IGEM:MIT/2005/Day 4 Discussion

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Day 4 discussion

A. Logistics

  • 1. After today: transition to ad hoc/team driven. Expect to need planning meeting (Mondays at 11, place TBS) and planning meeting (Fridays at 11). Team will drive meetings and needs.
  • 2. Default space Drew’s tea room? Drew’s antiroom as place to store stuff and maybe keep computer or two but space too small to sit everyone at once. Could also meet in BUG lounge near BioCafe, but need access keys after hours.
  • 3. In Kate’s space need to have advisor present at all times when team is working.

B. Biological Battery.

  • Bact Rhod: Will has emailed Ed Delong. He is happy to talk…team needs to email him to set up direct conversations. He will give 30’ presentation Tuesday on “potentially cool parts from sea critters.”
  • Follow up on electricity in eels?

C. Antibody signaling. Annie generated list of 7000 papers from Pubmed by searching bacterial + cholesterol receptor. Best 3 on first page:

  • 1. Use of bact expn’ cloning to localize epitope of ApoB, LDL. Used Western to map.
  • 2. Bact expn and purify of mAb frag for ApoE.
  • 3. 1990 publication: partial ApoE;;lacZ fusion expressed in coli retains affinity. Let TK know if there is article needed that can’t get from MIT. Alternativel, can ask library to get pdf.

Also found yeast surface display paper and posted ref on wiki. Talk to Dane Wittrup?

  • Seems like feasible project. Receptor for LDL known: gene for LDL receptor: 45kb, with introns? mRNA 5.3

Not clear if wanting to express antibody to LDL or express LDL receptor.

  • How to turn Ab binding into a signaling cascade: tyrosine kinases fused to anchor? I15010 fuses light domain with naturally occurring signaling path?

Big jump is how to make it happen with Ab binding.

  • How to det’n sequence of antibody? 10 cent version: Huge class of Antibody seq’s known
  • How to have binding of antibody become signal something? Gosh that seems useful. Drew will email Roger to ask if such a set-up has been made (he heard back: “Not one that works, Alas.”)
  • Can we get cells to change color upon binding of ligand to Ab? Screen for binders? Need promoter for GFP specific for signaling cascade?

D. Redesign

  • 1. Yeast chromosome redesign: What are first 2 or 3 steps to take?
    • Smallest chrom= 1 =225kb
      • Step zero: get up to speed on relevant yeast genetics/familiarize yourself with chromosome architecture/genome design….maybe by sequence comparison (Kellis in bldg 32) to other yeast (Gifford/Young to consider gene regulation….what makes a gene “essential”)
      • Step 1: redesign plan
      • Step 2: 22.5 kb chunks, make chromosomes (synthesize ourselves. Blue heron synthesis conservative and slow.), clone into yeast

2. Possible outcomes

  • Best case: rebuild chrom 1. Evolutionary distance masters, masters of this building technology, issues going fwd non/ess, insulation, genome architecture.
  • Worst case: “we’ve got a kickin’ design. No one can synthesize the darn parts, This sucks.”

3. Examples of bacterial chromosome redesign:

  • a. Blattner group: deleting nonessential seqs like Tns, has ripped out about 50 chunks adding up to ~25% of genome and cells OK under std lab condition but fitness changes.
  • b. TK: 875 kb florum no genetics, invent over summer?
  • c. Rebuild chrom by changing 3 stop codons to 2
  • d. Pam Smith/ Venter:/ hutch: mycoplasm . Human pathogen. Doubling time of 6 hrs. No defined medium. Bad bad and bad
  • e. Ron Davis (Stanford) proposed to NIH to rebuild yeast 5 years ago to
  • f. Reprogram bacteria to make spectacular dyes. “Paint by number”