Team:Hawaii/Meeting/2008-06- 5

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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)

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

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? 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

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

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.

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

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”