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 Biobrick submissions this year, we offer a complete set for switch evaluation on all cellular levels.
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.
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.

Design

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.
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 here, every sort of input may later be combined with our basic switching units.

general measurement principle

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.
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.

schematic estimated fluorescence spectra

Construction and Cloning

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 below. Unfortunately after two months of cloning we had to recognize that the plasmid in use did not work for us (see also pSB1A10 Falsification).
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

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.

Measurements based on submitted Biobricks

The Biobricks BBa_K494001-BBa_K494006 are constructed for easy design of a switch-evaluation system. Detailed information can be found here.

Switch evaluation in vivo

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.

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 OD₆₀₀ of 0.05. RFP/mCherry emission should be visible only in case of a working switch or inefficient termination.

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.

Bacterial cultures after incubation of 16h

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.
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.
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.

Design

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 provided this year, can again be used as the groundwork for constructing vectors for measurements.
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.

Measurements

We used the cell-free E. coli S30 extract system for circular DNA provided by Promega^[1], which is prepared by modifications of the Method Zubay et al.^[2]. The characterization of the kit can be obtained from the [http://partsregistry.org/Cell-free_chassis/Commercial_E._coli_S30 Parts Registry].
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.

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.
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.
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 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.

T7 RNA polymerase

E. coli RNA polymerase

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.
E. coli RPO was ordered saturated with σ70-factor.

Denaturing Polyacrylamide gel electrophoresis

Malachite green assay

Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA

In this year's DNA submission we contribute the malachite green binding aptamer which can be used as a transcription reporter in in vitro transcription experiments.
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.

Description of the malachit green assay. Antitermination allows the polymerase to produce the malachite green aptamer

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.

08.04.2010

Flo & Philipp

PCR

• samples:
  • R0011_His
  • R0011_Trp
  • Control
• protocol: protocol
  • templates: purified PCR products from 5.2.2010
  • primer G1004/1005
  • polymerase: Taq
  • programm: igempcr

Purification of PCR products with QIAquick PCR purification Kit

• protocol followed. exceptions: DNA-binding/unbinding with 3min 6000rpm followed by 60sec full speed

2% Agarose Gel

• in 1x TAE.

09.04.2010

Philipp & Flo

Gel of PCR products from 08.04.2010

• loaded: 10 µL sample+2 µL 6x GLD, 4/2 µL LMW standard
• 110 V, 90 min
• stained with Sybrgold, 20 min, 1:10.000 dilution in TAE
• Standard - Control - R0011_His - R0011_Trp - Standard(=low molecular weight (see Team:TU_Munich/Lab#Molecular_Biology Lab_Protocols))

15.04.2010

Philipp & Flo

[http://web.e14.physik.tu-muenchen.de/igem/index.php/Team:TU_Munich/Lab#Molecular_Biology PCR PCR] of B0014 and R0011

16.04.2010

Philipp & Flo

• Purification of PCR products from 15.04.2010 using QIA kit
  
  

• Concentrations measured with nanodrop:
  
  

• PCR of B0014
  
  • Purification with the Zymo Kit, Elution in 20 µL H2O
  • Concentration measured with nanodrop, 17.5 ng/µL --> worked
    
    

• Digestions of:
  
  

• Gel for purification of the cleaved plasmid
  • 2% Agarose in 1x TAE
  • 120 V, 90 min
  • stained with SybrGold
  • digestion, digestion, 1 kb ladder
    • Digestion worked (partly). band at 2000 bp (backbone) cut
      
      

• Purification of DNA from Gel
  • using the Zymo Kit

• Ligation of HisSig/TrpSig with R0011in 2 reactions
  
  

* approximated from the amount used in the digestion before

19.04.2010

• PCR of R0011-TrpSig and R0011-HisSig
  
  • Purification with the Zymo Kit, Elution in 30 uL H2O
  • Concentration measured with nanodrop: c(R0011-TrpSig)=20 ng/µL, c(R0011-HisSig)=12.5 ng/µl --> worked
    
    

• Gel for analysis of ligation and PCR
  • 2% Agarose in 1x TAE
  • 110 V, 90 min
  • stained with SybrGold 1:10000 20 min
  • pure R0011 PCR product used as control

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 ?!?

• Ligation of HisSig/TrpSig with R0011in 2 reactions
  
  

* approximated from the amount used in the digestion before

20.04.2010

• Gel for analysis of ligation and PCR (repeat of yesterday's gel)
  • 2% Agarose in 1x TAE
  • 130 V, 75 min
  • stained with SybrGold 1:10000 60 min
  • pure R0011 PCR product used as control
  • Excision and purification of marked bands at 200 bp using QIA Kit, elution in 30 µl H2O
    

• PCR of excised and purified bands of R0011-TrpSig and R0011-HisSig
  
  • complete samples (30 µl) used as templates
  • Purification with the Zymo Kit, Elution in 30 uL H2O
  • Concentrations of PCR-products: 0.5-1 ng/µl --> Gel excision or PCR didn't work

• Transformation (Woehlke-Lab)
  • 8 µl of ligation product pSB1K3-B0014 to 50 µl XL-10 competent cells
  • 200 µl plated on a Kanamycin-containing Plate
  • remaining 800 µl stored @4°C in S1-lab

21.04.2010

• Gel for analysis of ligation and PCR (repeat of yesterday's gel)
  • 2% Agarose in 1x TAE
  • 110 V, 90 min
  • stained with SybrGold 1:10000 80 min
  • pure R0011 PCR product used as control
  • Excision and purification of marked bands at 200 bp using Zymo 5 Kit, elution in 20 µl H2O
    

File:TUM2010 100421beschriftet.gif??

• PCR of excised and purified bands of R0011-TrpSig and R0011-HisSig
  
  • complete samples (20 µl) used as templates
  • Purification with the Zymo Kit, Elution in 25 uL H2O
  • Concentrations of PCR-products:
    • R0011-TrpSig: 22.5 ng/µl
    • R0011-HisSig: 9.5 ng/µl

--> worked!!!!!

