE_20xx_team.jpg" instead!<br />
==Editing your navigation bar==<br />
As the navigation section is the same for all wiki pages, it can be stored at one central page. In case your wiki was created according to this tutorial, the computer expert in your team can give the name of the corresponding page where you can edit the navigation. In this page you will find entries similar to "<nowiki>{{:Team:xxx/Templates/Button | text=Home | link=/}}</nowiki>". Each entry resembles one button or link to a page. Please note, that your team members might have changed the exact spelling. In the above example, you can just add a new button to your navigation by copying a new entry "<nowiki>{{:Team:xxx/Templates/Button | text=Home | link=/}}</nowiki>". Now replace "Home" by the text you want to appear in the button. The target of your button will be specified by "link=". Change "/" to "/Modeling" to link to the page "Modeling" in our namespace.<br />
==Using "Read more" sections==<br />
With the help of "Read more" sections you can give the user a better overview of the page. Your team member in charge of the wiki can tell you more details. In general, you only have to add two lines to your page to create a "Read more" box:<br />
*Place "<nowiki>{{:Team:xyz/Templates/ToggleBoxStart | text=Read more}}</nowiki> before the content you want to be inside the box. Note that you can change the text "Read more" to some custom words by change the text after text=<br />
*Place "<nowiki>{{:Team:xyz/Templates/ToggleBoxEnd}}</nowiki> behind the text that you want to be within the box.<br />
To give you an example, a simple toggle could look something like this:<br />
<nowiki>{{:Team:xyz/Templates/ToggleBoxStart | text=Read more}}</nowiki><br />
<nowiki>This is text will be hidden at the beginning, but can be read if the box is open.</nowiki><br />
<nowiki>{{:Team:xyz/Templates/ToggleBoxEnd}}</nowiki><br />
Your wiki is now ready and offers say-so-use "Read more" sections.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}}<br />
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{{:Team:TU_Munich/Templates/End}}</div>Hartlmuellerhttp://2010.igem.org/File:Toggle_switch.pngFile:Toggle switch.png2010-10-28T02:16:50Z<p>Hartlmueller: uploaded a new version of "Image:Toggle switch.png"</p>
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<div></div>Hartlmuellerhttp://2010.igem.org/Team:TU_Munich/ProjectTeam:TU Munich/Project2010-10-28T00:15:41Z<p>Hartlmueller: /* Putting it all together: the switching process */</p>
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<center><font size="5pt" color="#000000">'''bioLOGICS'''</font><font size="4pt" color="#000000">: Logical RNA-Devices Enabling BioBrick-Network Formation</font></center><hr color="black"><br><br />
= Vision=<br />
<br />
Until today, 13.628 biobrick sequences<sup>[[Team:TU_Munich/Project#ref1|&#91;1&#93;]]</sup> have been submitted to partsregistry, thereof 102 reporter units, 12 signaling bricks and xx sensing parts.<br />
Since then, people are trying to arrange these single biological building blocks in such a manner that allows producing special biotechnological products (metabolic engineering), developing biological sensory circuits (biosensors) and even giving microorganisms the ability to react on multiple environmental factors and serve both as disease indicator and drug. These examples and further promising ideas were implemented on previous iGEM-competitions.<sup>[[Team:TU_Munich/Project#ref2|&#91;2&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref3|&#91;3&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref4|&#91;4&#93;]]</sup> <br><br><br />
The idea of combining the outcome of several iGEM competitions to construct complex synthetic biological systems falls at the last hurdle - the fact, that each team uses a different principle how to access and functionally connect the respectively used biobricks. For example, it is a major challenge to create a system that uses several sensoring BioBricks from different iGEM-teams which in turn regulates reportering BioBricks from various teams. In order to combine and fully take advantage of these promising projects, our vision is to develop an adapter that allows interconnecting arbitrary biobricks on a functional level. Such a system easily allows to setup sensor-reporter circuits and interconnect them to complete biological chips... A further step towards artificial cells.<br><br><br />
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Generally speaking, the above descirbed adapter has to meet the following requirements:<br />
*'''Universality'''<br />
:The adapter has to be compatible to as many BioBricks as possible. This objective will guarantee that a large number of BioBricks can be connected.<br />
*'''Scalability'''<br />
:Once the basic design of the system is established, the construction of the system is supposed to be automated in silico. This way it will be possible to create an adapter connecting a large amount of BioBricks.<br />
*'''Biological orthogonality'''<br />
:Interfering with cellular components has to be as low as possible in order to avoid unwanted and perturbing side effects.<br />
*'''Logic'''<br />
:The adapter is supposed to not only associate different BioBricks, but to functionally connect BioBricks in a precisely determined manner (including operations such as AND/OR/NOT).<br />
<br><br />
Several biological logic units, devices and circuits have been developed so far<sup>[[Team:TU_Munich/Project#ref5|&#91;5&#93;]]</sup>, but to our knowledge, none was shown to meet all requirement listed above.<br />
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=Implementation=<br />
To functionally connect BioBricks, there are several possibilities including genetic switches, riboswitches and direct protein-protein interactions. We investigated several hypothetically principles, and decided to focus our practical work on the development of a RNA-RNA interaction-based switch. These switches are capable of changing between two states, a state of antitermination and termination, and make use of highly-specific RNA-RNA interaction. In principle such a switch can fulfill all requirements mentioned previously. The following text clarifies how these switches work in detail.<br />
==How to connect BioBricks==<br />
Our adapter is a system, that activates or disables BioBricks (output BioBricks) in response to the presence of other Biobricks (input Biobricks). Our approach uses a molecular network to put this into practice and consists of four major elements:<br />
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{|<br />
|-<br />
|[[Image:Networks.png|center|thumb|730px]]<br />
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In order to connect different BioBricks, our network requires four major types of components:<br />
*Input elements<br />
*Transmitter molecules<br />
*Logic gates<br />
*Output elements<br />
<br />
{{:Team:TU_Munich/Templates/InfoBoxStart}}'''Computer vs. molecular network - and our approach'''<br><br />
