Team:Gothenburg-Sweden/Lab Note

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                        <div class="h1">Preliminary Study</div>
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        <div class="logo"> <img src="http://2010.igem.org/wiki/images/f/fe/Gfp.png" width="40" height="40" align="left"></div> <strong><p>Fluorescent  Proteins:</strong><br>  Green Fluorescent Protein <a href="http://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#FP">(read more)</a></p><br>  
        <div class="logo"> <img src="http://2010.igem.org/wiki/images/f/fe/Gfp.png" width="40" height="40" align="left"></div> <strong><p>Fluorescent  Proteins:</strong><br>  Green Fluorescent Protein <a href="http://2010.igem.org/Team:Gothenburg-Sweden/Lab_Note/pre#FP">(read more)</a></p><br>  

Latest revision as of 12:03, 3 October 2010

Chalmers University of Technology

Team
FUSS

Fluorescent Proteins:
Green Fluorescent Protein (read more)


Plasmid backbone:
pSP-GM1 (read more)


SNF1 (modified subunits):
Snf1 (alfa), Snf4 (gamma) (read more)


FP positions:
N-terminal alfa with N-terminal gamma, N-terminal gamma with C-terminal gamma (read more)


Primer design:
Fusion primers with restiriction enzyme sites included at very ends, each annealing part was desinged with an melting temperature of around 60 C (read more)


Lab work by date
  • 2010-08-11
    Malin and Katarina

    The PCR reaction from yesterday was checked on a gel. α should be around 1500 bp but only a band around 750 bp was seen. We had a long meeting with supervisor Per about PCR reactions and the next couple of weeks in the lab.

    Karl and Kemal

    Ran PCR reaction α with different settings.
    α1: regular settings
    α2: 10 x diluted template 2
    α3: increased annealing time from 30 s to 60 s
    α4: decreased annealing temperature

    After PCR, the products were run on a gel. α1, α3 and α 4 had a band around 750 bp but nothing on α2.



  • 2010-08-10
    Peidi and Lokesh

    Checked the transformation plates but nothing had grown. Prepared PCR reaction Γ.

    Katarina and Kemal

    PCR product Γ was verified on a 0.7 % agarose gel. There was a band around 750 bp as expected and it was purified using the regular protocol. A new phusion mastermix was made and PCR reaction α was run with Γ as one of the template. Regular phusion PCR settings was used.

  • 2010-08-09
    Malin and Kemal

    Before checking if the ligation worked we need to transform and amplify in E.coli. Then we can miniprep the ligated plasmid and check on a gel. After that we can digest plasmid with PacI and BcuI and ligate the digested IV. Then another round of transformation and miniprep before we can transform yeast.

    Karl and Lokesh

    Transformed competent E.coli with plasmid ligated with III by heat shock. The cells were incubated at 37 °C over night.

  • 2010-08-07
    Lokesh

    Moved ligation tube to freezer.

  • 2010-08-06
    Malin, Julia and Lokesh

    III and IV were digested 3x protocol and were then put on a 1 % agarose gel together with Γ both for verification and purification. Correct bands were seen for purification except for Γ which was empty. In the verification lanes the digested PCR products were to light to be seen but they were seen in purification lanes. IIId and IVd were purified.

    IIId: 7.8 ng/µl
    IVd: 11.7ng/µl

    IIId will now be ligated with the cut plasmid (BamHI and HindIII). Ligation performed according to protocol with 156 ng insert and 52 ng plasmid. Ligation was incubated over night in fridge at 4-16° C.

    All 4 sporulation plates were checked but still no tetrads visible.

  • 2010-08-05
    Malin and Julia

    III and IV were digested using the regular protocol.
    III: HindIII and BamHI works with Buffer B
    IV: PacI and BcuI works with Buffer M

    A 1% gel was run with uncut and digested III and IV, control, I and Β. I worked well and will be purified. The Β lane showed no bands. None of the digested products were visible on the gel so a new gel was run trying to verify this result. The control shows a band around 1700 which is weird. This band is also visible on the III uncut but together with a band around 750 bp. No bands at the IV which is very strange since this PCR product has been verified before. Have something gone wrong with the purification?

    As a result PCR reactions III and IV will be redone. New PCR reactions of Γ and α were also performed in the same block.

