Endy:Screening plasmid/Notebook: Difference between revisions
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==Approach== | ==Approach== | ||
#Part 1 | #Part 1 | ||
#*Construction of High BG RFP and Hairpin constructs | #*'''Construction of High BG-promoter RFP and Hairpin constructs(completed)''' | ||
#*Characterization of Inducible GFP (spec out exps on plate reader / LH-FACS) | #*Characterization of Inducible GFP (spec out exps on plate reader / LH-FACS) | ||
#Part 2 | #Part 2 | ||
#*Characterization of Inducible RFP (same as inducible GFP exps) | #*Characterization of Inducible RFP (same as inducible GFP exps) | ||
#*High BG RFP and Hairpin experiments | #*'''High BG RFP-promoter and Hairpin experiments (completed)''' (Results: [[Endy:Screening plasmid/v1.0/High BG RFP|HighBG-promoter]],[[Endy:Screening plasmid/v1.0/Hairpin|Hairpin]]) | ||
#*Based on results from (2) test inducible RFP (<bbpart>I13520</bbpart>) at low and high induction on MOFLO | |||
#Part 3 | #Part 3 | ||
#*Rebuild ESP if necessary based on High BG RFP/Hairpin | #*Rebuild ESP if necessary based on High BG RFP/Hairpin | ||
#*Test ESP (same as inducible GFP exps) | #*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== | ==Current projects== | ||
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===Characterization of Inducible RFP=== | ===Characterization of Inducible RFP=== | ||
<bbpart>I13520</bbpart> | |||
===Characterization of Empty Screening Plasmid 1.0=== | ===Characterization of Empty Screening Plasmid 1.0=== | ||
Latest revision as of 11:46, 18 September 2007
Approach
- Part 1
- Construction of High BG-promoter RFP and Hairpin constructs(completed)
- Characterization of Inducible GFP (spec out exps on plate reader / LH-FACS)
- 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
- 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 GFP
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).