Logic gates in a molecular network are often compared to transistors used in a computer, where billions of transistors are incorporated<sup>[[Team:TU_Munich/Project#ref7|&#91;7&#93;]]</sup>. The main advantage on a computer chip is, all transistors share the same functional principle, and only the way connecting them in a special sequence allows specific addressing of only a subset of other transistors by an input. However, spatial fixed connections of molecular logic gates are not possible in a living cell. The "wiring" within a cell relies on the specific interaction between transmitter molecule and their corresponding logic gates, for example implemented by protein-protein/ligand-protein interactions or specific ligand-riboswitch interactions.<sup>[[Team:TU_Munich/Project#ref8|&#91;8&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref9|&#91;9&#93;]]</sup> As a result, in a cell, each occurring logic gate ("transistor") has to be different, at least in a special recognition site<sup>[[Team:TU_Munich/Project#ref10|&#91;10&#93;]]</sup> - for example like different transcription factors, recognizing different DNA-sites. Thanks to evolution, nature easily can invent a new transistor for each task - science achieves this only on a limited scale, and producing synthetic molecular logic gates artificially by either rational or evolutionary protein or riboswitch engineering, is limited to small circuits so far<sup>[[Team:TU_Munich/Project#ref11|&#91;11&#93;]]</sup>. Our project aims to establish a real molecular transistor, which shares the same functional principle for all logic gates. At the same time, we want to design a easily exchangeable recognition site, which can individually be designed by everyone! {{:Team:TU_Munich/Templates/InfoBoxEnd}}<br />
<br />
These elements can be combined to build up a molecular network (see illustration). Each input molecule (such as a BioBrick) produces a unique transmitter molecule. All transmitters belong to the same type of molecule and share a common design. However each transmitter molecule can only interact and activate a certain subset of logic gates. In other words, logic gates have to recognize as well as bind the corresponding transmitter molecules and are capable of producing a new output transmitter molecule. Depending on the type of the logic gate (AND, OR or NOT<sup>[[Team:TU_Munich/Project#ref6|&#91;6&#93;]]</sup>), an output transmitter is only created if both input transmitter molecules are present (AND), at least one of two input transmitters is present (OR) or if no input transmitter is present at all (NOT). Once a logic gates has produced a new output transmitter, these transmitters can in turn address another subset ("layer") of logic gates. In theory many layers of logic gates can be connected this way allowing the creation of large networks. Until this step, various transmitter molecules might have been produced. But in order to create a Biobrick output, the last layer logic gates finally generates transmitter molecules that will not active logic gates, but will rather interact with the cell metabolism to produce a BioBrick responds. In other words, the last layer of transmitter molecules is capable of regulating BioBrick formation.<br />
<br />
<br />
Summarizing, the network established a connection between input BioBricks and output BioBricks in a functional manner.<br />
Having addressed the basic layout of the molecular network, the next step is to determine what type of molecules can perform the required functions. We decided to use RNA, both as transmitter molecules and for constructing logic gates. Several advantages result from the utilization of RNA as the central element:<br />
*During the last years, many Biobricks were designed that are sensitive to various chemicals and substances. These BioBricks often function as a transcription factor that binds to a specific DNA sequence and consequently would be capable to produce a specific transmitter RNA molecule. Thus, in principle each BioBrick which involves transcription can be integrated in our network.<br />
*Since all logic gates are capable of producing transmitter RNA, they can also produce functional mRNA encoding any protein. This means, each BioBrick consisting of protein or RNA can be produced as an output of our network.<br />
*If RNA forms both, the transmitter molecule and the logic gates, they can specifically interact by RNA-RNA interaction, which is highly predictable compared to protein interactions. This allows to generate a library of transmitters and gates ''in silico''. Such a library is essential for the creation of large networks.<br />
*RNA production is fast and energy saving for a cell. Consequently, operating a network that only produces RNA rather than proteins will also be faster and more efficient for the host cell. Since our logic gates are based on transcription, translation and resource consuming protein production will only be required at the very last step. <br />
*As the half-time of RNA can be rather short, transmitter RNA will not accumulate within the cell and it is therefore less likely for the system to become saturated.<br />
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<br />
==Design and functional principle of logic gates==<br />
The concept introduced above provides a framework that can potentially serve as an universal adapter between different BioBricks. However, the [[Team:TU_Munich/Glossary#logic gate | logic gates]] have not been specified more precisely so far. This will be done in the following section.<br />
<br />
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Generally speaking, our logic gates are to posses the following characteristics:<br />
*Logic gates, such as AND, OR and NOT, have to be implemented by RNA-interaction based principles (see [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]).<br />
*All logic gates have to recognize their corresponding [[Team:TU_Munich/Glossary#Transmitter (bioLOGICS)| transmitter RNA´s]] and, in response, produce an output transmitter molecule.<br />
*Logic gates should follow a basic design rule, in such a way, that their creation can be automated ''in silico''.<br />
*The response efficiency of a logic gate toward a transmitter molecule should be comparable for all logic gates to provide calculable robustness and sensitivity. This will ensure comparable molecular concentrations and functionality of large networks.<br />
*The system has to be designed for ''in vivo'' utilization at the first place. As a reference we always assumed a temperature of 37 °C and an ''E. coli'' environment.<br />
{{:Team:TU Munich/Templates/ToggleBoxEnd}} <br />
In order to build logic gates for our bioLOGICS system we will first create a simple switch. A switch can be activated by one transmitter RNA and produce an output transmitter RNA. In contrast to a logic gate, a switch does not perform logic operations. However by combining switches, logic gates can be created. The following text will first describe how the developed switch works and secondly, how logic gates such as AND/OR/NOT can be created using these switches.<br />
