IGEM:MIT/2005/Natalie Discussion: Yeast

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.