    Karl and Kemal

    Reactions of Β were made. Β1 is a continuation of the 100 cycle PCR this time with primers. Β2 uses a megaprimer approach.

    All the PCR products were run on a 1 % agarose gel. III, IV and Γ were alright. α and Β2 showed a band around 750 bp.
    III: 531.3 ng/µl
    IV: 263.9 ng/µl

  • 2010-08-04
    Julia and Karl

    PCR products Β were loaded on a 0.7 % gel. No correct bands, only amplified templates around 750 bp and something else around 500 bp.

    PCR of SAMS will also be redone from scratch. PCR reactions 1, 2 and C are done, then loaded on a gel (2 % gel and low range ladder) and purified.
    1: 8.2 ng/µl
    2: 7.8 ng/µl
    C: 4.9 ng/µl
    Samples diluted to 1 ng/µl.

    PCR of Β was done again but this time in two steps and with the Finnzymes Tm which corresponds to an annealing temp at 85 °C. Firstly 10 cycles without primers were performed.

    Kemal and Lokesh

    Second round of PCR with primers were performed. Then new PCR reactions amplifying III and IV were done and also reaction I.
    III: 572 ng/µl
    IV: 660 ng/µl

    The sporplates were checked and very few tetrads were seen.

    Β samples were put on a gel but no correct bands visible. We suspect that the reverse primer is attaching in the wrong place since YFP and CFP is so similar which then would lead to 750 bp fragment.

  • 2010-08-03
    Malin and Lokesh

    PCR products II, III and IV were verified and purified from a 1 % agarose gel. III did not work and was not purified.

    Concentrations:
    II: 6.2 ng/µl
    IV: 5.6 ng/µl
    II and IV were diluted to 1 ng/µl.

    The new sporplates were examined but no tetrads visible. To check further the cells were stained with DAPI and examined in fluorescence microscope. Still no success.

    New PCR reactions were prepared. III was redone and Β was also done. Same settings as yesterday.

    Kemal and Karl

    PCR products were loaded on a 1% agarose gel for verification and purification. III worked this time and was purified from the gel using the protocol from QIAquick Gel Extraction Kit.
    III: 6.4 ng/µl

    New PCR of Β was run with two different block settings.
    Block A: increased annealing time to 1 min.
    Block B: increased annealing time to 58 °C.

  • 2010-08-02
    Kemal, Lokesh and Karl

    PCR products A, B and 3 were loaded on a gel and then purified from the gel using the protocol from QIAquick Gel Extraction Kit. α and IV were also run on a gel but only showed strong bands corresponding to amplified templates. On the α lane however a very light band was seen at the correct length. This will be further investigated.

    Malin and Julia

    The purified PCR products were measured with nanodrop:
    A1: 11.8 ng/µl
    A2: 10.2 ng/µl
    B1: 9.6 ng/µl
    B2: 17.1 ng/µl
    31: 15.0 ng/µl
    32: 16.3 ng/µl

    A, B and 3 were diluted to 1 ng/µl.

    PCR products 4 and 5 were loaded on a 1 % agarose gel together with IV that should be controlled. PCR products 4 and 5 were correct so they were purified from the gel using the protocol from QIAquick Gel Extraction Kit. IV however was not correct. Only a band around 750 bp was visible.

    Concentrations:
    4: 10.8 ng/µl
    5: 10.4 ng/µl
    4-5 were diluted to 1 ng/µl.
    New PCR reactions were started (II, III and IV). PCR settings that works well with the Phusion enzyme was set:
    Initial denaturation: 98 °C 3 min
    Denaturation: 98 °C 10 s
    Annealing: 55 °C 30 s
    Elongation: 72 °C 1.5 min+ 1s/cycle
    30 cycles
    Final extension: 72 °C 10 min
    4°C forever

    2010-07-30
    Julia and Katarina

    The concentration of the purified plasmids were measured with nanodrop:
    P1: 1.7 ng/µl
    P2: 3.5 ng/µl
    P3: 25.9 ng/µl
    P4: 42.8 ng/µl
    This is clear evidence that water should be used for eluation in the future!

    To make sure that we have the right products after purification a 1 % agarose gel was run with fastruler DNA ladder. The gel verified the plasmid size though the bands were light.

