Team:Hawaii/Meeting/2008-06-12

Revision as of 21:19, 12 June 2008 by Gracek (Talk | contribs)

Birds Eye View Team	Projects	Events	Resources
Sponsors	Experiments	Milestones	Protocols
	Notebook (t)	Meetings (t)

Agenda

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?)

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)

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

Grace

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)

[http://manoa.hawaii.edu/ ][http://manoa.hawaii.edu/ovcrge/ ][http://www.ctahr.hawaii.edu ]