Team LMU iGEM 2010
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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, Angelika Vetter
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| Contact
| igemlmu(at)yahoo(dot)de
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What we did
Short description of our work, our results and our supporters
The creation of certain constructs was necessary for our two systems for cell selection by means of apoptosis: “Cut’N’Survive” and “Jump-Or-Die”. We searched for sources of the DNA sequences we needed and found several supporters which are listed below.
Most genes and promoters were amplificated via PCR with overhang-primers with the BioBrick prefix or suffix. If the sequence contained a EcoR1-, Pst1-, Xba1-, Spe1- or Not1- restriction site, we used mutagenesis primers and fusioned both DNA parts by fusion PCR. All PCRs worked out, even the fusion PCRs.
The length of the PCR products were tested by agarose gel electrophoresis. We tried to sequence our PCR products, but obtained poor results and resorted to sequencing the plasmids.
In parallel, we made competent cells and multiplied ccdB (death gene)-vectors with different antibiotic resistances. All components were digested with the appropriate restriction enzymes. The samples were cleaned with a PCR clean up kit or dephosphorylated to reduce false ligations.
We ligated our constructs and several interim stages with the 3A-assembly according to our schedule. The ligations were transformed to E.coli DH5α strains and selected by antibiotics. Afterwards, some colonies were picked and we tested the insertion of the construct by colony PCR.
If the colony PCR resulted in bands of the right size, we extracted the plasmids from overnight cultures and sequenced the samples with forward and reverse BioBrick primers.
Unfortunately, not all BioBricks were cloned succesfully. However, we were able to produce 4 BioBricks, one of which represents a full construct while the other three are intermediates. The system wasn't completed on time, so we weren´t able to test them in eukarytic cell lines.
Besides, we found out that the CMV-promoter we ordered from the BioBrick registry (BBa_J52034) 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)
There's a second University in our beautiful Munich, even though it is "only" a technical University. Also check out their
wiki.
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