Team:Hawaii/Meeting/2008-06-12

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Contents

Agenda

  1. Part B project proposal discussion. (proposal sent via email Wed. for advisers to review before meeting)
    1. Krystle: signal peptides
    2. Grace: rbcl promoters
    3. Margaret: (teleconference in?)

Minutes

Present: SC, GP, GK, AB, KS, NW

Krystle: Signal Peptides (see project proposal)

  • Will the extra amino acids resulting from the Biobrick RE sites affect protein folding or excretion?
  • Look at amino acids of signal sequence
  • sec pathway may have trouble recognizing large amino acids; protein folding may also be problematic
  • Want hydrophillic amino acids at ends so signal sequence isn't folded into the protein
  • E. coli signal sequence - 20 amino acids + 2 lys residues; cleavage right before lys
  • Remember to recreate the sticky ends of the RE sites if signal peptides are synthesized (not PCR'd)
  • Need to fix primers: RE sites should always be on 5' end
  • Cite paper with isolation of nir promoter and primers used
  • Use of two transcriptional terminators in construct is standard
  • Lichenase would make a really good positive control -- make into a Biobrick?
  • Email Russian authors of paper that used lichenase for 5 μl of lichenase plasmid prep or E. coli w/ such a plasmid
  • Offer FedEx code if willing to ship to us
  • If the Russians don't send us lichenase, and no one has Clostridium that we can isolate it from, perhaps synthesize it?
  • Do experiments in parallel -- save time! Cyanos grow really slowly...
  • May want to stick our Biobricks into pRL1383a for now so we can begin experiments with PCC6803
  • Need to insert high copy oriV into pRL1383a
  • Make lots of plasmid in E. coli (with high copy oriV), cut out oriV and replace with Biobrick, transform E. coli, conjugate with PCC6803, let PCC6803 make more plasmid+Biobrick

Margaret: Plasmid (see project proposal)

  • Very well written proposal! :o)
  • How do we get more Biobrick base vectors when ccdB kills E. coli?
  • Norman will email iGEM people to find out. Perhaps get plasmid in E. coli rather than extract from filter paper?
  • ccdB is not necessary for selection; antibiotic resistance will do
  • If our vector has an insert that the organism already has, two sites of homologous recombination possible
  • Need to fix primers: RE sites should always be on the 5' end
  • NEB catalog for RE sites efficiency
  • Remember to check PCR products for RE sites
  • Detail 3 types of transformation and what you need from the transformations

Grace:rbc promoter

  • rbc promoter will still be made into a Biobrick part
  • Other parts of the project to be put on hold
  • Team has decided to pursue one single part B project
  • Compare nir with rbc promoter efficiency (partner with Krystle)

Gernot

  • SpeI is really expensive (4-5x cost others); Use other enzymes when possible (i.e. do a X-SP ligation rather than EX-S)
  • Team: find electroporation protocol for PCC6803 to decrease transformation time. Try it.
  • Flesh out expected results for each part of proposal so we can design experiments

Action Items

  • Everyone: Check primers, put in orders tomorrow
  • Everyone: Get all useful parts and put into E. coli
  • Strong and weak RBS
  • Base vector
  • Promoters
  • Krystle: Build CO2 incubator ASAP

Coming Up


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