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{{:Team:TU Munich/Templates/ToggleBoxStart1}}Read more{{:Team:TU Munich/Templates/ToggleBoxStart2}}<br />
[[Image:toggle_switch.png|500px|thumb|center|id="hideOnReadMore"| Rectangles present the composition of our functional units on the level of DNA. Fringed lines represent RNA produced by RNA polymerase. The stem loop structure depicts the switchable terminator. Terminator and target site are illustrated in blue and turquoise, respectively. Recognition sites are indicated in different colors, in this case red for the input transmitter and green for the output transmitter.Each switch and or later logical unit has to be flanked by a promotor and another constitutive terminator, to allow RNA-production by RNA-polymerase in a proper way. ]]<br />
{{:Team:TU Munich/Templates/ToggleBoxStart3}}<br />
===Switch===<br />
[[Image:TUM2010_switch-and-transmitter.jpg|550px|right|thumb|The basic strcutrue of a switch (left) and a transmitter RNA (right). See text for details.]]<br />
Roughly speaking, a switch can be regarded as an enhance transcriptional terminator. The enhancement can be described easier by dividing a switch into its components: <br />
*'''Target site'''<br><br />
:The target site is the functional core element of our switches, allowing a shift between an "on" and "off" state. Since we work on the level of RNA-production (transcription), a "switchable" transcriptional terminator is suitable for this purpose. By allowing or preventing formation of a transcriptional terminator, that is by switching between termination and antitermination it is possible to represent an "off" and an "on" state, respectively. Therefore, the target site is the 5' ending of the terminator and is required for a stable terminator formation. It should be noted that this principle was also observed in nature.<br />
:To highlight and illustrate the functional principle of our switches, only the part of the terminator which is involved in interacting with a transmitter molecule and which is responsible for shifting between "on" and "off" state is called target site. The remaining terminator sequence is called terminator in the following, even if both, target site and terminator build up the terminator structure occurring in nature. <br />
:The important aspect of our switches is the fact that all switches will hold the same identical target site. Therefore having found one functional "switchable" terminator, will allow almost unlimited upscaling since this terminator can be used for a large library of switches. This is the main difference to previous works done on this field, which always required developing a new shifting principle for each switch.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup><sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup> Beside this scalability, this principle provides a comparable on/off shifting rate (responds function) for all switches, avoiding complex fine tuning of molecular networks.<br />
:To sum it up, the target site, allows to switch between an "on" and "off" state. But so far, the switch is not capable of performing specific interaction with transmitter molecules. This is where the recognition site comes into play.<br />
*'''Recognition site'''<br />
:The recognition site defines which transmitter molecule can actually interact with the switch. Therefore, a unique recognition site is generated for each switch and is positioned right upstream of the target site. In principle the recognition can be any random sequence as long as it remains unique within the molecular network.<br />
Summing up, the recognition site allows a specific interaction between switches and transmitter molecules. Once this interaction is formed, an interaction between the transmitter and the target will actually switch the state of the terminator. This allows the specific arrange and interconnection of numerous of these switches by transmitter molecules, without changing the target site. Comparable to wires connecting many identical transistors, our target site remains the same.<br />
<br><br />
<br />
===Transmitter RNA´s===<br />
As desccribed above, transmitter RNA's are the input and output of bioLOGICS switches (compare [[Team:TU_Munich/Project#How_to_connect_BioBricks | How to connect BioBricks]]). These transmitters are short ssRNA molecules representing the "trigger" to shift switches between the "on" and "off" state. To fulfill this role, they need to posses the following properties:<br />
*A transmitter may only interact with certain switches. That is, a transmitter has to find the corresponding recognition site of a switch.<br />
*Once an interaction is established between a transmitter and a switch, a transmitter as to be capable of changing the secondary structure of a terminator and thus cause antitermination.<br />
Again, these two properties are fulfilled by two comnponents of the transmitter:<br />
*'''Identitiy site'''<br />
:This site is capable of forcing an interaction between the transmitter and the switch. Therefore it is complementary to the recognition site of this switch. As the recognition site is unique within a network, so it the identity site. However, the single identity site is not capable of changing the state off the switch. That is were the trigger site comes into play.<br />
*'''Trigger site'''<br />
:Once an interaction is created by the identity site, the trigger site is capable of actually shifting the switch since it is complementary to the target site of the switch. To fulfill this role, it is placed upstream at the 5' end of the identity site. As the target site is the same for all switches, the trigger site is the same for all signals. Therefore it is important, that similar to the identity site, a trigger site cannot function on its own. That is, a single trigger site cannot shift the state of a switch without the help of an identity site.<br />
<br />
Summing up, we applied the principle introduce for the switches to the transmitter molecules. In contrast to previous approaches on this field <sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup>, we introduced the described synthetic trigger site in such a manner that it is not able to change the state of the terminator on its own, but only in combination with the identity site. So the challenge is to arrange and optimize these elementary building blocks thermodynamically, that a trigger site is only able to switch in combination with its respective identity site. This was done by ''in silico'' design using [[TU Munich/Glossary#NUPACK| NUPACK]], presented in section [[TU Munich/Project#in silico design| in silico design]].<br />
<br />
<br><br />
<br />
===Putting it all together: the switching process===<br />
