Team:TU Munich/Project
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*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. | *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. | ||
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- | In order to build a logic gates for our bioLOGICS system we will first create a simple | + | In order to build a 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. |
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- | === | + | ===Switch=== |
- | Roughly speaking, a | + | 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: |
*'''Target site'''<br> | *'''Target site'''<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. | :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. | ||
: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. | :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. | ||
- | :The important aspect of our | + | :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|[12]]]</sup><sup>[[Team:TU_Munich/Project#ref13|[13]]]</sup><sup>[[Team:TU_Munich/Project#ref14|[14]]]</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. |
- | :To sum it up, the target site, allows to switch between an "on" and "off" state. But so far, the | + | :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. |
*'''Recognition site''' | *'''Recognition site''' | ||
- | :The recognition site defines which transmitter molecule can actually interact with the | + | :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. |
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. | 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> | ||
===Transmitter RNA´s=== | ===Transmitter RNA´s=== | ||
- | As desccribed above, transmitter RNA's are the input and output of bioLOGICS | + | 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: |
- | *A transmitter may only interact with certain switches. That is, a transmitter has to find the corresponding recognition site of a | + | *A transmitter may only interact with certain switches. That is, a transmitter has to find the corresponding recognition site of a switch. |
*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. | *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. | ||
Again, these two properties are fulfilled by two comnponents of the transmitter: | Again, these two properties are fulfilled by two comnponents of the transmitter: | ||
*'''Identitiy site''' | *'''Identitiy site''' | ||
- | :This site is capable of forcing an interaction between the transmitter and the | + | :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. |
*'''Trigger site''' | *'''Trigger site''' | ||
: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. | :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. | ||
- | Summing up, we applied the principle introduce for the | + | 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|[12]]]</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]]. |
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===Putting it all together: the switching process=== | ===Putting it all together: the switching process=== | ||
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- | [[Image:toggle_switch.png|500px|thumb|right| 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 | + | [[Image:toggle_switch.png|500px|thumb|right| 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. ]] 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]]. |
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. | 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. | ||
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- | ===From | + | ===From switches towards bioLOGICS logic gates=== |
- | 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 | + | 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. |
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". | 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". | ||
- | Since AND/OR/NOT are the most simple logic operations which can be implemented with the presented | + | 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. |
{| | {| | ||
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|[[Image:OR2.png|500px|thumb]] | |[[Image:OR2.png|500px|thumb]] | ||
|- | |- | ||
- | | *NOT is more complex to explain. In principle, it consists only of one | + | | *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. |
|- | |- | ||
|[[Image:NOT2.png|500px|thumb]] | |[[Image:NOT2.png|500px|thumb]] | ||
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! ratio of pairing [%] | ! ratio of pairing [%] | ||
|- align=center | |- align=center | ||
- | | | + | | switch (terminator secondary structure) |
| -30.10 | | -30.10 | ||
| - | | - | ||
|- align=center | |- align=center | ||
- | | transmitter + | + | | transmitter + switch |
| -72.88 | | -72.88 | ||
| 100 | | 100 | ||
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{|width=100% align=center | {|width=100% align=center | ||
|- | |- | ||
- | + | switch:''Comparison of free energy for secondary structure formation at 37°C, simulated by using NUPACK analysis.'' | |
|} | |} | ||
<br><br> | <br><br> | ||
{|width=100% | {|width=100% | ||
- | |[[Image:dotplot_switch.png|300px|thumb|center|Pairing probabilities of the His-Terminator based | + | |[[Image:dotplot_switch.png|300px|thumb|center|Pairing probabilities of the His-Terminator based switch simulated by NUPACK secondary structure analysis tool]] |
- | |[[Image:switch.png|300px|thumb|center|Illustration of the His-Terminator based | + | |[[Image:switch.png|300px|thumb|center|Illustration of the His-Terminator based switch secondary structure, generated by NUPACK utility tools]] |
|- | |- | ||
- | |[[Image:dotplot_signal+switch.png|300px|thumb|center|Pairing probabilities of the His-Terminator based | + | |[[Image:dotplot_signal+switch.png|300px|thumb|center|Pairing probabilities of the His-Terminator based switch in presence of the respective transmitter RNA, simulated by NUPACK secondary structure analysis tool]] |