    PCR product III was evaporated since it was so dilute. New concentration:
    IIId: 35 ng/µL

    The digested PCR product III and the digested plasmid were ligated with T4 ligase. The DNA concentration in total should not exceed 1 µg. The ratio insert:vector should be 3:1.

    Ligation protocol:
    150 ng insert
    50 ng vector
    3 µl 10x T4 buffer
    2.5 µl (2.5 U, between 1-5 U) T4 ligase
    Adjust volume with water up to 30 µl. Mix and incubate in 4-16 °C (refrigerator) overnight.
    The newly plated sporplates were moved to 30°C. The old sporplates were again studied in the microscope and no tetrads were seen.

    Kemal and Lokesh

    Since we have had a lot of trouble with the longer fusions we will start over from scratch with a new high fidelity polymerase called “Finnzumes Phusion”. New PCR reactions were run (A, B, 3, 4 and 5). IV and α were also performed using alternative settings (2 different PCR reactions described before) but with “old” templates.

  • 2010-07-29
    Julia and Katarina

    PCR product III is the final fusion protein where Snf4 is tagged with yfp. To be able to insert the construct into the plasmid we must digest it.

    Digestion: Mix DNA, buffer and water. Last add enzyme and mix. Incubate at 37° C for 1 hour.
    1 µg DNA
    2.5 µl Buffer
    1 U Enzyme
    Up to 25 µl H2O
    For PCR product III, the restriction enzymes BamHI and HindIII are used together with buffer B. After digestion (the protocol was scaled 3x) the sample was loaded on a 1 % agarose gel in 6 lanes with 1 kb ladder. The bands were cut out and weighed. The samples were purified from the gel using the protocol from QIAquick Gel Extraction Kit. The DNA was eluated with water.

    Concentration:
    IIId: 5 ng/µl (Total product 160 µl= 800 ng)

    Kemal and Lokesh

    The plasmids were also digested with BamHI and HindIII according to the same protocol as above. P1 and P2 were eluated with buffer EB and P3 and P4 with water.

    2 new sporulation plates were prepared by plating the diploid yeast cells on spore plates and kept at room temperature.

  • 2010-07-28
    Julia and Katarina

    The PCR products were run on a 1 % gel with 1 kb ladder. Γ may have worked. α and Β had a band around 750 bp and a weird light band around 500 bp. They obviously did not work. IV did not work at all.

    The sporulation plates were checked for spores. Cells were studied under a microscope. No tetrads visible.

  • 2010-07-27
    Julia and Katarina

    A 1.5 % gel was run with all of the fragments around 700 bp to control the validity of the PCR reactions. A low range ladder was used. PCR products 1-5 seems good. The results from yesterday were confirmed.

    A new PCR design was made with α, Β, Γ and IV. PCR was done in two steps to increase the chance of success for longer fusions.
    1. 7 cycles with short annealing time.
    2. 20 cycles with long annealing time (all other settings kept standard).

  • 2010-07-26

    A new experiment were designed called Γ which is the same as I but with longer annealing region. Also an experiment called Δ were designed which corresponds to II.

    Julia and Katarina

    PCR reactions were run with samples of 4, IV and Γ. IV was tried with two different PCR settings: one regular and one with two runs of PCR, the first without primers and only 5 cycles and the second one with regular settings.

    Kemal and Lokesh

    The PCR products were controlled on a 0.7% agarose gel. Only 4 worked. Γ was empty and IV had bands in the wrong place (amplified template).
    4: 534 ng/ µl

  • 2010-07-22
    Karl, Lokesh and Kemal

    A new mastermix was prepared using a different enzyme. Then the experiments from yesterday was redone using regular PCR settings:
    A, 5, 2, I
    Then some more experiments of IV was redone:
    2a, 2b and 2c
    The new primer 14 had arrived so PCR reaction 4 was also redone.

    All the PCR products were run on a 0.7 % agarose gel with GeneRuler 1 kb DNA ladder. The results were inconclusive. None of the expected bands were received. At this point we are not sure what to do since none of the PCR reactions worked.

  • 2010-07-21
    Malin, Julia, Karl and Lokesh

    Verification of the PCR products α1, α2, α3, α4, α5 and αK by running on a thin 0.7 % agarose gel (8 µl sample+ 2 µl loading buffer, GeneRuler 1 kb DNA ladder).