• Colony PCR
  • 7 Colonies picked and resuspended in 20 µl LB+Kana (each)
  • PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified)
  • 15 µl of each sample mixed with 3 µl GLPn and loaded to Gel

• Overnight cultures:
  • remaining 18 µl of samples 1, 3, 6, and 7 added to 5 ml LB + kanamycin
  • 37°C on Shaker

22.04.2010

• Gel for purification of PCR products R0011-TrpSig and R0011-HisSig (yesterday's result)
  • 2% Agarose in 1x TAE
  • 110 V, 90 min
  • stained with SybrGold 1:10000 30 min
  • pure R0011 PCR product used as control
  • Excision and purification of marked bands at 200bp using Zymo 5 Kit, elution in 20 µl H2O
    

• Miniprep
  • Result: about 4 µg Plasmid

23.04.2010

• Digestion of:
  
  

Incubated 90 min @ 37°C

• Gel for purification of the cleaved plasmid
  • 2% Agarose in 1x TAE (leftover from yesterday)
  • 140 V, 90 min
  • stained with SybrGold 40 min
  • 4 µl 1 kb ladder, 10 µl purified digestion + 2 µl GLPn, 10 µl purified digestion + 2 µl GLPn
    • Digestion worked (partly). band at 2400 bp cut out
      
      

• Purification of DNA from Gel
  • using the Zymo Kit
  • elution in 25 µl H2O
• A260/A230 and A260/A280 values were strange (see labbook)

26.04.2010

• Digestion of pSB1K3-B0014 with EcorI and XbaI
  • 10 µl template (No1, 50 ng/µl)
  • 5 µl BSA, 5 µl Buffer NEB#4
  • 1 µl EcoRI, 1 µl XbaI
  • 28 µl H2O
  • 1.5 h @ 37°C
  • Purification with Zymo5 Kit, elution in 20 µl H2O

• Ligation of Signals and PSB1K3-B0014
  • 3 µl of each sample, end volume 20 µl

• Preparation of Measurement Plasmid from Folder, Transformation
  • Plate 1022, Spots 1E, 1G, 2A: pSB1A10 with different Inserts, all inserts are Zinc-finger constructs with about 1.6 kb

• Transformation of XL10 with Ligation Products (8 µl each) and pSB1A10 (2 µl each)

• Preparation of Measurement Plasmid from Folder, Transformation
  • Plate 1022, Spots 1E, 1G, 2A: pSB1A10 with different Inserts, all inserts are Zinc-finger constructs with about 1.6 kb

• growing over night cultures of remaining PSB1K3-B0014-transformed cells
  • 2x 5 ml, 2x 1 ml

27.04.2010

• Plenty of cultures on both HisSig and TrpSig Ligation plates, but nothing on pSB1A10 plates! --> repeat DNA extraction, ask Prof. Simmel for new Distribution

• Colony PCR
  • 7 Colonies picked and resuspended in 20 µl LB+Kana (each)
  • PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified)
  • 10 µl of each sample mixed with 2 µl GLPn and loaded to Gel

Many colonies with pSB1K3-B0014, not one with pSB1K3-Sig-B0014

• Miniprep of PSB1K3-B0014
  • Samples I and II: 5 ml overnight cultures, centrifuged 10 min @ 3200 g, resuspended in 600 µl of the same culture
  • Samples III and IV: 600 µl overnight cultures
  • all samples mixed with 100 µl lysis buffer, Miniprep with Zyppy kit, each sample eluted in 50 µl H2O
  • Concentration measured (Nanodrop, LP=1mm, Factor 10, 4 µl sample)
    • cI=61.5 ng/µl
    • cII=33.5 ng/µl
    • cIII=103 ng/µl
    • cIV=108 ng/µl

--> Better results for 600 µl cultures without centrifuging!!!

• Preparation of Measurement Plasmid from Folder, Transformation
  • Plate 1022, Spots 1E, 1F, 1G, 1H, 2A: pSB1A10 with different Inserts, all inserts are Zinc-finger constructs with about 1.6 kb

28.04.2010

• No colonies on plates from Yesterday's transformations, but on the older plates (from monday) some colonies appeared
  • 7 Colonies picked and resuspended in 20 µl LB0 (each)
  • 1&2 from plate "1E", 3&4 from plate "1G", 5,6&7 from plate "2A", 8 LB0

• Colony PCR
  • PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified)
  • 15 µl of each sample mixed with 3 µl GLPn and loaded to Gel
  • 1% Agarose in 1xTAE, 95 V, after 50 minutes changed to 110 V

• Digestion of pSB1K3-B0014 with EcorI and XbaI
  • 10 µl template (No1, 50 ng/µl)
  • 5 µl BSA, 5 µl Buffer NEB#4
  • 1 µl EcoRI, 1 µl XbaI
  • 28 µl H2O
  • 1.5 h @ 37°C
  • heat inactivation 5min @60°C
• Dephosphorylation of restricted vector
  • Purification with Zymo5 Kit, elution in 20 µl H2O
  • loaded on gel (with 4 µl GLPn) (Gel shown above)
• Gel excision with Zymo Kit

29.04.2010

• Digestion of pSB1K3-B0014 with EcorI and XbaI
  • 10 µl template (NoIV, 108 ng/µl)
  • 5 µl BSA, 5 µl Buffer NEB#4
  • 1 µl EcoRI, 1 µl XbaI
  • 28 µl H2O
  • 1.5 h @ 37°C
  • heat inactivation 5min @60°C
• Dephosphorylation of restricted vector
  • Purification with Zymo5 Kit, elution in 20 µl H2O
  • loaded on gel (with 4 µl GLPn)

File:X

• Gel excision with Zymo Kit

• Digestion of R0011 with SpeI
  • 10 µl template (R0011, X ng/µl)
  • 5 µl BSA, 5 µl Buffer NEB#4
  • 1 µl SpeI
  • 29 µl H2O
  • 1.5 h @ 37°C
• Ligation
  • 5 µl R0011 (S-digested) with 12 µl TrpSig or HisSig, respectively (X-digested)
  • complete ligation (20 µl) loaded on Gel (with 4 µl GLPn)

• • Gel excision with Zymo Kit, eluted in 42 µl H2O

• Transformation
  • 50 µl XL-10 transformed with 2 µl of pSB1A10 prepared from IGem 2009 Distribution (13 µl left in pink Box @-20°C)

