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Team:Hawaii/Meeting/2008-06- 5
From 2008.igem.org
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== 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 | *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 |
| - | :: | + | :::*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 | *Krystle: bacterial export system, soluble proteins | ||
:: <b>Discussion Points</b> | :: <b>Discussion Points</b> | ||
| - | ::*it will be best to | + | ::*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 | |
| - | *Margaret: synthetic plasmid | + | :::possibilities: ''the nir promoter, inducible with nitrate'' or ''the tac promoter, an experimentally used strong promoter'' |
| + | *Margaret: synthetic plasmid Biobricks | ||
:: <b>Discussion Points</b> | :: <b>Discussion Points</b> | ||
| + | ::*pRL1383a may require integration into the bacterial chromosome to ensure retention | ||
| + | |||
| + | <strong>Additional Comments:</strong> | ||
| + | :*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 == | ||
| Line 38: | Line 45: | ||
:* Look into rbcR | :* Look into rbcR | ||
* Krystle | * 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? | :* 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
- Update progress on experiments performed this week
- competent cell creation (taken 2 days), and testing results
- Part B proposals
- Grace
- Krystle
- 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
- Discussion Points
- 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
- Discussion Points
- Margaret: synthetic plasmid Biobricks
- Discussion Points
- pRL1383a may require integration into the bacterial chromosome to ensure retention
- Discussion Points
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 
]