    The gel didn’t show the expected band lengths. A band around 700 bp could be seen, most strongly for α4 and α5. We suspect that there is something wrong with the ladder because we get inconclusive results. Since none of the PCR reactions for the last couple of days have worked we will redo the templates of α and IV: I, 2, 5 and A. These PCR reactions have worked before.

    Expected fragment length:
    A.2: 1938 bp
    5.2: 757 bp
    2.2: 762 bp
    I.2: 768 bp

    The PCR products were run on a thin 0.7 % agarose gel. We used both the FastRuler High Range Ladder and the GeneRuler 1 kb DNA ladder. 5.2 and 2.2 showed the expected bands around 750 bp. I.2 and A.2 did not work at all, nothing visible on the gel. We also run some of the old PCR products to see what the result would be using a new ladder. We run α2, α5, 4c (IV) and 4c*(IV). Here some template amplifications were visible but sadly none of the expected fragment lengths. Both ladders showed the same result so there is probably nothing wrong with the GeneRuler ladder except that it is not showing strong color.

  • 2010-07-20
    Malin, Julia, Lokesh and Karl

    Verification of yesterdays PCR reactions by running on a 0.7 % agarose gel (8 µl sample+ 2 µl loading buffer, GeneRuler 1 kb DNA ladder)

    None of the reactions worked.

    Gel 1 (Block A): Nothing visible on gel, ladders only very light so these samples were run on a new 0.7 % agarose gel but this time thinner to get better resulotion (0.58 g agarose + 80 ml TBE). This time no expected fragments were seen, but bands corresponding to primer dimers were clearly seen.
    Gel 2 (Block B): A few small bands were seen but no bands of expected length.

    We decided to stop working on IV until proper feedback on the experimental setup was received. Instead we continued with α.

    Doing another PCR round of α (SAMS peptide):
    α: PCR fusion of cfp-SAMS and yfp: Adds BamHI on N-terminus of cfp and HindIII on C-terminus of yfp and keeps the stop codons. Expected fragment length: 1502 bp.

    α1: regular protocol.
    α2: regular protocol.
    α3: 1.25 µl extra primer 9.
    α4: 1.25 µl extra primer 12.
    α5: 1.25 µl extra primer 9 and 1.25 µl primer 12.
    αK: Control
    Regular PCR-settings

  • 2010-07-19
    Karl, Peidi and Kemal

    Doing a new experimental setup for IV:
    IV. PCR fusion of cfp and Snf1: Adds SpeI on N-terminus of cfp and PacI on C-terminus of Snf1 and keeps the stop codon. Expected fragment length: 2643 bp.

    13 different tubes were prepared and run at two different PCR-settings. Block A: Regular settings except double annealing time (1 min). Block B: Regular settings except 58° C annealing temperature.

    C : Control
    1a: same as 2* (template 5 5 x diluted)
    1b. same as 3* (template 5 10 x diluted)
    1c: same as 4* (template 5 100x diluted)
    2a: increased product A concentration to 5 ng/µl.
    2b: increased product A concentration to 10 ng/µl.
    3a: same as 5* (2x primer 7)
    3b: same as 6* (template 5 diluted 5x, 2x primer 7)
    3c: same as 7* (template 5 diluted 10x, 2x primer 7)
    3d same as 8* (template 5 diluted 100x, 2x primer 7)
    4a: 2x primer 7.
    4b: 2x primer 7, 5x template A.
    4c: 2x primer 7, 10 x template A.

  • 2010-07-16
    Lokesh, Kemal, Katarina and Peidi

    Verification of PCR reactions IV* (8 tubes) and α on a 0.7 % agarose gel (5 µl sample+ 2µl loading buffer, GeneRuler 1 kb DNA ladder).

    No bands visible. None of the PCR reactions worked, just small bands visible corresponding to primers. The α PCR reaction messed the tubes up. That might be an explanation why there were no bands on the gel for α. A new experimental setup must be made tomorrow.

    PCR products I and 2 were run with a low range ladder to see that those products are ok. That gel was satisfactory.

  • 2010-07-15
    Malin, Julia, Kemal and Karl

    Concentrations of PCR products:
    4.2. 356.2 ng/µl
    I. 338.3 ng/µl
    II. 303.6 ng/µl
    III. 337.5 ng/µl
    IV. 280.9 ng/µl

    All PCR-products were diluted to 1 ng/µl.

