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From 2012.igem.org

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Lab Notebook

A compilation over what we did in our lab, including the standard protocols we used.

Summary

Our lab work started in May and after the first successful transformations we came across our greatest issue: The LovTap of our distribution-kit isn't what it is supposed to be. A long time of troubleshooting followed as Lov is our main component. Finally, we switched to the wild-type Lov instead of using the BioBrick (which was broken for us and the teams who sent us samples of their Lov distribution-kit plasmids), which has two PstI cutting sites to be removed. Our work with the other parts could be resumed and we started the restrictions and ligations of our final constructs.

Results

Lov-Ccdb - Lov Kills: Ccdb (gyrase inhibitor) induces cell-death when released by Lov after activation with ~450nm light. For this project, we finished the constructs pLac-RBS32-Lov-Ccdb-TT-pSB1C3, as effective part, and pLac-RBS32-Ccdb-TT-pSB1C3, our control vector.

Lov-LacZα - Lov Blues: When treated with blue light, Lov releases a part of β-Galactosidase, which completes the enzyme (other parts are floating in the cytosol) and induces the conversion of X-Gal to a blue-coloured product. We finished the corresponding part pLac-RBS32-Lov-LacZalpha-TT-pSB1C3 and the control vector pLac-RBS32-LacZalpha-TT-pSB1C3 for this.

Lov-MazF - Lov Cuts: If activated through light, the nuclease MazF becomes active. For this aspect we created the parts MazF-pSB1C3 and MazF-TT-pSB1C3. The issue itself isn't completed at time.

Fusion Kit: We created the following parts to create fusion proteins with Lov: Lov-pSB1C3 - with 1 PstI restriction site pLac-RBS32-LOV-pSB1C3 - with 1PstI restriction site

All Lov samples we use still contain two PstI restriction sites inside the Lov. We are currently working on a mutagenesis PCR to remove them, but this issue hasn't been completed yet.

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. The failure on our LovTAP plasmid was misinterpreted: We thought we had problems with the restriction, PCR or our transformation, but in fact our sample of the iGEM LovTAP biobrick was not functional.

08.06. - 03.07.

PCR, transformation and restriction troubleshooting to find the issue in our LovTAP transformation. All tests and modifications of our protocols failed for the LovTAP plasmid, but worked out with other biobricks and sample plasmids. So we finally send the LovTAP iGEM plasmid to a company for sequencing.

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 as the found sequence didn't match the desired one at all.

later this month...

Further troubleshooting and optimization of our standard procedures. We also tried to figure out if only our distribution plate contained the broken LOV, so we asked other iGEM teams to provide us with samples from their plates. We did transformations and sequencing, but these LOV plasmids showed the same pattern of degeneration our LOV did.

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.

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.

Mutagenesis PCR No 1: pLac-LOV-pSB1C3 and LOV-pSB1C3 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