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Team LMU iGEM 2010
Who we are
We are a team constisting of biologists, biochemists, chemists and bioinformatics and four faculty advisors... The LMU team is competing for the first time.
Advisors
Prof. Dr. Angelika Böttger: Department Biologie II/ Cell and Developmental Biology
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Prof. Dr. Kirsten Jung: Head of the Division of Microbiology
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Prof. Dr. Thorsten Mascher: Department Biologie I/ Microbiology
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Dr. Achim Tresch: Computational Biology and Regulatory Networks
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Undergrads
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Erik Fumi: Chemistry and Biochemistry, Pathway
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Who is in charge of what?
| Press
| Nicolas Keller, Marisa Kurz
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| Sponsoring
| Sabine Wagner, Franziska Haefele
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| Pathway
| Tobias Bauer, Emanuel Stiegeler, Benny Clanner
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| ApoControl
| Julia Bartels, Jara Radeck, Mengzhe Grace Wang
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| Homepage
| Tatyana Goldberg, Jens Popken
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| Coordination/Organisation
| Jara Radeck
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| Contact
| igemlmu@yahoo.de
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What we did
Short description of our work, our results and our supporters
To establish our two systems “Cut’N’Survive” and “Jump-Or-Die” for selecting cells by apoptosis, we first set out to build the necessary BioBricks. We searched for sources of the DNA parts involved and several labs kindly provided us with the plasmids and sequences in need, as listed at the end.
Most genes and promoters were amplificated per PCR with overhang-primers with the BioBrick prefix or suffix. If the sequence contained a EcoRI-, PstI-, XbaI-, SpeI- or NotI- restriction site, mutagenesis primers were used and the amplified DNA parts were then fused by fusion PCR. All PCRs worked in the end in spite of several problems with the touch down PCR and the fusion PCR.The PCR products were then tested by agarose gel electrophoresis if they were of the right lengths.
In parallel, we made competent cells and multiplied the ccdB (death gene)-vectors with different antibiotic resistances.
All components were then digested with the appropriate restriction enzymes. The PCR products were cleaned with a PCR purification kit and the ccdB-vectors were dephosphorylated to reduce false ligations.
We ligated our constructs and several intermediates with the 3A-assembly according to the schedule. The ligations were transformed to E.coli DH5α strains and selected by antibiotics. The colonies were later picked and the insertion of the construct was confirmed by colony PCR.
If the colony PCR showed bands of the right size, the plasmids were extracted from overnight cultures and sequenced with forward and reverse BioBrick primers.
In the end we succeeded in obtaining four correctly sequenced BioBricks. But since the sequences of other necessary parts turned out wrong, neither of our systems could be completed and we were not able to test them in eukarytic cell lines.
In addition, we found out that the CMV-promoter we ordered from the BioBrick registry was in fact a lacI gene, which resulted in the procrastination of our plan. We have sent the correction to the registry to facilitate future teams.
Our notebook with detailed descriptions can be found here: ApoControl notebook
The protocols we used are listed here: Protocols
The Biobricks we submitted:
- BBa_K368004: attP+eGFP+SV40PA
- BBa_K368011: eGFP+SV40PA
- BBa_K368016: TEVrecognition site+N-degron+SF3b155
- BBa_K368019: TEV-Protease+p14*+TEVrecognition site
Supervisors
- Prof. Dr. Angelika Böttger :
- prevTRE (tet-on CMV promoter; inducible by doxycycline in special cell lines)
- supported the construction ideas and would have given us the cells and mediums we would have needed
- SV40PA (Polyadenylation site): gave us a vector containing it
- Human Bak: her assistant Erika Clement gave us appropriate cDNA
- Prof. Dr. Thorsten Mascher:
- Helped with primer design, agarose gel electrophoresis apparatuses and trouble shooting
- Prof. Dr. Kirsten Jung:
- Helped with ideas and fundraising
- Dr. Susanne Gebhard:
- Helped with trouble-shooting and materials
- Kemal Akman
- Helped and supported our sub-project ProSearch
Plasmid and sequence sources
As our project for human practice, we carried out a survey about synthetic biology on the general public at the Oktoberfest, LMU Munich and the Municipal Secretary: Human practice
Where we are from
We are students at Ludwigs-Maximilians-University in Munich, Germany.
our bio-center (photos courtesy of www.stbam2.bayern.de)
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