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

For our two systems “Cut’N’Survive” and “Jump-Or-Die” of selecting cells by apoptosis, we first had to build the constructs. Therefore we searched for sources of the DNA sequences we needed and found several supporters 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 EcoR1-, Pst1-, Xba1-, Spe1- or Not1- restriction site, we used mutagenesis primers and fusioned both DNA parts by fusion PCR. Fortunately all PCRs worked in spite of several problems with a touch down PCR and the fusion PCRs.

The PCR products were tested by agarose gel electrophoresis and the right amount of basepairs. We tried to sequence our PCR products, but we got poor results and were told to sequence the plasmids.

In parallel, we made competent cells and multiplied ccdB (death gene)-vectors with different antibiotic resistances. All components were restriction 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 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, some BioBrick sequences were always 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

• Dr. Arnim Weber: submitted us a vector with human Bak
• 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
• Knop, M (Heidelberg): (Knop et al.: Efficient protein depletion by genetically controlled deprotection of a dormant N-degron (2009))
  • 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:
  • eGFP (BBa_I714891)
  • CMV-Promoter (BBa_J52034: this part was wrong: its lacI !!!)
  • ccdB amp, cam, tet, kan in E.coli DH3

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.

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