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Team:Hawaii/Meeting/2008-06-12
From 2008.igem.org
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Normanwang (Talk | contribs) (New page: {{Team:Hawaii/Header}} == Agenda == # Part B project proposal discussion. (proposal sent via email Wed. for advisers to review before meeting) ## Krystle: signal peptides ## Grace: rbcl ...) |
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== Minutes == | == Minutes == | ||
| - | Present: | + | Present: SC, GP, GK, AB, KS, NW |
| - | + | '''Krystle: Signal Peptides''' [[Team:Hawaii/Project/Part_B | (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''' [[Team:Hawaii/Project/Part_A | (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 | ||
| + | '''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) | ||
| + | :* | ||
== Action Items == | == Action Items == | ||
Revision as of 21:19, 12 June 2008
| 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
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)
Action Items
- <Person A>: Task
Coming Up
[http://manoa.hawaii.edu/ 
][http://manoa.hawaii.edu/ovcrge/ 
][http://www.ctahr.hawaii.edu 
]
