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Team:Bonn/Notebook
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=== Summary === | === 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 | + | 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 === | === LOVTAP === | ||
We used 2 month for the troubleshooting of our LOVTAP sample from the distribution plate. | 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 | + | 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 | + | In the end we used LOVTAP-pCal-n (modified) instead of the Biobrick. |
=== Highlights by date === | === Highlights by date === | ||
| Line 69: | Line 69: | ||
* Ligations to LacZalpha-TT-pSB1C3 and ccdB-TT-pSB1C3 | * Ligations to LacZalpha-TT-pSB1C3 and ccdB-TT-pSB1C3 | ||
| - | Midi-Preparation of | + | Midi-Preparation of LOVTAP-pCal-n (wt) - we later used this construct for testing reasons only. |
| - | *we got | + | *we got LOVTAP-pCal-n(wt) and LovTAP-pCal-n(mod) from the Sosnick Lab and used it instead of the BioBrick. |
===== 07.08. ===== | ===== 07.08. ===== | ||
| Line 82: | Line 82: | ||
* Sequencing of our first constructs (LacZα-TT-pSB1C3 and ccdB-TT-pSB1C3) for verification. | * Sequencing of our first constructs (LacZα-TT-pSB1C3 and ccdB-TT-pSB1C3) for verification. | ||
* First successful PCR of wildtype-LOV (test for future experiments) | * First successful PCR of wildtype-LOV (test for future experiments) | ||
| - | * Midi-Preps of | + | * Midi-Preps of LOVTAP-pCal-n (Mutant) and mazF-pBAD |
===== 16.08. ===== | ===== 16.08. ===== | ||
| - | Work on constructs pLac-RBS32- | + | Work on constructs pLac-RBS32-ccdB-TT-pSB1A3 and pLac-RBS32-LacZα-TT-pSB1A3 started |
===== 21.08. ===== | ===== 21.08. ===== | ||
| Line 91: | Line 91: | ||
===== 22.08. - 28.08. ===== | ===== 22.08. - 28.08. ===== | ||
| - | First trial of ligation to form the new constructs pLac-RBS32-LOV-Ccdb-TT-pSB1C3 and pLac-RBS32-LOV- | + | First trial of ligation to form the new constructs pLac-RBS32-LOV-Ccdb-TT-pSB1C3 and pLac-RBS32-LOV-ccdB-TT-pSB1C3 |
===== 29.08. ===== | ===== 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. | + | 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. ===== | ===== 23.08. - 28.08. ===== | ||
| Line 105: | Line 105: | ||
===== 06.09. ===== | ===== 06.09. ===== | ||
| - | * Successful PCR-amplification of | + | * Successful PCR-amplification of mazF fragment |
| - | * Constructs | + | * Constructs mazF-pSB1C3 and mazF-TT-pSB1C3 completed |
===== 10.09. - 11.09. ===== | ===== 10.09. - 11.09. ===== | ||
| Line 113: | Line 113: | ||
===== 13.09. - 24.09. ===== | ===== 13.09. - 24.09. ===== | ||
New constructs: | New constructs: | ||
| - | * pLac-RBS32-LOV- | + | * pLac-RBS32-LOV-ccdB-TT-pSB1C3 |
* pLac-RBS32-LOV-LacZalpha-TT-pSB1C3 | * pLac-RBS32-LOV-LacZalpha-TT-pSB1C3 | ||
=== Protocols === | === Protocols === | ||
You can find the protocols we used at our [[Team:Bonn/Protocols|protocols page]] | You can find the protocols we used at our [[Team:Bonn/Protocols|protocols page]] | ||
Latest revision as of 01:24, 27 September 2012
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Contents |
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
