Endy:Screening plasmid/Notebook

Approach

1. Part 1
   • Construction of High BG-promoter RFP and Hairpin constructs(completed)
   • Characterization of Inducible GFP (spec out exps on plate reader / LH-FACS)
2. Part 2
   • Characterization of Inducible RFP (same as inducible GFP exps)
   • High BG RFP-promoter and Hairpin experiments (completed) (Results: HighBG-promoter,Hairpin)
   • Based on results from (2) test inducible RFP (<bbpart>I13520</bbpart>) at low and high induction on MOFLO
3. Part 3
   • Rebuild ESP if necessary based on High BG RFP/Hairpin
   • Test ESP (same as inducible GFP exps)
     • Test the flipped FP constructs, cassie's constructs, and also the other version of the ESP where we cut out ccdb and re-ligated.

Current projects

High BG RFP

• Endy:Screening plasmid/v1.0/High BG RFP - Figure out why we are seeing a high BG level of RFP in the empty SP

Hairpin effectiveness

• Endy:Screening plasmid/v1.0/Hairpin - characterize effectiveness of the hairpin to stabilize the mRFP1 transcript

Characterization of Inducible RFP

<bbpart>I13520</bbpart>

Characterization of Empty Screening Plasmid 1.0

• Endy:Screening plasmid/v1.0/RNAseE sites - characterize effectiveness of the RNAseE sites
• Endy:Screening plasmid/v1.0/Growth rates - characterization of growth rates under different induction levels / conditions
• Endy:Screening plasmid/v1.0/Steady state - evaluate expression levels at various time points following induction.
• Endy:Screening plasmid/v1.0/Characterization protocol

To ensure our screening system was operating as expected we measured the expression levels of GFP and mRFP1 when the plasmid did not contain a part (e.g. input equals output).

Glycerol Boxes

• Endy:Screening plasmid/v1.0/Glycerol box
• Endy:Screening plasmid/v2.0/Glycerol box