[[Image:TUM2010_switching-process.jpg|620px|right|thumb|The basic strcutrue of a switch (left) and a transmitter RNA (right). See text for details.]]The functional principle of the designed switches is illustrated in the figure. The switch is positioned on DNA upstream of a desired output transmitter. So in the absence of a triggering transmitter molecule, transcription will be canceled by the formation of a RNA stem loop in the nascent RNA-chain. This will cause the RNA polymerase to stop transcription and fall off the DNA and consequently no output RNA will be produced. This process only relies on [[Team:TU_Munich/Glossary#Termination| rho-independent termination]].<br />
On the other hand, in the presence of a [[Team:TU_Munich/Project#RNA_transmitters | input transmitter]], this small functional RNA inhibits the stem loop formation by complementary base-pairing and hence avoids termination of transcription. In detail, the identity site (red part on transmitter) binds the recognition site (red part on switch) and serves as [[Team:TU_Munich/Glossary#Toehold|toehold]], which will thermodynamically allow the trigger site (turquoise part on transmitter) to perform a [[Team:TU_Munich/Glossary#Strand Displacement| strand displacement]] and open up the stem loop structure. Consequently the polymerase can read all the way through and form the output RNA.<br>Summing up, we use this concept to create a switch that can be toggled by a transmitter RNA molecule and in response, is able to produce another transmitter RNA.<br />
<br><br />
<br><br />
<br><br />
<br />
===From switches towards bioLOGICS logic gates===<br />
As described, each switch can be accessed by a specific RNA-transmitter molecule, illustrating the input. In turn, another RNA-transmitter molecule will be produced if the switch shifts its state. This output transmitter of one switch can serve as input transmitter for the next switch by meaningful selection and design of the respective recognition sites. This easily allows arranging several switches in specific sequences and faulty wiring - the corner stone of a logical network.<br />
<br />
To ease the building of logical networks, applying mathematical logics, e.g. Boolean logics like in computational science would be worthwhile. It is possible to establish general Boolean operators with our switches and thus build "logical modules". <br />
Since AND/OR/NOT are the most simple logic operations which can be implemented with the presented switches, and all remaining operations can be expressed by these three operators[ZITAAAT Wiki oder so)we exemplary designed them.<br />
<br />
{|<br />
|-<br />
| *AND consists of a parallel circuit of two switches<br />
|-<br />
|[[Image:AND2.png|500px|thumb|center]] <br />
|-<br />
| *OR is implemented by connecting two switches in series<br />
|-<br />
|[[Image:OR2.png|500px|thumb|center]]<br />
|-<br />
| *NOT is more complex to explain. In principle, it consists only of one switch which contains its respective signal molecule intrinsic, so via intramolecular interaction, antitermination is the initial state. The signal is composed of the same components as usual to allow interconnection with other logic gates.<br />
|-<br />
|[[Image:NOT2.png|500px|thumb|center]]<br />
|}<br />
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<br />
==Network construction==<br />
Designing complex biological networks based on either traditional protein engineering or our new bioLOGICS is still a complex task. We developed a software which allows the fast construction of a bioLOGICS based networks. <br><br />
To read more about this, look at our [https://2010.igem.org/Team:TU_Munich/Software Software page]<br />
<br />
=Our Objective=<br />
Putting the implementation described above into pratise, will be a major challenge. For this year's iGEM competition our goal is to do the first step: design and build a switch that can be toggled by a RNA molecule. To be precise, we want to apply the design rules of our switch to modify a transcription terminator, in such a way, that it interacts with a second RNA molecule and as a result is no longer capable of forming a stem loop. This objective will require intensive ''in silico'' designing and modeling of switches based on different terminators and their corresponding transmitters. In connection to this theoretical part, we also have to test and verify the switches. For this step, we establish custom-made assays, ''in vitro'' and ''in vivo''.<br />
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Once the objective mentioned above is accomplished, these basic RNA/RNA-interactions have to be modified in such a manner that the described identity/trigger site pattern for the transmitter and the complementary recognition/target site switch composition has to be established. The most important requirement is to is to optimize these modules that the transmitter is only able to switches specifically, meaning only in the presence of both, identity AND trigger site. <br />
<br><br />
Once the objective mentioned above is accomplished, the creation of an OR gate will be rather simple since it only requires two switches. However the creation of an AND or NOT gate and optimizing the logic gates to improve their responds function will remain the goal of future work. Also the creation of small networks and the correct integration of BioBricks as input and output molecules will be future challenges. Furthermore, we wanted to rather focus on the development and the testing of our structural design of the switches, rather than developing a variety of new BioBricks.<br />
<br />
==''In silico'' design==<br />
As desribed above, our switches are based on certain design rules. However, there still are different strucutral paramters that need to be tested and optimized (length of recognition site and target site, choice of terminator, etc.).<br />
We used [[Team:TU_Munich/Project#in silico design |''in silico'' design]] and [[Team:TU_Munich/Modeling| modeling]] ) to test different parameters. Furthermore we tried to use the [[Team:TU_Munich/Glossary#Antitermination|antitermination principle]] observed in nature, such as [[Team:TU_Munich/Glossary#Attenuation| attenuation]] in ''E. coli'' or [[Team:TU_Munich/Glossary#Tiny Abortive RNA´s| tiny abortive RNA´s]] of T7-phage.<br />
==Evaluation and Measurements==<br />
To evaluate the functionality of our molecular switches, we first had to establish several assays. Therefore, we improved an existing [[Team:TU_Munich/Lab#In vivo Measurements |''in vivo'' assay]] and developed an [[Team:TU_Munich/Lab#In vitro Transcription | ''in vitro'' assay]] for this purpose. For more information please refer to the [[Team:TU_Munich/Lab | lab]] section.<br />
<br><br />
<br><br />
Summarizing, the main challenges are <br />
* to find a suitable terminator construct and design a complementary trigger unit, which is only functional in combination with a specificity site - meaning an optimization of the '''thermodynamically parameters''' (see[[Team:TU_Munich/Project#in silico design| in silico design]])<br />
* to investigate whether the transmitter/switch interaction reaction is on a timescale to be competitive to terminator formation - meaning an comparison of '''kinetic parameters''' (see [[Team:TU_Munich/Modeling|Modeling page]])<br />
