"

From 2012.igem.org

Home Team Official Team Profile Project Parts Submitted Modeling Notebook Attributions

Contents 1 Notebook for week of July 23 to July 29 1.1 Design of Converter Plasmid 1.2 Design of Reporter Plasmid 1.3 Spinach Aptamer 1.4 CBB5 Caffeine Inducible Promoters 1.5 Inducible Odor

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.

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