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.
- Resemble yeast chromosome. It is circular and can linearize. Has telomere, centromeres, and cloning sites.
- 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.
- Seem to do okay with glucose starvation.
- 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.
- Maltose binding protein -- change binding sites
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.