* to proof antitermination can be also be caused by synthetically RNA-interaction (see [[Team:TU_Munich/Glossary#Antitermination| Antitermination in nature]] and [[Team:TU_Munich/Project#Results| ''in vivo'' and ''in vitro'' measurements]] )<br />
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=Results=<br />
Every network starts with a basic unit. While our declared aim is to enable networks allowing fine-tuning of gene expression beyond the regular on/off, exploring such an on/off switch/signal pair is the first step towards a functional network. We constructed several units and tested their efficiency, robustness and reproducibility ''in vivo'', ''in vitro'' and ''in silico''. Furthermore we developed a software which allows easy constructions of networks based on our designed logic gates. Conclusive elaboration of a few first RNA-based logic units is the major contribution of our iGEM team.<br />
<br />
==in silico Design of Switching and Trigger Unit==<br />
===attenuation principle===<br />
A random sequence was derived from xxx. A complementary sequence, reaching within the terminator´s stem loop was stepwise shortened to find the length, where the formation of the terminator is thermodynamically favored compared to the strand displacement by the signal. The trigger sequence was defined by selecting the shortest unit which still is able to "destroy" the stem loop. Subsequently, the trigger unit was tested in regard of not being able to resolve the stem loop on its on. As table xxx illustrates, the terminator is thermodynamically favorite toward the trigger unit, but in combination with the specificity site, binding becomes possible.<br />
<br />
==Diffusion and RNA Folding Dynamics==<br />
We estimated the diffusion time for our constructs and modeled the folding dynamics of our bioLOGICS switches including the switching process with a stochastic RNA folding program. We were able to provide better insight in their folding dynamics and proved that they are able to interrupt termination. We also optimized the switches and the corresponding signals. Furthermore, we combined the switches what resulted in a logic gate. See our [[Team:TU Munich/Modeling|Modeling page]] for further details.<br />
<br />
==''in vivo'' Functionality Screening==<br />
Since our logic gates are intended to function in living cells, ''in vivo'' measurements were essential. In a set of experiments we concentrated on two different switches based on known [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|attenuators]] from nature: the [[Team:TU_Munich/Modeling#Switch|HisTerm]] and [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Focusing on fluorescent proteins for quantifiable input and output we designed a functional and robust screening system. For greater detail see [[Team:TU_Munich/Lab#Experiment_Design|Experimental Design]]. Unfortunately, setting up a working screening system failed twice. Only in redesigning and improving the screening plasmid pSB1A10 we succeeded, but lost precious time.<br />
<br />
Ultimately, the two switches displayed remarkable differences in their terminator efficiency, but neither of them responded to their corresponding signal. However, screening one transmitter signal cannot disprove our principle system. Limited by time, we hope for future teams to take up our work and to use our improved test system that we submitted to the parts registry, for performing successful in vivo measurement.<br />
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Considering the high complexity of ''in vivo'' measurements compared to other experimental challenges, a robust and easy to handle test system for [[Team:TU_Munich/Glossary#PoPS-based devices| PoPS-based devices]] is desirable. As described in [[Team:TU_Munich/Lab#Experiment_Design|Experimental design]], we used fluorescent proteins: RFP or mCherry to measure the amount of produced output and eGFP for normalization. Our first attempt, using the screening plasmid pSB1A10, yielded no interpretable results. Switching the fluorescent protein to mCherry did not work either, but after several experimental setups we determined a transcriptional problem causing no reporter protein expression regardless of the inserted part. Thereby we demonstrated the screening plasmid pSB1A10 to be [[Team:TU_Munich/Biobricks#Falsification| malfunctioning]]. <br />
Finally a new design based on pSB1A10 lead to a functional and robust screening system (compare [[Team:TU_Munich/Parts#Screening system: Backbone BBa_K494001| Screening system: Backbone BBa_K494001]]). A second promoter with identical induction properties inside the BioBrick cloning site enforces transcription of the PoPS-based device and the mCherry output.<br />
<br />
Exemplary, the graph below on the right shows the positive control, induced and uninduced at OD<sub>600</sub>=0.7 followed by 16 h incubation at 25 °C. Clearly visible are eGFP and mCherry fluorescence in the induced samples. The uninduced control showed no fluorescence at all, demonstrating the PBad promoter to be tight and providing very low basal transcription. A major advantage for the screening system. This newly designed screening approach renders the characterization of PoPS-based devices in general and switches in particular easy and robust. The low basal transcription furthermore fulfills one of the most important requirements for the designed switches, since output transmitters may only be produced in presence of an input transmitter. This helps to avoid strong "background" noise, which would extremely harden the successful interconnection of several switches. <br />
<br><br />
[[Image:TUM2010_PosControlklein.JPG|200px||thumb|left|Bacteria containing positive control]]<br />
[[Image:TUM2010_graphPosControl1.png|355px|thumb|center|Emission spectra of induced (green/red) and uninduced(black) positive control BBa_K494002 ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
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Due to the time limitations of the iGEM completion we had to focus our efforts on few switches after designing the screening system. Relying on the functionality of systems occurring in nature, we choose the [[Team:TU_Munich/Modeling#Switch|HisTerm]] as well as the [[Team:TU_Munich/Modeling#Switch|TrpTerm]]. Both switches are based on known natural [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|attenuators]]. Testing synthetic and none-naturally switchable terminators in vivo are goals for future work.<br />
Delorme et al. reported the His-Terminator to be a remarkable effective Terminator with more than 99% termination efficiency.<sup>[[Team:TU_Munich/Project#ref12|&#91;12&#93;]]</sup> The exemplary measurement below on the right confirms the high terminator efficiency. In fact, we could not detect any mCherry fluorescence in any cells containing the [[Team:TU_Munich/Modeling#Switch|HisTerm]]. Even induction of the corresponding signal transmitter RNA via IPTG did not alter the Terminator efficiency. Again time was the limiting factor and prevented us from testing more than one corresponding transmitter, although the [[Team:TU_Munich/Modeling| Modeling]] highly suggested the necessarily of finding an optimized transmitter lenght. Thus, the results are insufficient either to prove or to disprove the functionality of the [[Team:TU_Munich/Modeling#Switch|HisTerm]] or our concept in general.<br />
