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From 2012.igem.org

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Lab Notebook

This is an overview on what we did in our lab, including our standard lab protocols.

Summary

Our lab work started in May and after a few first successful transformations we came across our greatest issue: all our experiments with the LOVTAP plasmid from the distribution failed. A long time of troubleshooting followed since LOV is our main component. Finally, we switched to a non-BioBrick LOV with two PstI sites instead of using the distribution. Our work with the other parts could be resumed and we finished the final ligation for the LOV-ccdB and Lov-LacZalpha fusion constructs. But we could not remove the two PstI restriction sites. Additionally we removed one PstI restriction site from the single LOV domain for the fusion system.

LOVTAP

We used 2 month for the troubleshooting of our LOVTAP sample from the distribution plate. The first experiments with our LOVTAP plasmid were misinterpreted. We thought our problems lay in the restrictions, PCR or our transformation. In fact, restriction and transformation worked well with all the other plasmids. We also sent out the plasmid for sequencing. We could not get sequencing results matching any part of the LOVTAP plasmid at all but had a contamination with another sequence. Finally we asked two other German iGEM teams (LMU Munich and Darmstadt) for a sample of their distribution plate LOVTAP. This samples did not lead to good sequencing results either and showed the same pattern of degeneration. In the end we used LOVTAP-pCal-n (modified) instead of the Biobrick.

Highlights by date

May 2012

23.05.

Starting our lab work with creating chemo-competent DH5α bacteria.

31.05.

First successful transformation of iGEM plasmids (pLac, Lac1, LOVTap, J23119, LacZα, ccdB, RBS32 and the double terminator). XL1-blue bacteria turned out to be much more competent than our DH5α, so we used these from now on.

June 2012

04.06.

Midi-Preps of iGEM plasmids from our distribution kit.

06.06. - 08.06.

First restrictions, some of them were successful.

08.06. - 03.07.

PCR, transformation and restriction troubleshooting to find the issue in our LOVTAP transformation.

July 2012

04.07.

Sequencing results of all our biobricks are available: Sequence is okay for RBS32, LacZα, pLac and our double-terminator. A frameshift was found in LacI plasmid. LOVTAP was contaminated with another sequence.

later that month...

Further troubleshooting and optimization of our standard procedures.

August 2012

06.08.

First application of the 3A assembly, following the official iGEM protocol. (we used a different one before).

• Restrictions of LacZalpha-pSB1AK3, ccdB-pSB1A2, TT-pSB1AK3, pSB1C3
• Ligations to LacZalpha-TT-pSB1C3 and ccdB-TT-pSB1C3

Midi-Preparation of LOVTAP-pCal-n (wt) - we later used this construct for testing reasons only.

• we got LOVTAP-pCal-n(wt) and LovTAP-pCal-n(mod) from the Sosnick Lab and used it instead of the BioBrick.

07.08.

Transformation of constructs prepared the day before.

08.08.

• Restriction analysis of our first completed constructs: LacZα-TT-pSB1C3 and ccdB-TT-pSB1C3
• Started work on constructs J23119-lacI-pSB1C3 and pLac-RBS32-pSB1K3

10.08.

• Sequencing of our first constructs (LacZα-TT-pSB1C3 and ccdB-TT-pSB1C3) for verification.
• First successful PCR of wildtype-LOV (test for future experiments)
• Midi-Preps of LOVTAP-pCal-n (Mutant) and mazF-pBAD

16.08.

Work on constructs pLac-RBS32-ccdB-TT-pSB1A3 and pLac-RBS32-LacZα-TT-pSB1A3 started

21.08.

Successful PCR-amplification of mod-LOV

22.08. - 28.08.

First trial of ligation to form the new constructs pLac-RBS32-LOV-Ccdb-TT-pSB1C3 and pLac-RBS32-LOV-ccdB-TT-pSB1C3

29.08.

Gel-purification of the pSB1C3 fragment, which was cut with EcoRI and SpeI before, for usage in the pLac-RBS32-LOV-pSB1C3 construct.

23.08. - 28.08.

Creation/finalization of constructs J23119-LacI-pSB1C3 and J23119-LacI-pSBA3

September 2012

05.09. - 07.09.

Formation of construct pLac-RBS32-LOV-pSB1C3

06.09.

• Successful PCR-amplification of mazF fragment
• Constructs mazF-pSB1C3 and mazF-TT-pSB1C3 completed

10.09. - 11.09.

Succesful mutagenesis PCR: pLac-LOV-pSB1C3 and LOV-pSB1C3, first of two PCRs to make the LOV part BioBrick compliant

13.09. - 24.09.

New constructs:

• pLac-RBS32-LOV-ccdB-TT-pSB1C3
• pLac-RBS32-LOV-LacZalpha-TT-pSB1C3

Protocols

You can find the protocols we used at our protocols page