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Team:Bonn/Notebook
From 2012.igem.org
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=== Summary === | === Summary === | ||
| - | Our lab work started in May and after the 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 as Lov is our main component. Finally, we switched to a non-Biobrick Lov instead of using the BioBrick, which has two PstI cutting sites to be removed. Our work with the other parts could be resumed and we could finish the final ligation for the Lov-Ccdb and Lov-LacZalpha fusion constructs. | + | Our lab work started in May and after the 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 as Lov is our main component. Finally, we switched to a non-Biobrick Lov instead of using the BioBrick, which has two PstI cutting sites to be removed. Our work with the other parts could be resumed and we could finish 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. |
=== Highlights by date === | === Highlights by date === | ||
Revision as of 23:03, 26 September 2012
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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: all our experiments with the LovTAP Plasmid from the distribution failed. A long time of troubleshooting followed as Lov is our main component. Finally, we switched to a non-Biobrick Lov instead of using the BioBrick, which has two PstI cutting sites to be removed. Our work with the other parts could be resumed and we could finish 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.
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