- | |[[Image:signal+switch.png|300px|thumb|center|Illustration of the His-Terminator based | + | |[[Image:signal+switch.png|300px|thumb|center|Illustration of the His-Terminator based switch secondary structure in presence of the respective transmitter RNA, generated by NUPACK utility tools]] |
|- | |- | ||
- | |[[Image:dotplot_triggerunit+switch.png|300px|thumb|center|Pairing probabilities of the His-Terminator based | + | |[[Image:dotplot_triggerunit+switch.png|300px|thumb|center|Pairing probabilities of the His-Terminator based switch in presence of the transmitter RNA´s trigger site only, simulated by NUPACK secondary structure analysis tool]] |
- | |[[Image:trigger+switch.png|300px|thumb|center|Illustration of the His-Terminator based | + | |[[Image:trigger+switch.png|300px|thumb|center|Illustration of the His-Terminator based switch secondary structure in presence of the transmitter RNA´s trigger site only, generated by NUPACK utility tools]] |
|- | |- | ||
|[[Image:dotplot_identity+switch.png|300px|thumb|center|Pairing probabilities of the His-Terminator based NOT-gate in presence of the transmitter RNA´s identity site only, simulated by NUPACK secondary structure analysis tool]] | |[[Image:dotplot_identity+switch.png|300px|thumb|center|Pairing probabilities of the His-Terminator based NOT-gate in presence of the transmitter RNA´s identity site only, simulated by NUPACK secondary structure analysis tool]] | ||
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! ratio of paaring [%] | ! ratio of paaring [%] | ||
|- align=center | |- align=center | ||
- | | | + | | switch (NOT-gate secondary structure) |
| -68.18 | | -68.18 | ||
| - | | - | ||
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==Diffusion and RNA Folding Dynamics== | ==Diffusion and RNA Folding Dynamics== | ||
- | We modeled our bioLOGICS toggle switches theoretically to prove that our they are able to interrupt termination. We also optimized the switches and the corresponding signals and furthermore took a look a combination of | + | We modeled our bioLOGICS toggle switches theoretically to prove that our they are able to interrupt termination. We also optimized the switches and the corresponding signals and furthermore took a look a combination of switches that result in logic gate. See our [[Team:TU Munich/Modeling|Modeling page]] for further details. |
==''in vivo'' Functionality Screening== | ==''in vivo'' Functionality Screening== | ||
- | 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 | + | 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. |
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. | 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. | ||
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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. | 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. | ||
- | 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 | + | 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> | ||
[[Image:TUM2010_PosControlklein.JPG|200px||thumb|left|Bacteria containing positive control]] | [[Image:TUM2010_PosControlklein.JPG|200px||thumb|left|Bacteria containing positive control]] | ||
[[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]] | [[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> | ||
- | Due to the time limitations of the iGEM completion we had to focus our efforts on few | + | 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. |
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|[12]]]</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. | 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|[12]]]</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. | ||
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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 | + | 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> |
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|[14]]]</sup> | 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|[14]]]</sup> | ||
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VisionUntil today, 13.628 biobrick sequences[1] have been submitted to partsregistry, thereof 102 reporter units, 12 signaling bricks and xx sensing parts.
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.[2][3][4] Generally speaking, the above descirbed adapter has to meet the following requirements:
ImplementationTo 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. How to connect BioBricksOur 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:
In order to connect different BioBricks, our network requires four major types of components:
Computer vs. molecular network - and our approach
Logic gates in a molecular network are often compared to transistors used in a computer, where billions of transistors are incorporated[7]. 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.[8][9] As a result, in a cell, each occurring logic gate ("transistor") has to be different, at least in a special recognition site[10] - 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[11]. 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! 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[6]), 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.
Design and functional principle of logic gatesThe concept introduced above provides a framework that can potentially serve as an universal adapter between different BioBricks. However, the logic gates have not been specified more precisely so far. This will be done in the following section. Generally speaking, our logic gates are to posses the following characteristics:
SwitchRoughly speaking, a switch can be regarded as an enhance transcriptional terminator. The enhancement can be described easier by dividing a switch into its components:
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.