30.04.2010

PCR R0011-HisSig and R0011-TrpSig --> 13 ng/µl x 20 µl

04.05.2010

• Digestion of pSB1K3-B0014 with EcorI and XbaI
  • 10 µl template (NoIII, 103 ng/µl)
  • 5 µl BSA, 5 µl Buffer NEB#4
  • 1 µl EcoRI, 1 µl XbaI
  • 28 µl H2O
  • 1.5 h @ 37°C
  • Purification with Zymo5 Kit, elution in 15 µl H2O
  • loaded on gel (with 3 µl GLPn)

• Gel excision with Zymo Kit
• Digestion of R0011-HisSig and R0011-TrpSig with EcorI and SpeI
  • 10 µl template (PCR-product)
  • 5 µl BSA, 5 µl Buffer NEB#4
  • 1 µl EcoRI, 1 µl SpeI
  • 28 µl H2O
  • 1.5 h @ 37°C
  • Purification with Zymo5 Kit, elution in 20 µl H2O

• Ligation
  • 4 µl R0011-Signal (E/S-digested) with 4 µl pSB1K3-B0014 (E/X-digested)
  • 15 min @ RT, 20 min heat inactivation @ 65°C

• Transformation
  • 50 µl XL-10 transformed with 10 µl of Ligation mix

• Overrnight liquid cultures of pSB1A10-RFP made from
  • 1 and 2: picked clones from original plates from *Do 29.04.2010
  • 3a: picked clone from copy plate from *Fr 30.04.2010
  • 3b: resuspended clone N° 3 from *Fr 30.04.2010

--> each in 600 µl LB+Carbenicillin (=Ampicillin) @37°C

05.05.2010

• Miniprep of pSB1A10; 4 samples (1, 2; 3a; 3b)
  • eluted in 50 µl H2O each
  • Concentrations:
    • c1=37.5 ng/µl
    • c2=56.5 ng/µl
    • c3a=46.5 ng/µl
    • c3b=30 ng/µl

• Digestion of pSB1A10 with EcorI and PstI, 4 samples (1, 2; 3a; 3b)
  • 15 µl template
  • 5 µl BSA, 5 µl Buffer NEB#3
  • 1 µl EcoRI, 1 µl PstI
  • 23 µl H2O
  • 1.5 h @ 37°C
  • heat inactivation 5min @60°C
  • Purification with Zymo5 Kit, elution in 15 µl H2O
  • loaded on gel (with 3 µl GLPn)

Insert @ 1 kb as expected, but vector @ 2 kb and not @ 5 kb as expected!!!! --> 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!

• Digestion of HisTerm and TrpTerm with EcorI and PstI
  • 5 µl template
  • 5 µl BSA, 5 µl Buffer NEB#3
  • 1 µl EcoRI, 1 µl PstI
  • 33 µl H2O
  • 1.5 h @ 37°C

• Clones picked: 7 from each Plate (pSB1K3-R0011-TrpSig-Boo14 and pSB1K3-R0011-HisSig-Boo14)
• Colony PCR
  • PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR)
  • 15 µl of each sample mixed with 3 µl GLPn and loaded to Gel
  • 2% Agarose in 1xTAE, 130 V, 90 min

06.05.2010

• Digestion of pSB1K3 with EcorI and XbaI
  • 20 µl template (sample III, 103 ng/µl)
  • 5 µl BSA, 5 µl Buffer NEB#3
  • 1 µl EcoRI, 1 µl XbaI
  • 18 µl H2O
  • 1.5 h @ 37°C
  • heat inactivation 5min @60°C
  • loaded on gel (with 10 µl GLPn) in 4 lanes

• Gel excision with Zymo Kit (lanes 1&2) and with Qiaquick Kit (lanes 3&4)
  • c1=4.5 ng/µl
  • c2=3.5 ng/µl
  • c3=2 ng/µl
  • c1=7 ng/µl
• A260/A230 and A260/A280 values were strange (see labbook)

• Ligation
  • 4 µl R0011-Signal (E/S-digested) with 10 µl pSB1K3-B0014 (E/X-digested, from 23.04.)
  • 15 min @ RT, 20 min heat inactivation @ 65°C

• Transformation
  • 50 µl XL-10 transformed with 7 µl of Ligation mix

07.05.2010

• Clones picked: 7 from each Plate (pSB1K3-R0011-TrpSig-Boo14 and pSB1K3-R0011-HisSig-Boo14)

---Too damn stupid to do a PCR!!!---

• replated picked clones on new plates, incubated at RT

10.05.2010

• Colony PCR of picked clones from Fr 07.05.2010
  • PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified)
  • 15 µl of each sample mixed with 3 µl GLPn and loaded to Gel
  • 2% Agarose in 1xTAE, 120 V, 110 min
  • stained in SybrSafe 50 min

Interpretation: 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. Possible explanation: Ligation worked, but not with R0011-Signal-construct but with R0011 or Signal. Which?

Prefix: 20 bp; Suffix: 21 bp; X-S-scar: 6 bp

--> 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.

11.05.2010

• Ligation
  • 4 µl Signal (E/S-digested; from ) with 5 µl pSB1K3-B0014 (E/X-digested; from)
  • 15 min @ RT

• Transformation
  • 50 µl XL-10 transformed with 7 µl of Ligation mix

12.05.2010

• Colony PCR of picked clones from Tu 12.05.2010
  • PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR, modified)
  • 15 µl of each sample mixed with 3 µl GLPn and loaded to Gel
  • 2% Agarose in 1xTAE, 120 V, 110 min
  • stained in SybrSafe 60 min

File:TUM2010 100512beschriftet.png

Prefix: 20 bp/29 bp after PCR; Suffix: 21 bp/30 bp after PCR; X-S-scar: 6 bp

14.05.2010

• Digestion of pSB1K3 with EcorI and XbaI
  • 10 µl template (sample III, 103 ng/µl)
  • 2 µl BSA, 2 µl Buffer NEB#4
  • 1 µl EcoRI, 1 µl XbaI
  • 4 µl H2O
  • 1 h @ 37°C
  • loaded on gel (with 4 µl GLPn) in 1 lane

File:TUM2010 100514beschriftet.png

• Gel excision with Zymo Kit

• Digestion of HisSig and TrpSig with EcorI and SpeI
  • 10 µl template ("1:100")
  • 2 µl BSA, 2 µl Buffer NEB#3
  • 1 µl EcoRI, 1 µl SpeI
  • 4 µl H2O
  • 1.5 h @ 37°C
  • Purification with Zymo 5
  • or heat inactivated (20 min @ 80°C)

• Ligation

• Transformation
  • 50 µl XL-10 transformed with 10 µl of Ligation mix
  • 50 µl untransformed cells plated on Kana-plate as control

17.05.2010

• Plates from Friday:
  • plenty colonies on control plate --> XL10 cells are impure!
  • use DH5a from now on!!!!!

