Team:Hawaii/Meeting/2008-06-12

From 2008.igem.org

(Difference between revisions)
(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 ...)
(Minutes)
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== Minutes ==
== Minutes ==
-
Present: <Person A>, <Person B>
+
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

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Notebook (t) Meetings (t)

Contents

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

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/ Sponsor_UHM.gif][http://manoa.hawaii.edu/ovcrge/ Sponsor_OVCRGE.gif][http://www.ctahr.hawaii.edu Sponsor_CTAHR.gif]