Transmitter RNA´sAs desccribed above, transmitter RNA's are the input and output of bioLOGICS switches (compare 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:
Again, these two properties are fulfilled by two comnponents of the transmitter:
Summing up, we applied the principle introduce for the switches to the transmitter molecules. In contrast to previous approaches on this field [12], 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 NUPACK, presented in section in silico design.
Putting it all together: the switching process
![]() 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. On the other hand, in the presence of a 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 toehold, which will thermodynamically allow the trigger site (turquoise part on transmitter) to perform a strand displacement and open up the stem loop structure. Consequently the polymerase can read all the way through and form the output RNA. From switches towards bioLOGICS logic gatesAs 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. 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". 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.
Network constructionDesigning 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.
Our ObjectivePutting 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.
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.
In silico designAs 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.). We used in silico design and modeling ) to test different parameters. Furthermore we tried to use the antitermination principle observed in nature, such as attenuation in E. coli or tiny abortive RNA´s of T7-phage. Evaluation and MeasurementsTo evaluate the functionality of our molecular switches, we first had to establish several assays. Therefore, we improved an existing in vivo assay and developed an in vitro assay for this purpose. For more information please refer to the lab section.
ResultsEvery 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. in silico Design of Switching and Trigger Unit
attenuation principleA 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.
tiny abortive principleubiquitous terminatorsCloseDiffusion and RNA Folding DynamicsWe modeled our bioLOGICS toggle switches theoretically to prove that our they are able to interrupt termination. We also optimized the switches and the corresponding signals and furthermore took a look a combination of switches that result in logic gate. See our Modeling page for further details. in vivo Functionality ScreeningSince 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 [[1]] from nature: the HisTerm and TrpTerm. Focusing on fluorescent proteins for quantifiable input and output we designed a functional and robust screening system. For greater detail see 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. 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. Considering the high complexity of in vivo measurements compared to other experimental challenges, a robust and easy to handle test system for PoPS-based devices is desirable. As described in 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 malfunctioning. Finally a new design based on pSB1A10 lead to a functional and robust screening system (compare 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. Exemplary, the graph below on the right shows the positive control, induced and uninduced at OD600=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.
Attaining only 90% terminator efficiency, the natural Trp [[3]] is known be less effective than the HisTerm.[13] The graph on the right depicts the to our designed TrpTerm characteristic efficiency of about 40 %, notably below the natural standard. Allowing 60% transcription in the “off” state excludes the TrpTerm from possible candidates for a scalable network of logic gates, due to the mentioned required "yes or no" function (see Implementation and how to connect Biobricks). Thus the TrpTerm is inoperative as intended, but may still be useful in other contexts. Similar to the HisTerm, the 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.
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 lab book. in vitro Screening
in vitro translationBeside optimization of the reporter proteins in use, a major problem occuring in the experiments was the low capacity of the kit. The signal intensity is very low, which made it difficult to observe any signal intensity alterations, so no conclusion could be drawn from these measurements. in vitro transcriptionE.coli RPOT7 RPOCloseSoftwareAlthough 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 Software page.
Outlook... future plans will also work with Synthetic Terminators, which will retrieve additional informations what drives the process of Termination ... References[1] http://partsregistry.org/cgi/partsdb/Statistics.cgi [2] https://2009.igem.org/Team:Imperial_College_London/M1 encapsulation [3] https://2009.igem.org/Team:TUDelft [4] https://2008.igem.org/Team:Heidelberg [5] Smolke and so on.... [6] http://en.wikipedia.org/wiki/Logic_gate#Symbols [7] http://en.wikipedia.org/wiki/Moore's_law [8] http://en.wikipedia.org/wiki/Protein_interaction [9] http://en.wikipedia.org/wiki/Riboswitch [10] http://en.wikipedia.org/wiki/Binding_sites + http://en.wikipedia.org/wiki/Recognition_site [11] irgend ein damn review über directed evolution and so on [12] Delorme, Ehrlich and Renault, Regulation of Expression of the Lactococcus lactis Histidine Operon. Journal of Bacteriology, Apr. 1999, p. 2026–2037 [13] Trun and Trempy(2003): Fundamental Bacterial Genetics, Wiley-Blackwell, Chapter 12 [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 [15] [16]
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