Team:Hawaii/Meeting/2008-06-12

Agenda

 * 1) Part B project proposal discussion. (proposal sent via email Wed. for advisers to review before meeting)
 * 2) Krystle: signal peptides
 * 3) Grace: rbcl promoters
 * 4) 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 &mu;l 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