• Transformation
  • 50 µl DH5a transformed with 10 µl of Friday's Ligation mix
  • plated on Kana-Plates; Overnight @ 37°C

• DNA Isolation from BioBrick Distribution 2010
  • 10 µl H2O added to Well 1A of plate 1 containing pSB1A10 with RFP-insert
  • 2 µl used for Transformation of 50 µl DH5a-cells
  • plated on Carbenicillin (=Amp-analogon)-plates, Overnight @ 37°C

• Polyacrylamide Gel prepared for tomorrow
  • 1 big denaturing Gel with 20 pockets

18.05.2010

• Colony PCR of picked clones
  • PCR of 2 µl of each sample, 2 µl as negative control (Program: ColonyPCR)
  • 10 µl of each sample mixed with 10 µl Formamide loading buffer and loaded to Polyacrylamide Gel

• Polyacrylamide Gel

Samples: 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

• 5 µl of each samples mixed with 5 µl formamide loading dye and loaded to gel(except Ladder and colonyPCR)
  • LMW: 3 µl LMW (Korbinian) + 3 µl Formamide loading Dye
  • LMW2: 5 µl LMW Quickload (with GLP) + 10 µl Formamide loading Dye
• stained in SybrSafe

Important Mistake! See below Gel!
IMPORTANT MISTAKE: DENATURING GELS NOT USEFUL FOR dsDNA!!! REPEAT WITH NATIVE GEL, IGNORE INTERPRETATION!!!

(*R0011 and B0014 look normal

• ColonyPCR: bands that look like B0014 in all clones (and in control) --> Religation?
• Signals at the wrong size: should be about 75 bp, look like 200 bp!!!
• terminators completely strange: should be around 100 bp!

--> are all of our sequences just wrong??? What are we going to do? Order everything new?)

19.05.2010

• Gel from Korbinian
• 5 µl of each samples mixed with 5 µl formamide loading dye and loaded to gel(except Ladder and colonyPCR)
  • LMW: 3 µl LMW (Korbinian) + 3 µl Formamide loading Dye
  • colony PCR: from Tuesday, 8 µl sample with 8 µl Formamide loading Dye
• stained in SybrSafe 20 min

• Colony PCR
  • 4 colonies picked from each Plate (Ligations from yesterday; Signal-B0014)
  • 15 µl of each Sample mixed with 3 µl GLPn and loaded to Gel:
  • 3% Agarose in 1x TBE, 130 V

File:TUM2010 100519beschriftet.png600px

20.05.2010

• Digestion
  • template
  • 2 µl BSA
  • 2 µl Buffer
  • 1 µl of each enzyme
  • water to reach 20 µl

• • incubated @37°C for 1.5 h
  • digested inserts heat inactivated (20 min @ 80°C)
  • digested plasmids loaded on gel (with 4 µl GLPn) in 1 lane

• Gel excision with Zymo Kit
  • c(pSB1A10, I)=4.5 ng/µl
  • c(pSB1A10, II)=1.5 ng/µl ?!?!?!
  • c(pSB1K3 E/P)=7 ng/µl
  • c(pSB1K3_B0014 E/X)=2.5 ng/µl

• Gel of PCR products
  • 3% Agarose in 1x TBE; 2h @130 V

• Ligation
  • templates
  • 2 µl T4-buffer 10x
  • 1 µl T4-Ligase
  • Water to reach 20 µl

• Transformation
  • 50 µl DH5a transformed with 10 µl of Ligation mix
  • 50 µl DH5a transformed with 2 µl of pSB1K3_B0014
  • 50 µl DH5a transformed with 2 µl of pSB1K3_RFP
  • 50 µl DH5a transformed with 2 µl of pSB1A10_RFP

21.05.2010

• Colony PCR
  • 4 colonies picked from each Plate (pSB1K3_HisSig_B0014, pSB1K3_TrpSig_B0014, pSB1K3_HisSig_B0014 double ligation, pSB1K3_TrpSig_B0014 double ligation)
  • each clone resuspended in 20 µl LB0, 3 µl used as template for PCR
  • 15 µl of each Sample mixed with 3 µl GLPn and loaded to Gel:
  • 3% Agarose in 1x TBE, 130 V

25.05.2010

• Colony PCR
  • 4 colonies picked from each Plate
    • pSB1K3_HisSig_B0014
    • pSB1K3_TrpSig_B0014
    • pSB1K3_HisSig_B0014 double ligation
    • pSB1K3_TrpSig_B0014 double ligation
    • pSB1A10_TrpTerm
    • pSB1A10_HisTerm
  • each clone resuspended in 20 µl LB0, 2 µl used as template for PCR
  • 15 µl of each Sample mixed with 3 µl GLPn and loaded to Gel:
  • 3% Agarose in 1x TBE, 220 V (double Gel, 35 cm)
  • stained in SybrSafe

600px

• overnight cultures made of
  • HisSig 3, DL1, DL4
  • TrpSig DL2, DL4
  • HisTerm/TrpTerm 1,2,3

26.05.2010

• Miniprep of cultures set up 25.05.2010
  • HisSig 3, DL1, DL4
  • TrpSig DL2, DL4
  • HisTerm/TrpTerm 1,2,3

• Restriction
  • analytical: E/P HisSig(3, DL2); TrpSig(DL2, DL4): 1.5 h 37 °C
  • prep: E/X HisSig(3, DL2); TrpSig(DL2, DL4): 1.5 h 37 °C
  • prep: E/S R0011: 1.5 h 37 °C, inactivation 20 min @ 80 °C
    • total volume each 20 µl, 10 µL template
• Gel: 1% Agarose, TAE - 1,5 h 110 V
  • bands excised: all E/X cleaved vectors

• Ligation:
  • HisSig_3 (E/X) with R0011 (E/S)
  • HisSig_DL2 (E/X) with R0011 (E/S)
  • TrpSig_DL2 (E/X) with R0011 (E/S)
  • TrpSig_DL4 (E/X) with R0011 (E/S)
    • batches
    • total volume 20 µL
    • 2 µL R0011 (E/S)
    • 2 µL T4 buffer
    • 1 µL T4 Ligase
    • 15 µL vector

