Team:Hawaii/Meeting/2008-06- 5


 * Hawaii/Header}}

Agenda
9am St. John 515
 * 1) Update progress on experiments performed this week
 * 2) competent cell creation (taken 2 days), and testing results
 * 3) Part B proposals
 * 4) Grace
 * 5) Krystle
 * 6) 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? 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