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[[Image:TUM2010_HisSwitchklein.JPG|200px|thumb|left|Bacteria containing HisTerm]][[Image:TUM2010_HisSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing HisTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
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Attaining only 90% terminator efficiency, the natural Trp [[https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation|Attenuator]] is known be less effective than the [[Team:TU_Munich/Modeling#Switch|HisTerm]].<sup>[[Team:TU_Munich/Project#ref13|&#91;13&#93;]]</sup> The graph on the right depicts the to our designed [[Team:TU_Munich/Modeling#Switch|TrpTerm]] characteristic efficiency of about 40 %, notably below the natural standard. Allowing 60% transcription in the “off” state excludes the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] from possible candidates for a scalable network of logic gates, due to the mentioned required "yes or no" function (see [[Team:TU_Munich/Project#Implementation| Implementation and how to connect Biobricks]]). Thus the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] is inoperative as intended, but may still be useful in other contexts. Similar to the [[Team:TU_Munich/Modeling#Switch|HisTerm]], the [[Team:TU_Munich/Modeling#Switch|TrpTerm]] also did not react to the induction of the corresponding signal. Under circumstances, termination efficiencies altered by the transmitter are on a low range and not resolvable within observed 40% basal transcription. <br />
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[[Image:TUM2010_TrpSwitchklein.JPG|200px|thumb|left|Bacteria containing TrpTerm]][[Image:TUM2010_TrpSwitchGraph1.png|355px|thumb|center|Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing TrpTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm]]<br />
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Making use of our improved screening system we also carried out some ''in vivo'' kinetic measurements in addition to the end-point measurements above. In contrast to the ''in vitro'' experiments we did not obtain significant results for the characterization of our switches. As the switching process is many times faster than protein synthesis our ''in vivo'' kinetics include the synthesis of mCherry as well as its maturation. Therefore we centered our attention on end-point experiments. For more information browse the [[Team:TU_Munich/Lab#Lab_Book|lab book]]. <br><br />
Considering our ''in vivo'' measurements, neither of the tested switches showed any effect regarding the signal induction. But due to the small number of tested switches and signals this can hardly be regarded as disprove of concept. In particular in light of the recent findings by Sooncheol proving antitermination in principle using a T7 system.<sup>[[Team:TU_Munich/Project#ref14|&#91;14&#93;]]</sup><br />
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==''in vitro'' Screening==<br />
To minimize the amount of disturbing factors we decided to countercheck our ''in vivo'' results with a set of ''in vitro'' measurements. While the ''in vitro'' systems are no doubt much less complex than living cells, the work with these set-ups proved to be quite as difficult.<br />
Just as with the ''in vivo'' measurements we could prove our switching system neither right nor wrong, leaving enough work for future iGEM teams.<br />
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===''in vitro translation''===<br />
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Beside optimization of the reporter proteins in use, the major problem occuring in the experiments was the low capacity of the kit. The signal intensity was very low, which made it difficult to observe any signal intensity alterations, so no conclusion could be drawn from these measurements.<br />
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===''in vitro'' transcription===<br />
We used two completely independent ''in vitro'' systems: Using ''E.coli'' RNA Polymerase we analyzed the His and Trp switches that had already been tested ''in vivo''. In a second set-up, we used the well-established T7 RNA Polymerase and switch based on the T7 terminator as well as several signal sequences.<br />
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====T7 System====<br />
In contradiction to the results of Kang and coworkers and other groups, in our ''in vitro'' set-up the T7 terminator did not seem to terminate at all. The negative control (Promoter_Terminator_malachite binding aptamer) showed a similar increase in fluorescence as the positive control (Promoter_random sequence_malachite binding aptamer). <br />
[[Image:TUM2010_T7Result1.png|350px||thumb|left|''in vitro'' transcription measurement of T7 terminator with no signal(upper left), nonsense signal (upper right) and two different designed signals (below)]]<br />
[[Image:TUM2010_T7Result2.png|350px||thumb|right|''in vitro'' transcription measurement of T7 terminator with nonsense signal(upper left), and three different designed signals (remaining traces)]]<br><br />
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[[Image:TUM2010_T7Result3.png|350px||thumb|left|''in vitro'' transcription measurement of positive control(upper left and T7 terminator with three different designed signals (remaining traces)]]<br />
Furthermore denaturing Polyacrylamide Gel Electrophoresis (PAGE) confirmed that there was no observeable termination of transcription. The addition of a signaling sequence led to a significantly lower increase in fluorescence, which can be attributed to the fact that both DNA sequences, switch and signal, compete for RNA Polymerases.<br />
However, there is almost no difference between the designed signals and random sequences, which is not a big surprise since there can be no antitermination if the terminator itself does not work.<br><br />
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Possible explanations for the contradiction between our results and those of Kang and coworkers might be the experimental set-up and the RNA Polymerases we used. Different variants of T7 RNA Polymerase might respond in different ways to terminator structures, and the termination might be influenced by the presence or absence of cofactors, depending on the purification methods used in producing the Polymerase.<br><br><br />
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This set-up offers a lot of possible experiments for the future, which we would have loved to conduct with a just a bit more time...<br />
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====''E.coli'' System====<br />