• Transformation of ligations
  • DH5a

• PCR
  • His and TrpSig
  • His and TrpTerm
  • R0011
  • B0014
    • 50 µL total volume
      
    • 1 µL template
      
    • 1 µl G1004
      
    • 1 µl G1005
      
    • 0.2 µL Taq
      
    • 5 µl Taq standard buffer
      
    • rest water

27.05.2010

• Colony PCR
  • 3 colonies picked from each Plate
    • pSB1K3_R0011_HisSig_B0014 (N° 3 from yesterday)
    • pSB1K3_R0011_HisSig_B0014 (N° DL1 from yesterday)
    • pSB1K3_R0011_TrpSig_B0014 (N° DL2 from yesterday)
    • pSB1K3_R0011_TrpSig_B0014 (N° DL4 from yesterday)
    • "N6"
    • "N15"
  • each clone resuspended in 20 µl LB0, 2 µl used as template for PCR
  • 15 µl of each Sample mixed with 3 µl GLPn and loaded to Gel:
  • 3% Agarose in 1x TBE, 220 V (double Gel, 35 cm)
  • stained in SybrSafe

Prefix: 29 bp after PCR; Suffix: 30 bp after PCR; X-S-scar: 6 bp

• overnight cultures made of
  • HisSig 3_1, DL1_3
  • TrpSig DL4_1, DL4_3
  • N15 1&2
  • HisTerm/TrpTerm (picked colonies from yesterdays plates #2 each)

• Purification of yesterday's PCR
  • elution in 50 µl H2O
    • c(HisSig)=5.5 ng/µl
    • c(TrpSig)=10 ng/µl
    • c(HisTerm)=6.5 ng/µl
    • c(TrpTerm)=6.5 ng/µl
    • c(R0011)=13 ng/µl
    • c(B0014)=12.5 ng/µl
  • 5 µl loaded on gel with 1 µl GLPn; 3% Agarose in 1x TBE, 220 V (double Gel, 35 cm)

28.05.2010

• Miniprep of cultures from 27.05.2010, Elution in 50 uL nuclease free water
  • (1)HisSig 3_1
  • (2)HisSig DL1_3
  • (3)TrpSig DL4_1
  • (4)TrpSig DL4_3
  • (7)HisTerm#1
  • (8)HisTerm#2 (7 ml culture)
  • (9)TrpTerm#1
  • (10)TrpTerm#2 (7ml culture)
  • (5)N15-1 (=BBa_I719005)
  • (6)N15-2 (=BBa_I719005)
    • ()=numbers on gel

• analytical digest (E/P)
  
  • of all samples. total volume 20 uL, 5 uL template used for Term-constructs, 10 uL teplate for all others

• Agarose Gel
  • 3% broad range agarose in TBE. Run in TBE, 140 V, 1.50 h
  • stained with SybrGold, 45 min
  • signals look fine
  • terminators also (without pre/suffix 97/104 bp)
  • T7 promoter without pre/suffix has a length of 23 bp + cut pre/szffix ca at 60 bp -->buffer?

31.05.2010

• Digestions
  • 15N-1 (BBa_I719005, 19.5 ng/uL) with S/P
  • HisSig/TrpSig (6.5/10 ng/uL) with X/P

2 h 37 °C

heat inactivation of insert-digestions

• Gel: 1% Agarose in 1x TAE

1 h 25 min, 115 V

stained with SybrGold, 40 min

Band at ~2100b cut und purified using the zymo kit

• ligation
  • HisSig (4 µL of digest) with purified plasmid (with BBa_I719005)
  • TrpSig (2 µL of digest) with purified plasmid -=-

reason: concentration of His Sig before digest was 1/2 of TrpSig

• transformation

of DH5a with Ligation batches, HisSig1-3, HisSig3-1, TrpSig DL4-1, TrpSig 4-3

01.06.2010

• Colony PCR

of Ligations transformed into DH5a yesterday

4 Colonies of each Ligation

• • T7-HisSig
  • T7-TrpSig

• Gel: 3% broad range Agarose in 1xTBE

1.5 h 140 V

stained with SybrGold

calculation for the expected size of the fragments

part	size (bp)
HisSig	32
TrpSig	34
T7 promoter	23
prefix	20
suffix	21
X/S scar	6

in PCR we get additional bp due to the primers - +9 at pre/suffix=+18 bp

overall size of the fragments expected to come out of the PCR: T7_HisSig: 120 bp, T7_TrpSig: 122 bp

• ON cultures

5 ml cultures of pSB1K3_R0011_HisSig_B0014 (1_3 & 3_1) and pSB1K3_R0011_TrpSig_B0014 (DL4_1 & DL4_3)

1 ml cultures of each colony monitored in Colony PCR

02.06.2010

• Miniprep of yesterdays cultures using Zymokit, elution by nuclease-free water
• Concentration determination
• analytic digestion
• results on gel:

• Sequencing
  

JobNr. Barcode Last change Date/Time Last message / Files 6549287 AE2739 02.06.2010 / 13:51:12 HisSig 1-3-forward G1004

 	  	We just received your order. Many thanks.

6549288 AE2738 02.06.2010 / 13:51:12 HisSig 3-1-forward G1004

 	  	We just received your order. Many thanks.

6549289 AE2737 02.06.2010 / 13:51:12 TrpSig DL4-1-forward G1004

 	  	We just received your order. Many thanks.

6549290 AE2736 02.06.2010 / 13:51:12 TrpSig DL4-3-forward G1004

 	  	We just received your order. Many thanks.

6549291 AE2735 02.06.2010 / 13:51:12 HisTerm-forward G1004

 	  	We just received your order. Many thanks.

6549292 AE2734 02.06.2010 / 13:51:12 TrpTerm-forward G1004

 	  	We just received your order. Many thanks.

