Team:LMU-Munich/Team

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

Prof. Dr. Kirsten Jung: Head of the Division of Microbiology

Prof. Dr. Thorsten Mascher: Department Biologie I/ Microbiology

Dr. Achim Tresch: Computational Biology and Regulatory Networks

Kemal Akman: Bioinformatics

Undergrads

Julia Bartels: expected BSc Biology, ApoControl, Pathway

Tobias Bauer: BSc Biology, Pathway

Alexander Buschle: BSc Biology, ApoControl

Benjamin Clanner: Chemistry and Biochemistry, pharmaceutical sciences, Pathway

Maria Katharina Drexler: BSc Biology, ApoControl

Erik Fumi: Chemistry and Biochemistry, Pathway

Tatyana Goldberg: BSc Bioinformatics, ApoControl

Franziska Häfele: expected BSc Biology, ApoControl

Corinna Hofer: BSc Biology, ApoControl

Nicolas Keller: Biology, Law, ApoControl, Pathway

Laura Kleinknecht: BSc Biology, ApoControl

Christina.jpg Christina Krönauer: expected BSc Biology, Pathway

Marisa Kurz: Chemistry and Biochemistry, Pathway

Jens Popken: Biology, Pathway

Jara Radeck: Biology, ApoControl

Emanuel Stiegeler: BSc Biology, Pathway

Angelika Vetter: Biology, ApoControl

Sabine Wagner: Biology, ApoControl, Pathway

Mengzhe Wang (Grace): Biology, ApoControl

Who is in charge of what?

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 correctly sequenced BioBricks. But since other necessary BioBrick sequences 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 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

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

Plasmid and sequence sources <b>

• Dr. Arnim Weber: provided us with human Bak plasmid
• Dr. Philipe Soriano: (Raymond CS et al: High-Efficiency FLP and PhiC31 Site-Specific Recombination in Mammalian Cells (2007))
  • Sequences of attB and attP site
  • PhiC31o was bought via Addgene
• Dr. Michael Knop:(Heidelberg): (Knop et al.: Efficient protein depletion by genetically controlled deprotection of a dormant N-degron (2009))
  • provided us with:
  • TEVrecognition site+N-degron+SF3b155
  • TEV-Protease+p14*+TEVrecognition site
• 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
• Prof. Dr. Andreas Brachmann:
  • Sequenced our samples
• Partsregistry provided us with:
  • eGFP (BBa_I714891)
  • CMV-Promoter (BBa_J52034: this part was wrong: its lacI !!!)
  • ccdB amp, cam, tet, kan in E.coli DH3

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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