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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 built the necessary constructs. Therefore we searched for sources of the DNA parts involved and several labs kindly supported us by sending the plasmids and sequences we needed, which are listed below.
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, we used mutagenesis primers and fused both DNA parts by fusion PCR. Fortunately all PCRs worked 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 are of the right length. We sequenced the plasmids due to poor results from PCR products' sequencing.
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 sequenced BioBricks, though some BioBrick sequences turned out wrong. so that we only got 4 BioBricks although every ligation leaded to colonies after the transfection. Neither of our constructs was completed, so that we weren´t able to test them in eukarytic cell lines.
In addition, we found out that a CMV-promoter we ordered from the registry was in fact a lacI gene and therefore some BioBricks just couldn´t be right.
Our notebook with detailed descriptions can be found here: ApoControl notebook
The protocols we used are listed here: Protocols
The Biobricks we could submit:
- BBa_K368004: attP+eGFP+SV40PA
- BBa_K368011: eGFP+SV40PA
- BBa_K368016: TEVrecognition site+N-degron+SF3b155
- BBa_K368019: TEV-Protease+p14*+TEVrecognition site
Sources, helpers and supporters:
- 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
We went to the Oktoberfest, LMU Munich and the municipal secretary and asked a few questions about synthetic biology: 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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