Team:Hawaii/Meeting/2008-06- 5

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{{Team:Hawaii/Header}}
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:Hawaii/Header}}
== Agenda ==
== Agenda ==
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== Minutes ==
== Minutes ==
Present: GK, KS, MR, SC, GP, KLS, NW, LG <br>
Present: GK, KS, MR, SC, GP, KLS, NW, LG <br>
-
Presentations:<br>
+
<strong>Presentations:</strong><br>
-
Grace: lux operon night light
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*Grace: lux operon night light
-
: <b>Discussion Points</b>
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::<b>Discussion Points</b>
-
:*rbc may be too strong a promoter for use in the proposed construct
+
::*rbc may be too strong a promoter for use in the proposed construct
-
:*test the strength of RBC and explore if it is leaky by creating a lacZ/GFP fusion protein
+
::*test the strength of RBC and explore if it is leaky by creating a lacZ/GFP fusion protein
-
::*use the fusion protein plasmid from SC, use amplifier to get the RBCL promoter, cut out the fusion site and put into pCC1383. -> 'It can be made into a biobrick if it works.'
+
::*use the fusion protein plasmid from SC, use amplifier to get the RBCL promoter, cut out the fusion site and put into pCC1383.  
-
:*expression of lux as controlled by lac may not be enough without available lactose
+
::::-> ''It can be made into a biobrick if it works''
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::**question: Is lactose membrane permeable for S. PCC6803?  [http://cyano.genome.jp/cgi-bin/cyorf_view.cgi?ORG=syn&ACCESSION=slr1202 YES] PCC6803 encodes two lactose permease proteins, lacF and lacG.
+
::*expression of lux as controlled by lac may not be enough without available lactose
-
::**may need to find a membrane permeable substance that can bind to the lac promoter
+
:::*question: Is lactose membrane permeable for S. PCC6803?  [http://cyano.genome.jp/cgi-bin/cyorf_view.cgi?ORG=syn&ACCESSION=slr1202 YES] ''PCC6803 encodes two lactose permease proteins, lacF and lacG.''
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Krystle: bacterial export system, soluble proteins  
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:::may need to find a membrane permeable substance that can bind to the lac promoter
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: <b>Discussion Points</b>
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*Krystle: bacterial export system, soluble proteins  
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:*it will be best to focus on pilA and slr2016, signal sequences that have already been experimentally confirmed
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:: <b>Discussion Points</b>
 +
::*it will be best to start with pilA and slr2016, signal sequences experimentally proven to cause lichenase secretion
 +
::*an appropriate promoter needs to be found
 +
:::possibilities: ''the nir promoter, inducible with nitrate'' or ''the tac promoter, an experimentally used strong promoter''
 +
*Margaret: synthetic plasmid Biobricks
 +
:: <b>Discussion Points</b>
 +
::*pRL1383a may require integration into the bacterial chromosome to ensure retention
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Margaret: synthetic plasmid biobricks
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<strong>Additional Comments:</strong>
-
: <b>Discussion Points</b>
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:*Dr. Callahan stated it may be helpful to use Accuzyme from bioline as our polymerase.  The likelihood of mistakes has been almost zero as tested in his lab.
 +
:*Plate a high concentration of E.coli and Synechocystis on BG-11 + 5% LB agar with no antibiotics.  Let it sit there for two days in the light.  Then, scrape the plate and transfer onto BG-11 only agar with spectinomycin and streptomycin. In a week or two, there should be colonies of  Synechocystis.  Take colonies off the selection plate and streak onto another spec/strep plate.  It will take a week or two to see if you have colonies initially.
== Action Items ==
== Action Items ==
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:* Look into rbcR
:* Look into rbcR
* Krystle
* Krystle
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* Margaret:
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:*Get lichenase sequence
 +
:*Pick a strong promoter
 +
:: look into the nir promotor and tac promoter
 +
:*Work on getting the signal sequence biobrick to ligate in frame with desired protein sequences.
 +
:*Learn about fusion proteins and getting things “in frame”
 +
 
 +
* Margaret
:* Does RSF1010 replicate autonomously in PCC6803?
:* Does RSF1010 replicate autonomously in PCC6803?
* All:
* All:

Latest revision as of 08:13, 6 June 2008

Hawaii/Header}}

Contents

Agenda

9am St. John 515

  1. Update progress on experiments performed this week
    1. competent cell creation (taken 2 days), and testing results
  2. Part B proposals
    1. Grace
    2. Krystle
    3. Margaret (teleconference in from US mainland)

Minutes

Present: GK, KS, MR, SC, GP, KLS, NW, LG
Presentations:

  • Grace: lux operon night light
Discussion Points
  • rbc may be too strong a promoter for use in the proposed construct
  • test the strength of RBC and explore if it is leaky by creating a lacZ/GFP fusion protein
  • use the fusion protein plasmid from SC, use amplifier to get the RBCL promoter, cut out the fusion site and put into pCC1383.
-> It can be made into a biobrick if it works
  • expression of lux as controlled by lac may not be enough without available lactose
  • question: Is lactose membrane permeable for S. PCC6803? [http://cyano.genome.jp/cgi-bin/cyorf_view.cgi?ORG=syn&ACCESSION=slr1202 YES] PCC6803 encodes two lactose permease proteins, lacF and lacG.
may need to find a membrane permeable substance that can bind to the lac promoter
  • Krystle: bacterial export system, soluble proteins
Discussion Points
  • it will be best to start with pilA and slr2016, signal sequences experimentally proven to cause lichenase secretion
  • an appropriate promoter needs to be found
possibilities: the nir promoter, inducible with nitrate or the tac promoter, an experimentally used strong promoter
  • Margaret: synthetic plasmid Biobricks
Discussion Points
  • pRL1383a may require integration into the bacterial chromosome to ensure retention

Additional Comments:

  • Dr. Callahan stated it may be helpful to use Accuzyme from bioline as our polymerase. The likelihood of mistakes has been almost zero as tested in his lab.
  • Plate a high concentration of E.coli and Synechocystis on BG-11 + 5% LB agar with no antibiotics. Let it sit there for two days in the light. Then, scrape the plate and transfer onto BG-11 only agar with spectinomycin and streptomycin. In a week or two, there should be colonies of Synechocystis. Take colonies off the selection plate and streak onto another spec/strep plate. It will take a week or two to see if you have colonies initially.

Action Items

  • Grace
  • Align PCC6803 rbc promoter with other PCC6803 promoters and other rbc promoters to confirm -10 and -35 consensus sequences
  • What metabolites are needed for luxCDABE expression?
  • What transcription factors/repressors/activators are known to interact with rbc promoter?
  • Can PCC6803 take up IPTG?
  • What is the lifespan of lacI in vivo without degradation signal? With?
  • Look into rbcR
  • Krystle
  • Get lichenase sequence
  • Pick a strong promoter
look into the nir promotor and tac promoter
  • Work on getting the signal sequence biobrick to ligate in frame with desired protein sequences.
  • Learn about fusion proteins and getting things “in frame”
  • Margaret
  • Does RSF1010 replicate autonomously in PCC6803?
  • All:
  • Primer and BioBrick orders should be compiled by 6/12

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]

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