• Gel 3% broad range agarose in 1x TBE

07.06.2010

• Sequenbcing results from GATC
  • HisSig DL1-3 is ok
  • HisSig 3-1 is ok
  • TrpSig DL4-1 is ok
  • TrpSig DL4-3 is ok
  • TrpTerm + HisTerm bad runs... --> new sequencing order with Primer 100 bp upstream (within GFP)

Files can be found stored in our GATC account

• Sequencing@GATC: both Term-constructs with primer pGFP-FP provided by GATC

• Restrictions
  • psB1A10_TrpTerm/HisTerm with Nsi1, Aat2
  • pSB1K3_R0011_HisSig/TrpSig_B0014 with Pst1, Aat2
  • T7 bb with Spe1, Pst1
  • PCRProducts: HisSig/TrpSig with Pst1, Xba1, 2 h @37°C

all plasmid digests done sequential as enzymes do not have 100% activity in the same buffer, each reaction 1.5 h@37°C

psB1A10_TrpTerm/HisTerm and T7 bb dephosphorylated the last 30 min

• Ligation
  
• Transformation

liquid culture (10 ml) of pSB1K3_R0011_HisSig/TrpSig_B0014

08.06.2010

• Sequencing results from GATC: His/TrpTerm with pGFP-FP primer
  • HisTerm worked
  • TrpTerm worked

checked with blast2seq

• Colony PCR

8 colonies from each plate of T7His, T7Trp, MonsterHis, MonsterTrp as ligations resulted in many colonies

• 3% Agarose Gel in 1xTBE

for all colony PCR reactions

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

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

• over night cultures

09.06.2010

• Miniprep of 4 Monster_His, 4 Monster_Trp, 3 T7_His and 3 T7_Trp cultures
• Analytical digestion of plasmids mentioned above
• gel of Monster_Plasmid digestion

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?

• gel of T7_Plasmid digestion

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.
occured trouble:

• • 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

10.06.2010

• ordered

Promega E.coli S30 in vitro transcription/translation kit

Spe1, Aat2 from NEB, 500 U each

• analytical Digestion

of Ligation colonies from MonsterHis/trp 1-3 and T7His/Trp 1,2,5/1,2,3

2h digestion

Monster: 6 µL DNA template with Aat2/Spe1 in Buffer 4/Bsa

T7-Signal: 6 µL DNA template with E/P in Buffer 3

• Agarose Gels

used standards: lmw, 2-log click here

Gel1: 1% Agarose in 1xTBE for Digestions of Monsterplasmid

run in big chamber @ 200 V for 1 h 20 min

Gel2: 3% Agarose (broad range) in 1xTBE for Digestions of T7-Signal

run in small chamber @140 V for 1 h 35 min

Conclusions:

1. all T7-Signal ligations loaded on the gel worked
2. 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...

11.06.2010

• 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

fragments expected are 5087 and 176. original size of plasmid is 5263. This is a Try to differ between 5087 and 5263 bp

Band @ 5000 bp of Trp_Term purified, obviously digestion was 100%. Bad point is that HisTerm includes an Nsi1 cleavage site...

14.06.2010

• Ligation

10 µL pSB1A10_TrpTerm Aat2/Nsi1 0.5 ng/uL

1 µL R0011_TrpSig_B0014 Aat2/Psb1 11 ng/uL

2 µL T4 ligase buffer

1 µL T4 Ligase

6 µL H2O

10 min RT

• Transformation of DH5a with

Ligation

T7His#1

T7Trp#1

• over night cultures of

pSB1K3_R0011_TrpSig_B0014

pSB1K3_R0011_HisSig_B0014

pSB1A10_TrpTerm

pSB1A10_HisTerm

15.06.2010

• Over night cultures
• Aliquots of the Promega in vitro expressions kit from E. coli S30 extract:

40 µL with aa mix including all aa.

16.06.2010

Fluoresence measurements using in vitro kit

• in vitro kit sample
• adding psBA1A10 Trp_Term --> constant over time, no significant changes compared to kit alone --> high efficiency of AraC
• 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)
• adding psB1K3 R0011_TrpSig_B0014

Cell culture 5 ml culture for

• psBA1A10 Trp_Term/HisTerm
• psB1K3 R0011_TrpSig/Hissig_B0014

17.06.2010

Cloning
Digestion of Trp-Sig with E/P and psB1A10 Trp_Term with E/P

• Gel purification of psB1A10 Trp_Term E/P cut

• Heat inactivation of Trp-Sig E/P cut
• Ligation for 10 min @ RT and Transformation in DH5-a cells

18.06.2010

cloning

• Transformation (about 20 colonies) --> picking 5 colonies
• colony PCR
• Gel
  

2 % broad range agarose, 1 h 120 V Sample 2, 4, 5 shows probably Trp-Signal + Pre/Suffix --> send sample 2 for sequencing!

• control digestion of all 10 picked psB1A10-TrpSig in 1% broad range agarose, > 3 h, 120 V

--> digestions worked, but again, no insert can be found, despite gel was at maximum resolution ( 3h 120 V, see LMW)

in vitro measurements 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!
Next steps:

• Try in vivo measurements, just using psb1A10_xTerm without Signal (thus just measuring plasmid) to proof if kit or measuring plasmid causes this problem!

22.06.2010

Transformation

• psb1A10_HisTerm/TrpTerm into BL21 (DE3) RIL
• psb1A10_Monster-Trp No. 7, 8, 10 (Positiv control) into DH5-a

Liquid culture

• psb1A10_TrpSig (Positiv control) No. 2

23.06.2010

• Miniprep

pSBN1A10_TrpSig - 40 µL, 12.5 ng/µL -->very low amount of DNA...

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)

• cultures
  • 5 ml cultures of DH5a

with MonsterTrp, #7,8,10 (Carbamp)

• 50 ml cultures of BL21 (DE3) RIL

pSB1A10_HisTerm

pSB1A10_TrpTerm

• Arabinose Stock

0.2%

24.06.2010

• Miniprep

MonsterTrp #7,8,10

positive Control

concentrations are too low for sequencing --> again we have to set up 5 ml cultures for tomorrow

• measurements

25.06.2010

Plasmid purification and sequencing

• Monster_Trp 7, 8, 10 and psB1A1ß_TrpSig plasmids are isolated (concentrations up to 110 ng/ul) and sent for sequencing
• psB1A1ß_TrpSig liquid culture was completely pink! still not clear what happend (wrong labeling of digested psB1A10_Trpterm?) --> wait for sequencing details

Fluorescence Measurements

• 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?
• 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!!!

28.06.2010

• Sequencing results

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:
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".

