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Team:Hawaii/Meeting/2008-06-12
From 2008.igem.org
| Projects | Events | Resources | ||
|---|---|---|---|---|
| Sponsors | Experiments | Milestones | Protocols | |
| Notebook (t) | Meetings (t) |
Contents |
Agenda
- Part B project proposal discussion. (proposal sent via email Wed. for advisers to review before meeting)
- Krystle: signal peptides
- Grace: rbcl promoters
- 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 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
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
- <Person A>: Task
Coming Up
[http://manoa.hawaii.edu/ 
][http://manoa.hawaii.edu/ovcrge/ 
][http://www.ctahr.hawaii.edu 
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