From 2012.igem.org

Notebook for week of July 23 to July 29

Design of Converter Plasmid

• Razan
  • 7/24- Amplified pACYC backbone successfully with new primers for both control and converter plasmids.
  • 7/26- Performed overlap PCR of the 2 inserts (CRE and LasR for converter and GFP and LasR for control).
  • 7/27- Performed gibson assembly on the overlap PCR of the inserts and the pACYC backbone amplifications for both pconverter and control converter with successful transformation!

Design of Reporter Plasmid

• Ben
  • Prepped pReporter and pReporter.permaflipped DNA.
  • PCR:
    • pReporter: JE.rrnB.F, JE.GFPTerm.R
    • pReporter.permaflipped: JE.rrnB.F, JE.GFPTerm.R
    • Results: One correct-size band for pReporter.permaflipped; none for pReporter. Submitted for sequencing. Made frozen stock. Will retry colony PCR for pReporter.
  • Colony PCR: (retrying with Phusion + DMSO instead of Taq MM because I noticed JE.rrnB.F has poor secondary structure--probably why no products have shown up in these colony PCRs)
    • pReporter: JE.rrnB.F, JE.GFPTerm.R
    • Results: Correct-size band. Made frozen stock, prepped, and submitted for sequencing.

Spinach Aptamer

• Logan
  • 7/23 - Grew cultures of 5' and 3' mCherry-SpA constructs, SpA, and mCherry alone for plate reader exp. Submitted 3' construct for sequencing.
  • 7/24 - Attempted Cuvette exp in plate reader: added 25uM DFHBI to solution, induced constructs in BL21 AI cells with 1 mM IPTG, 0.2% arabinose. Result: No fluorescence detected. (bad plate reader?)

CBB5 Caffeine Inducible Promoters

• Peter
  • 7/23- Cotransformation of PRA301-ndmApro LacZ reporter plasmid (with spectinomycin reporter) and ORF1 -4,5,6 plasmid minipreps. Ran out of PRA301-ndmApro plasmid, so only ORF1-4,5 were cotransformed. ORF1- 6 was transformed by itself as a control. Plated on kanamycin/XGal plates.
  • 7/24- Cotransformation failed. Likely due to too much plasmid, mixed with poor transformation.

Inducible Odor