Team:Hawaii/Meeting/2008-06- 5

From 2008.igem.org

(Difference between revisions)

Latest revision as of 08:13, 6 June 2008

Hawaii/Header}}

Agenda

9am St. John 515

Update progress on experiments performed this week
1. competent cell creation (taken 2 days), and testing results
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/ ][http://manoa.hawaii.edu/ovcrge/ ][http://www.ctahr.hawaii.edu ]

@@ Line 1: / Line 1: @@
-{{Team:Hawaii/Header}}
+:Hawaii/Header}}
 == Agenda ==
@@ Line 12: / Line 12: @@
 == Minutes ==
 Present: GK, KS, MR, SC, GP, KLS, NW, LG <br>
-Presentations:<br>
+<strong>Presentations:</strong><br>
-Grace: lux operon night light
+*Grace: lux operon night light
-: <b>Discussion Points</b>
+::<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''
-::**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.''
-Krystle: bacterial export system, soluble proteins
+:::may need to find a membrane permeable substance that can bind to the lac promoter
-: <b>Discussion Points</b>
+*Krystle: bacterial export system, soluble proteins
-:*it will be best to focus on pilA and slr2016, signal sequences that have already been experimentally confirmed
+:: <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
-Margaret: synthetic plasmid biobricks
+<strong>Additional Comments:</strong>
-: <b>Discussion Points</b>
+:*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 ==
-* &lt;Person A&gt;: Task
+* 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 ==

Team:Hawaii/Meeting/2008-06- 5

From 2008.igem.org

Latest revision as of 08:13, 6 June 2008

Contents

Agenda

Minutes

Action Items

Coming Up