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Compared to the T7 System, the ''E. coli'' RPO system produced poor increases in fluorescence, indicating little RNA synthesis. It was shown that the presence of a terminator decreases, as expected, the production of downstream RNA. This result was also confirmed by denaturing PAGE. However, due to the poor changes in fluorescence we were not able to actually characterize the behaviour of our switches ''in vitro'', and the small RNA concentrations did not allow a quantitative interpretation of our gels. A major problem with this method was the low concentration of the ordered Polymerase resulting in a much weaker overall signal as comparable measurements using the T7 Polymerase. <br><br><br />
In future experiments we might try to work with smaller volumes in order to reach higher concentration of RPO and of the synthesized RNA molecules, so measuring in 96 well plate readers might be a good choice. <br />
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==Software==<br />
Although we could not show the full functionality of bioLOGICS in the lab we still want to demonstrate the potential of our approach. Hence we implemented the idea behind our logic gates in a program which illustrates how bioLOGCIS theoretically would allow the construction of complex information processing networks interconnecting BioBricks. For further details take a look at our [[Team:TU Munich/Software|Software page]].<br />
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=Outlook=<br />
...<br />
future plans will also work with [[Team:TU_Munich/Glossary#Synthetic Terminator| Synthetic Terminators]], which might retrieve additional informations on what drives the process of Termination<br />
...<br />
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=References=<br />
<html><a name="ref1"></a></html>[1] http://partsregistry.org/cgi/partsdb/Statistics.cgi<br />
<html><a name="ref2"></a></html>[2] https://2009.igem.org/Team:Imperial_College_London/M1 encapsulation<br />
<html><a name="ref3"></a></html>[3] https://2009.igem.org/Team:TUDelft<br />
<html><a name="ref4"></a></html>[4] https://2008.igem.org/Team:Heidelberg<br />
<html><a name="ref5"></a></html>[5] Maung Nyan Win and Christina D. Smolke, Science Oct. 2008 Vol. 322. no. 5900, pp. 456 - 460<br />
<html><a name="ref6"></a></html>[6] http://en.wikipedia.org/wiki/Logic_gate#Symbols<br />
<html><a name="ref6"></a></html>[7] http://en.wikipedia.org/wiki/Moore's_law<br />
<html><a name="ref6"></a></html>[8] http://en.wikipedia.org/wiki/Protein_interaction<br />
<html><a name="ref6"></a></html>[9] http://en.wikipedia.org/wiki/Riboswitch<br />
<html><a name="ref6"></a></html>[10] http://en.wikipedia.org/wiki/Binding_sites + http://en.wikipedia.org/wiki/Recognition_site<br />
<html><a name="ref6"></a></html>[11] irgend ein damn review über directed evolution and so on<br />
<html><a name="ref12"></a></html>[12] Delorme, Ehrlich and Renault, Regulation of Expression of the Lactococcus lactis Histidine Operon. Journal of Bacteriology, Apr. 1999, p. 2026–2037<br />
<html><a name="ref13"></a></html>[13] Trun and Trempy(2003): Fundamental Bacterial Genetics, Wiley-Blackwell, Chapter 12 <br />
<html><a name="ref14"></a></html>[14]Sooncheol Lee, Huong Minh Nguyen and Changwon Kang, Tiny abortive initiation transcripts exert antitermination activity on an RNA hairpin-dependent intrinsic terminator. Nucleic Acids Research, 2010, 1–9<br />
<html><a name="ref6"></a></html>[15] <br />
<html><a name="ref6"></a></html>[16]<br />
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<!-- The idea behind our project is to change the way BioBricks have been used up to now. Over the years, many receptors and signals have been constructed as BioBricks during the annual iGEM competition, but still it is not possible to interconnect these Bricks in a complex biological network resuting in a cell, that is able to respond to its environment giving differenciated responses depending on the input signals. (Beispiel: cambridge hat das gemacht, xx dies, aber eine zelle kann nicht beides...<br><br />
We plan to create biological switches, that can function as locial gates inside a cell. Our switches rely on RNA/RNA-interactions, regulating transcriptional termination. This is a major advance of our concept, as regular switches rely on complex regulation including proteins and/or metabolites. Thus, our switches shall offer a greater robustness and their behaviour should be easier to predict. [[switch|Read more]] (hier sollte noch das hochskalieren erwähnt werden...<br><br />
These switches can further be used to build up a logical network inside a bacterial cell, enabling every scientist to connect as many functionalities (in form of BioBricks) as designated. We plan to offer simulation on each specifically designed network.<br />
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<br><br>Over the years, many teams participating in the iGEM competition spent their time on constructing receptors and systems to detect a certain input that a variety of gorgeous oppurtunities is available so far.[[Image:TUM2010 network.png|thumb|300 px|right|Our visioon: A logic network inside the cell]] Nevertheless, until now it is not possible to link all those functionalities and build up a network giving differenciated responses to several of those input signals, where the molecular response depends on the complex composition of the environment a cell faces. We would like to offer this possibility to everyone.<br />
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The logic network we want to apply will be based on devices, that can be easily upscaled and therefor offer the chance to build networks of any wanted complexicity. Our devices rely on pure RNA/RNA interactions and thus their behaviour is well predictable.<br />
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The concept we rely on for our design of RNA-switches is based on the principle of [https://2010.igem.org/Team:TU_Munich/Glossary#Attenuation/ '''attenuation'''].<br />
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= Experiments =<br />
We designed several experiments to test our switches, all of them based on fluorescence measurements. We designed experiment setting for measurements ''in vivo'' as well as ''in vitro''. Our ''in vitro'' measurements relied on two different experiment set-ups. While the first was based on a commercial ''E. coli''-lysate, the latter was reporting on a transcriptional level only, eliminating most of the possible side-effects one could expect in the complex behaviour of a living cell or cell-lysate. [[Experiments_main|Read more]]<br />
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= Results =<br />
We ...blablabla<br />
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Text that will present our results...<br />
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= thing to move =<br />
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'''bioLOGICS: Logical RNA-Devices Enabling BioBrick-Network Formation'''<br />