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

• 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)
  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.
  as a result, in vitro expression did somehow not work, reasons are unclear, maybe too low DNA-concentrations.
  positvie control psB1A10_TrpSig was pink again, we have to wait the results from GATC

29.06.2010

01.07.2010

• in vitro measurement

using Invitrogen kit

measuring kinetics for 3 h @ 37°C

40 µL + 5 µL pSB1A10_TrpSig (126 ng/µL) + 0.5 µL 100x L-Arabinose (=0.2%) + 4.5 µL H2O

observations: GFP signal grows, after 30 min it crashes. RFP grows

emission spectra for GFP and RFP result in no spectrum

looks strange, a problem might be evaporation of liquid and hence scattering of light which produces artefacts

• over night cultures

pSB1A10_TrpSig (DH5a), 5 ml for miniprep

pSB1A10 XS (DH5a), 5 ml for miniprep

02.07.2010

• Miniprep

pSB1A10_XS: 30 µL 10 ng/µL

pSB1A10_TrpSig: 30 µL 10 ng/µL

• Transformation

BL21 with pSB1A10_XS (positive control without insert and no without any bio brick site left)

05.07.2010

• over night cultures

250 ml of DH5a pSB1A10_XS

20 ml BL21 pSB1A10_XS

• in vitro transcription measurement planned

check In_vitro_Measurements

06.07.2010

• In vivo measurement

BL21 pSB1A10_XS - positive control (to check the measurement plasmid...)

GFP, RFP Fluorescence

induced with 0.2% arabinose in (1), uninduced (2)

at OD 0.15: GFP/RFP emissions spectra /100706/spectra/gfp10 and rfp10

2.5 h kinetic measurement GFP/RFP /100706/kinetics/

OD 0.7 (1) and 0.64 (2) after 2.5 h --> GFP/RFP emissions spectra /100706/spectra/gfp11,rfp11,gfp21,rfp21

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

observation: measurement plasmid is totale verarsche. RFP is not expressed at all, or this protein is not rfp. whatever.

No Lab work this week, everybody is busy studying for their exams...

No Lab work this week, everybody is busy studying for their exams...

No Lab work this week, everybody is busy studying for their exams...

No Lab work this week, everybody is busy studying for their exams...

09.08.2010

• overnight cultures inoculated from Glycerolstock J06702(mCherry generator) in pSB1A2 from Christoph.

10.08.2010

• MiniPrep of pSB1A2-mCherry using ZymoKit
• Digestion
  • pSB1A2-mCherry E/P
  • pSB1A2-mCherry X/P
  • pSB1A10-HisTerm S/P
  • pSB1A10-TrpTerm S/P
  • pSB1A10-HisTerm E/P
• Purified with agarose gel (1%)
  • Gel Doc broken => no picture
  • Description: mCherry cut was ok, Plasmid was cut at least once (linear DNA), generally contaminated with genomic DNA
• Ligation
  • 50 ng Plasmid and 34 ng Insert
  • ca. 30min @ RT
• Transformation of DH5a cells with ligation samples

(=> no colonies the next day)

• overnight cultures
  • pSB1A2-mCherry from Christoph`s stock
  • pSB1A10-HisTerm from earlier plate
  • pSB1A10-TrpTerm from earlier plate

(=> pSB1A10-TrpTerm and pSB1A10-HisTerm did not grow until next day)

11.08.2010

• MiniPrep of pSB1A2-mCherry using ZymoKit
• analytic gel of Mini preps and ligation of the previous day
  • preps still hold genomic DNA
  • mCherry Plasmid runs at ca. 2400 bp

• Digestion
  • pSB1A2-mCherry E/P
  • pSB1A2-mCherry X/P
• Purified with agarose gel (1%)
  • Gel Doc broken => no picture
  • Description: mCherry cut was ok, stil contaminated with genomic DNA
• Ligation
  • Plasmid (from previous day) and mCherry-Insert
  • ca. 30 min @ RT
• Transformation of DH5a cells with
  • ligation samples (=> no colonies the next day)
  • pSB1A10-HisTerm
  • pSB1A10-TrpTerm

• overnight cultures
  • pSB1A2-mCherry from Christoph`s stock

12.08.2010

• MiniPrep of pSB1A2-mCherry using ZymoKit
• analytic gel of Mini preps and ligation of the previous day
  • preps still hold genomic DNA
  • mCherry Plasmid runs at ca. 2400 bp
  • Gel (1%)

• Digestion
  • pSB1A2-mCherry X/P
  • pSB1A10-HisTerm S/P
  • pSB1A10-TrpTerm S/P
• Purified with agarose gel (1%)

• Ligation
  • Plasmid and mCherry-Insert
  • ca. 30 min @ RT
• Transformation of DH5a cells with
  • ligation samples (=> no colonies the next day)
  • pSB1A10-HisTerm
  • pSB1A10-TrpTerm

• overnight cultures
  • pSB1A2-mCherry from Christoph`s stock
  • pSB1A10-HisTerm
  • pSB1A10-TrpTerm

Caution: ran out of gas => not steril?

13.08.2010

• MiniPrep using ZymoKit
  • pSB1A2-mCherry
  • pSB1A10-HisTerm
  • pSB1A10-TrpTerm
• analytic gel of Mini preps and ligation of the previous day
  • low concentration
  • Gel (1%)

16.08.2010

• Concentrating MiniPrep-Samples using ZymoKit
  • pSB1A2-mCherry
  • pSB1A10-HisTerm
  • pSB1A10-TrpTerm

• Digestion
  • pSB1A2-mCherry E/P
  • pSB1A2-mCherry X/P
• Purified with agarose gel (1%)

=> no mCherry band!!!

