IGEM:Harvard/2007/Meetings/Week 6

[[Media: shloweek6igem2007.ppt | Stephanie's Powerpoint]] S.Lo's Notes
 * Quorum
 * Switch fluorophores on constructs
 * Actually do dilution/extinction for fluorescence ...
 * Make graphs without normalizing for autofluorescence?
 * Keep fluorescence in one assay, run OD in parallel (unfortunate, but probably more accurate)
 * Make graphs with controls / noninduced
 * Watch RFP carefully ... try to 'bridge' these past experiments ... keep some controls/things constant
 * Alain - be careful about such long Vacufuges ...
 * Fec
 * Use Quorum constructs for positive control (the ~3000 RFU) to see full range
 * In a way, this also calibrates all our experiments
 * Use E0240 (not that sketchy, apparently) for GFP fluorescence next ligation
 * PennState working on pdz and nickel domains
 * MACS
 * AIDA-1 construct: MACS turned out well, about 200 colonies (versus 5 RFP colonies); assay worked well
 * Use AIDA to construct libraries
 * FACS results
 * Still haven't grown that well
 * Compatible with standard biobrick restriction sites
 * Have varied amount of antibody added (except for AIDA - exact protocol, except addition of 10 minutes to incubation, about 5uL of extra antibody each, repeat of second wash step ... first time worked; this protocol is promising then)
 * Sammy
 * Recieved Primers for 2-step PCR
 * Have two products: first part of OmpA, second part: BB cutting site, strep and his at beginning as positive control, or random 10mer or 15mer (haven't gotten the randomers yet)
 * PCR for strep and his = good results
 * Cut first product with Spe1 and second with Xba, ligate
 * Ligation yielded low DNA concentrations this past weekend
 * Instead, have transformation inside protocol to increase DNA product
 * Looked at diversity - 15 and 10mers looked similar, compared through sequencing and BLAST - not much diversity?
 * Mike - the design of primers - accidental match to end of OmpA; two Biobrick sites flanking randomer site ... actually chopping randomer before ligation
 * Need to redesign primers (and more double-checking before ordering of primers)
 * Target?
 * Alain suggests Calmodulin - already known binding sites; works only in presence of calcium; already have beads; well-characterized, structure known - seems like the perfect protein - believes we have antibodies, all that we need except library
 * Alex
 * Used different ligation strategy
 * Every step - addition of ethanol and things ... extractions ... keep volume down and DNA concentrated?
 * Transformation - results from chemically competent cells about same as before (30 colonies per plate)
 * OneStep Electrocompetent cells (from freezer) gave only 2 colonies
 * Arsenic-sensitive promoters
 * Split normal Fec promoter into 2 different FecI (sigma factor)
 * Use directed mutagenesis to knock binding affinity