Team:Hawaii/Meeting/2008-06-12

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Latest revision as of 21:46, 12 June 2008

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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?)

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

Coming Up

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

@@ Line 10: / Line 10: @@
 == Minutes ==
-Present: &lt;Person A&gt;, &lt;Person B&gt;
+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 &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''' [[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
+:* 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 ==
-* &lt;Person A&gt;: Task
+* 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 CO<sub>2</sub> incubator ASAP
 == Coming Up ==