• Purification using Zymo Concentrator Kit
  • pSB1A10-HisTerm S/P
  • pSB1A10-TrpTerm S/P

• Ligation
  • Plasmid (from earlier date) and mCherry-Insert (from 11.08)
  • ca. 30 min @ RT
  • used new ligase and new ligase buffer

• Transformation of DH5a cells with
  • ligation samples (=> colonies found the next day)
  • pSB1A10-HisTerm
  • pSB1A10-TrpTerm
  • pSB1A10-RFP (BioBrick Standard)

• overnight cultures
  • pSB1A2-mCherry from Christoph`s stock
  • pSB1A10-HisTerm
  • pSB1A10-TrpTerm

17.08.2010

• MiniPrep using ZymoKit
  • pSB1A2-mCherry
  • pSB1A10-HisTerm
  • pSB1A10-TrpTerm

• Digestion
  • pSB1A2-mCherry X/P
  • pSB1A10-HisTerm S/P
  • pSB1A10-TrpTerm S/P

=> heating block went up to 50°C

• Purified "digestion" samples with ZymoKit => stored for next day

• Picked 12 colonies from previous day's ligation

=> Colony PCR => Gel (2%)

• Purified with agarose gel (1%)
  • Gel Doc broken => no picture
  • Description: mCherry cut was ok, stil contaminated with genomic DNA
• Ligation
  • Plasmid (from previous day) and mCherry-Insert
  • ca. 30 min @ RT
• Transformation of DH5a cells with
  • ligation samples (=> no colonies the next day)
  • pSB1A10-HisTerm
  • pSB1A10-TrpTerm

• overnight cultures
  • pSB1A10-RFP (plate from previous day)
  • pSB1A2-mCherry
  • pSB1A10-HisTerm
  • pSB1A10-TrpTerm

PROBLEM: mCherry has a SgrA1-cleavage site! These constructs cannot be used. Starting all over, cloning the linker sequence first...

18.08.2010

• MiniPrep using ZymoKit
  • pSB1A2-mCherry
  • pSB1A10-HisTerm
  • pSB1A10-TrpTerm
  • pSB1A10-RFP

• Analytical agarose gel (1%):

• Digestion
  • pSB1A10-HisTerm SgrAI/PstI
  • pSB1A10-TrpTerm SgrAI/PstI
  • pSB1A10-RFP SgrAI/PstI

=>RFP has a SrgAI cleaving site. Discarded RFP digestion.

• preparativ agarose gel (1%):

• Soubilization of SrgAI-PstI Linker

• Ligation
  • Plasmid His-Term(Trp-Term) and Linker
  • ca. 30 min @ RT
• Transformation of DH5a cells with
  • ligation samples

• overnight cultures
  • pSB1A2-mCherry
  • pSB1A10-TrpTerm

19.08.2010

• MiniPrep using ZymoKit
  • pSB1A2-mCherry
• analytic agarose gel (1%) from various mCherry Preps

• Picked 6 colonies from pSB1A10-HisTerm-linker and pSB1A10-TrpTerm-linker each (previous day's ligation)

=> Colony PCR => Gel (1.5%)

• overnight cultures (600µl)
  • pSB1A2-mCherry
  • pSB1A2-R0011
  • pSB1A10-HisTerm-linker (#7, 11, 12)
  • pSB1A10-TrpTerm-linker (#1, 2, 4)

20.08.2010

• MiniPrep using ZymoKit
  • pSB1A2-mCherry
  • pSB1A2-R0011
  • pSB1A10-TrpLinker (picked Colonies)
  • pSB1A10-HisLinker (picked Colonies)

• analytical Digestion
  • pSB1A10-HisLinker SgrAI /EcoRI
  • pSB1A10-HisLinker NsiI
  • pSB1A10-TrpLinker SgrAI /EcoRI

• analytical agarose gel (1.5%)

• preparativ Digestions
  • pSB1A2-mCherry EcoRI /PstI
  • pSB1A2-mCherry XbaI /PstI
  • pSB1A2-R0011 SpeI /PstI
  • pSB1A10-HisLinker SpeI /PstI
  • pSB1A10-HisLinker EcoRI /PstI
  • pSB1A10-TrpLinker SpeI /PstI
  • pSB1A10-TrpLinker EcoRI /PstI
  • PCR_BB1006 XbaI /PstI

• agarose gel (1.5%):

• agarose gel (1.0%):

• agarose gel (1.0%):

23.08.2010

• Ligation
  • pSB1A10-HisLinker SpeI /PstI + mCherry XbaI /PstI
  • pSB1A10-HisLinker EcoRI /PstI + mCherry EcoRI /PstI
  • pSB1A10-TrpLinker SpeI /PstI + mCherry XbaI /PstI
  • pSB1A10-TrpLinker EcoRI /PstI + mCherry EcoRI /PstI
  • pSB1A2-R0011 SpeI /PstI + PCR_BB1006 XbaI /PstI

• Transformation of DH5a cells

24.08.2010

• Picked 2 colonies per plate (previous day's ligation)
  • R0011_B1006Sig
  • Trp1_mCherry
  • Trp2_mCherry
  • His7_mCherry
  • His11_mCherry
  • pSB1A10_mCherry

• Colony PCR of picked colonies with prefix/suffix primers

• analytical agarose gel 1 (1.5%)

• analytical agarose gel 1 (1.5%)

Trp=R0011, R0011= Trp :)

Faint bands at the correct length can be guessed.

• overnight cultures (5 ml)
  
  • pSB1A10_TrpTerm_mCherry_linker
  • pSB1A10_HisTerm_mCherry_linker
  • pSB1A10_mCherry_linker

25.08.2010

• Miniprep using Zymo Miniprep-Classic Kit:
  • pSB1A10_TrpTerm_mCherry_linker
  • pSB1A10_HisTerm_mCherry_linker
  • pSB1A10_mCherry_linker

• analytical digestions
  • pSB1A10_TrpTerm_mCherry_linker EcoRI /PstI
  • pSB1A10_HisTerm_mCherry_linker EcoRI /PstI
  • pSB1A10_mCherry_linker EcoRI /PstI

• analytical agarose gel 1 (1.0%)

• Picked 2 colonies per plate (day before yesterday's ligation)
  • R0011_B1006Sig
  • Trp1_mCherry
  • Trp2_mCherry
  • His7_mCherry
  • His11_mCherry
  • pSB1A10_mCherry

• Colony PCR of picked colonies with prefix/suffix primers
  • Program: colonypcr

• analytical agarose gel 1 (1.5%)

• analytical agarose gel 1 (1.5%)

• overnight cultures (5 ml)
  
  • pSB1A10_TrpTerm_mCherry_linker
  • pSB1A10_HisTerm_mCherry_linker
  • pSB1A10_mCherry_linker

26.08.2010

• Miniprep using Zymo Miniprep-Classic Kit:
  • pSB1A10_TrpTerm_mCherry_linker
  • pSB1A10_HisTerm_mCherry_linker
  • pSB1A10_mCherry_linker
  • pSB1A2_R0011_B1006

• PCR
  • Trp-Signal (R0011_Sig_B0014)