    Verified PCR products I-IV and 4.2 on a 0.7 % Agarose Gel (8 µl sample + 2µl loading buffer, GeneRuler 1 kb DNA ladder)
    PCR products I, II and III were good but 4.2 showed no bands at all. After primer design control we found a problem with primer 14, which then was ordered again. IV had only a band around 700 bp instead of 2600. This might be template 5, which then has been amplified. If the settings are changed maybe it will work. Firstly the template 5 will be diluted 0x, 5x, 10x and 100x (4 tubes: 1*, 2*, 3* and 4*) and these dilutions will also be run with higher primer concentration 7 (4 tubes: 5*, 6*, 7* and 8*). 2.5 µl primer 7 will be added instead of 1.25 µl primer 7.

    New PCR reactions were run:
    α: PCR fusion of cfp-SAMS and yfp: Adds BamHI on N-terminus of cfp and HindIII on C-terminus of yfp and keeps the stop codons. Expected fragment length: 1502 bp.
    K4. Control.

    IV *: PCR fusion of cfp and Snf1: Adds SpeI on N-terminus of cfp and PacI on C-terminus of Snf1 and keeps the stop codon. Expected fragment length: 2643 bp.
    Half volume 25 µl. Regular PCR settings except 56 °C annealing temperature.
    1*;- 8*
    K1*. Control for 1-4*.
    K2*. Control for 5-8*.

  • 2010-07-14
    Julia and Katarina
    Ran PCR 1-5.
    1. Amplification of cfp-sams compatible: Add restriction enzyme site BamHI on N-terminus +SAMS on C-terminus.
    Expected fragment length: 748 bp.
    2. Amplification of yfp-SAMS competible: Adds restriction enzyme site for HindIII on C-terminus and SAMS tail on N-terminus.
    Expected fragment length: 762 bp.
    3. Amplification of yfp-snf4 compatible: Adds restriction enzyme site BamHI on N-terminus and Snf4 tail on C-terminus.
    Expected fragment length: 755 bp.
    4. Amplification of cfp-Snf4 compatible: Adds restriction enzyme site HindIII on C-terminus and Snf4 tail on N-terminus.
    Expected fragment length: 769 bp.
    5. Amplification of cfp-snf1 compatible: Adds restriction enzyme site SpeI on N-terminus and Snf1 tail on C-terminus.
    Expected fragment length: 757 bp.
    K2. Control K2.

    Julia, Katarina and Malin
    Verified PCR 1-5: measured concentration by nanodrop and checked if the fragments were the correct length by running on a 2 % agarose gel (8 µl sample+2 µl loading buffer, Fermentas FastRuler Low Range).
    PCR product 1, 2, 3 and 5 worked and gave expected fragment lengths. Number 4 however did not work. Only a fragment around 80 bp was visible, probably a primer dimer.

    Concentrations:
    1. 443.9 ng/µl
    2. 371.0 ng/µl
    3. 361.7 ng/µl
    4. --
    5. 377.6 ng/µl
    A. 451.1 ng/µl (snf1)
    B. 438.7 ng/µl (snf4)
    C. 408.9 ng/µl (SAMS)
    1-5 and A-C were diluted to 1 ng/µl.

    Ran PCR of I, II, III, IV and 4.
    4.2. Amplification of cfp-Snf4 compatible: Adds restriction enzyme site HindIII on C-terminus and Snf4 tail on N-terminus. Expected fragment length: 769 bp. (second try)
    I. PCR fusion of cfp and SAMS: Adds BamHI on N-terminus of cfp and gives a SAMS C-terminus. Expected fragment length: 768 bp.
    II. PCR fusion of yfp and Snf4 1: Adds BamHI on N-terminus of yfp and removes stop codon. Expected fragment length: 1694 bp.
    III. PCR fusion of yfp and Snf4 2: Adds BamHI on N-terminus of yfp and adds HindIII on C-terminus of Snf4 plus adds stop codon. Expected fragment length: 1708 bp. Final Product!
    IV. PCR fusion of cfp and Snf1: Adds SpeI on N-terminus of cfp and PacI on C-terminus of Snf1 and keeps the stop codon. Expected fragment length: 2643 bp.
    K3. Control