'''Abstract'''<br />
Among the goals of iGEM is the creation of synthetic biological parts and their utilization to achieve novel features and behavior in biological systems. The emphasis of our project is put on this latter, "systems" aspect of iGEM. More precisely, we aim at the development and experimental demonstration of a scalable approach for the realization of logical functions in vivo.<br />
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By developing a computational biological network based on RNA logical devices we will offer everyone the opportunity to 'program' their own cells with individual AND/OR/NOT connections between BioBricks of their choice. Thereby, BioBricks can finally fulfill their original assignment as biological parts that can be connected in many different ways. We will achieve this by engineering simple and easy-to-handle switches based on predictable RNA/RNA-interactions regulating transcriptional termination. These switches represent a complete set of logical functions and are capable of forming arbitrarily complex networks.<br />
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== The Experiments ==<br />
===Fluorescent proteins as reporter===<br />
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Our initial idea to prove our concept of antitermination was to use flourescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks ''in vivo'' as well as ''in vitro'', the latter using a translation kit based on e.coli lysate. <br>We decided to use the flourescent proteins GFP and RFP, as their spectra do not overlap and we would not measure any FRET. We would use GFP fluorescence as internal control and RFP fluorescence as signal to detect termination/antitermination by our switch we cloned in between the coding sequences of the proteins. Both protein sequences are under the control of one (L-arabinose induced) promoter.<br />
[[Image:TUM2010_gfprfp_schalter_klein.gif|center|our idea]]<br />
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When measuring the termination of our BioBricks and the antitermination by their corresponding signal-RNA, we should be able to observe an increasing RFP emission compared to the GFP emission upon induced signal-RNA production in the cells/in the kit:<br><br />
[[Image:TUM2010_Expected_emission_spextra.png|center|our idea]]<br />
Wiith these measurements, it should also be possible to observe differences in efficiency of termination as well as antitermination between our designed switches. <br><br><br />
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===Measurements with the malachite green aptamer as reporter===<br />
A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramaticly (about 3000 times) upon binding of a specific RNA-aptamer.<br><br>#<br />
[[Image:TUM2010_Malachitgruen-2.png|500px|center|our idea]]<br />
---concept to be desribed, as well as literature---<br />
<ref>refs</ref><br />
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To study the switches on the transcriptional level gives the advantage, that we would have less interferences and possible artefacts. Also, we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems.<br><br />
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[[Image:TUM2010_Malachit_emission.png|200px|thumb|left|Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA]]We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.<br>Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level.<br />
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Results <br />
==Flourescent proteins==<br />
Unfortunatly, we had to change the reporter construct two times during our experiments as several problems occured in our measurements:<br><br><br />
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===First Try: based on the measurement plasmid pSB1A10===<br />
At the beginning, we decided to use the reporter plasmid [http://partsregistry.org/Part:pSB1A10 pSB1A10] from the registry. It consists of the fluorescent proteins eGFP and mRFP1. Each sequence includes a ribosome binding site and a stop-codon; the two genes are divided by a cloning side including the BioBrick cleavage sites.[[Image:ScreeningPlasmid1.0.PNG|300px|thumb|right|pSB1A10]]<br> In front of the eGFP sequence, the plasmid includes an arabinose-inducable promoter. The plasmid also contains an ampicilline resistence.<br><br />
We cloned our switches into the cloning site of the measurement plasmid and used an empty cloning site as control; our signal-RNAs we cloned into the [http://partsregistry.org/Part:pSB1K3 pSB1K3] vector, together with the BioBricks R0011 (Lac promoter) and B0014 (double terminator of transcription). Afterwards, we cut pSB1K3 with Aat2 and Pst1 and pSB1A10 with Nsi1 and Aat2 and ligated those fragments of each plasmid that contained our Bricks to get a Monsterplasmid.<br>We had to do so, as both plasmids contain the same ori mechanism. In addition, having both the switch and the signal RNA transcribed from the same plasmid gives us a high local concentration of the signal, once its transcription is induced.<br><br />
[[Image:TUM2010_Messplasmid_nr1.png|left|the measurement plasmid]]<br />
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We transformed BL21(DE3) cells with the plasmid. We set up cultures, induced the arabinose promoter and measured the GFP and mRFP1 excitation/emission spectra within time.<br><br><br />
Unfortunatly, we were ot able to detect any RFP signal, not even in the positive control with no switch in between the GFP/RFP sequences.<br><br><br />
From these experiments, we concluded, that the mRNA of the RFP variant used was instable and rapidly degraded by RNases, so the RFP was not synthesized in the cells. This was also the conclusion from XXX...<br />
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As solution to this, we decided to design a measurement plasmid ourselves:<br><br />
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===Second Try: A measurement plasmid of our own design===<br />
To design our own plasmid to overcome the problems that occurred in our first try gave us tghe possibility to overcome several other problems:<br><br />
#<br />
#<br />
#<br />
[[Image:TUM2010_Construct_no2.png|400px|our construct|center]]<br />
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===Third Try: One promoter for each protein===<br />
We decided to use the measuremnt plasmid we developed in our second try but to clone another L-arabinose induced promoter into the plasmid, in front of our switch followed by the mCherry sequence.<br><br />
<br>[[Image:TUM2010_Construct_no3.png|left|400px|Construct #3]]In this way, we still can use GFP fluorescence as internal control, because both protein transcription is under the control of a promoter of identical design.<br />
Though we are still not able to tell exactly why our previous measurements did not work, but with this construct we measured the first time fluorescence of the mCherry protein in our positive control.<br />
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