IGEM:MIT/2005/Natalie Discussion: Yeast

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Redesign Yeast Chromosome

  • SGD = database for yeast (Google it)

Check out: http://db.yeastgenome.org/cgi-bin/search/featureSearch to get info like:

    • Chrom 1 = 117 ORFs, 13 essential genes
    • Chrom 3 = 182 ORFs, 16 essential genes
    • Chrom 6 = 141 ORFs, 27 essential genes
  • Chrom 1 is the shortest chromosome of the 16.
  • Yeast artificial chromosomes are available.
    • Resemble yeast chromosome. It is circular and can linearize. Has telomere, centromeres, and cloning sites.
      • This could be a way to move essential gene from chromosome and test from there.
  • 11/13 genes on Chrom1 (essential) examined for expression data
    • Seem to do okay with glucose starvation.
      • Look at kinds of genes on Chrom, and see what happens.
  • Chromatin structure is sensitive.
  • Drug marker (kanomysin resist) in the PCR product, transform into yeast, select for cells with drug marker. Haploid might not get anything --kill yeast. Diploid = spore it, 8-10 days. Technically difficult to do. Random spore analysis might work. To confirm, southern blot to detect.
  • Feasibility depends on what we choose as goal.
    • Discovery project for sure.
    • If goal for Nov. is for astounding re-engineering, project might be hard.
    • Time scale may be more like a grad-student project.
    • Might be good for learning microarrays.

Antibody signalling with Yeast

  • Antibod frag fused to protein -> exported
    • Fusion protein -> surface of cell
    • Also tethering protein
      • held together with sulfide bonds
  • Would it be possible to put antibody frag into bacteria
  • Bacteria are tiny = # proteins that can be expressed on their surface is less.
  • Surface-expressed E.coli out there that have been manipulated
    • Maltose binding protein -- change binding sites
      • Hellinga paper about Maltose.

Signalling

  • Multimerization signal - need polypeptides on surface to generate signal
  • Antibody on outside would signal dimerization?
    • Know that it's possible to mix-match DNA binding proteins, modular.
  • Scaffold protein holds some components of transudction pathway, limits cross talk.
  • E.coli would be great to work with for this because fewer components to pathways.
  • Yeast vs. E.coli
    • More E.coli biobricks than Yeast (5 or 6 in registry and are all fluorescence)
    • More starting material w/ E.coli
    • Yeast is eukaryotic --can be more useful for application.
    • Bacteria could be first step.
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