    Karl and Malin
    Transferred diploid cells of snf1Δ mutants to two sporulation plates and incubated in 30° C. (For 2-7 days)

  • 2010-07-13
    Julia and Lokesh
    Ran the gel with products of our first three PCRs along with the templates we made for the two fluorescent proteins. We also prepared the sample solutions of DNA templates with appropriate concentration (1ng/µl) for the next PCR reactions.
    Verified the PCR products and the digested miniprepped EYFP and ECFP by running an agarose gel which showed good results. Expected fragment lengths were received. The SAMS-peptide however was too short to be visible on the gel (67 bp).
    Prepared for PCR 1-5. The miniprepped ECFP and EYFP were diluted to a stock concentration of 1 ng/ ml to use for the PCR. The concentrations of the first PCR product were measured using nanodrop and then diluted to 1 ng/ml.
    snf1: 20.5 ng/ml
    snf4: 122.6 ng/ml
    SAMS: 114.9 ng/ml

    Karl and Adnan
    We made duplicates of the haploid Snf4∆ and wildtype strains on new plates in case the original plate would be contaminated. Prepared a sporulation medium that was autoclaved and poured into empty growth plates to stay in room temperature over night.


  • 2010-07-12
    Katarina and Julia
    Amplification of genomic SNF1 DNA by PCR. All primers were resuspended in mq-water according to the volume on the lable, the final concentration 100 pg/ml. The primers were then diluted 10x and the SAMS-peptide templates were diluted 1000x. PCR reactions were run amplifying subunits Snf1 and Snf4 and also the SAMS-peptide. YPD medium and agar plates were prepared (yeast).

    Adnan and Kemal
    Miniprep purification of EYFP and ECFP plasmides from overnight culture. The concentrations were measured using Nanodrop:
    ECFP1: 99 ng/ml
    ECFP2: 89.9
    EYFP1: 104, 7
    EYFP2: 172,5
    The concentrations were verified with electrophoresis. The plasmids were cut with restriction enzymes EcoRI and Pst1.
    ECFP, plasmid pSB1AK3, fragment sizes 750 bp and 3200 bp.
    EYFP, plasmid pSB1A2, fragment sizes 750 bp and 2050 bp.


  • 2010-07-11
    Katarina
    Made overnight cultures with colony 3 and 4 plasmid (not single cultures) and twp each of EYFP and ECFP.


  • 2010-07-10
    Julia
    Moved plated bacteria from incubator to refrigerator.

  • 2010-07-09
    Malin and Julia
    The SOC medium was autoclaved. Competent E.coli cells were transformed with our FPs through heat shock and plated on petri dishes. 20 new LB-amp plates were made.
    Adnan and Katarina
    Picked up primers at Chalmers and checked if they were ok. The concentration of the genomic DNA (SNF1 gene) were measured. Prepared a master mix so that the PCR-reactions could be started Monday morning.


  • 2010-07-08
    Malin, Julia and Peidi
    The two BioBricks EYFP and ECFP were extracted from the kit plates and stored in the freezer. A SOC-medium was prepared.

  • 2010-07-07
    Adnan, Katarina and Julia
    As the gels from Tuesday showed ambigous results we decided to redo the miniprep of the plamids. We used the overnight preparations of the two unused colonies (3 & 4). The miniprep went smoothly except for major difficulties regarding untrustworthy pipettes. The pipettes marked with green tape are the ones to use from now on together with the tips with the same markings! A gel was molded and this time we decided to use a full gel to get a better resolution. The results on the gel were very good. The plasmids had clearly been cut in one or two places and the fragment sizes seem to correspond to the expected sizes.

  • 2010-07-06
    Malin, Adnan and Peidi
    Colony 1 and 2 were picked to do a miniprep. The plasmids were extracted and a plasmid integrity check was performed. By cutting with BamHI, one cut were made to get the size of the plasmid. Then we cut with ClaI which is a two-cutter that gives a fragment that is 600 bp and one that is 6800. The gel which should confirm this was not satisfactory since the 600 band was missing.

  • 2010-07-05
    Malin and Katarina
    An overnight culture were made from 4 different colonies of E.coli containing the plasmid pSB-GM1.

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