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From 2008.igem.org

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Contents 1 Agenda 2 Minutes 3 Action Items 4 Coming Up

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

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