BioSketchHarvard iGEM 2005 4 What has been done: CollinsMod Testing the circuits Collins switch & thermo-sensitive circuits GFP and mCherry as reporters Using a FACS analyzer Cloning experiments PCR cloning of double-reporter
BioSketchHarvard iGEM 2005 5 Testing the Circuit The (Final) Circuit: Treatments: IPTG or heat treatment (expected to reduce reporter expression). UV treatment (expected to increase reporter expression). PL*PL*PL*PL*lacIts cI cI P trc gfpmut3b PL*PL*PL*PL*UVHeat
BioSketchHarvard iGEM 2005 6 Expected Results of IPTG/Heat Treatment Collins switch was always tested @ 37C. The switch on pTS241 is (supposed to be) turned off @ 40C. The switch on pTS265 is (supposed to be) turned off @ 37C. Bacterial mCherry and GFP were both used as reporters. Strain 0mM IPTG 30C* 2mM IPTG 30C* 0mM IPTG 40C/37C Collins switch (pTS) + reporter +/-- 40C thermo-sensitive switch (pTS241) + reporter +/--- 37C thermo-sensitive switch (pTS265) + reporter +/--- GFP reporter (pWG) +++ mCherry reporter (pWCh) +++
BioSketchHarvard iGEM 2005 8 UV Treatment Should Turn ON Switches UV intensities used 0, 6, 12, 24, 48J/m2 Plated- and UV-treated cells were allowed to grow to confluence, which took two nights @ 30C. Results Inconclusive: There was no visible difference between UV-treated and untreated cells.
BioSketchHarvard iGEM 2005 9 A FACS Analyzer to Quantify Results A FACS analyzer will permit rapid and high-resolution quantification of results. Experiment planned for FACS analyzer @ Dana Farber Spoke with the flow cytometry expert @ the Bauer Center ~12 samples for pilot experiment Fluorophore: GFP Density: ~1 x 10 7 cells/ml Filter: 0.22um Millipore Millex-GV membrane Tentative appointment made for Aug 9.
BioSketchHarvard iGEM 2005 10 Cloning the Double-Reporter pTSGC GFP is repressed by LacI (and turned ON by IPTG/heat) mCherry is repressed by CI (and turned ON by UV) Sequencing for P(trc)-GFP plasmid is pending. PCR has been successful in preparing P(L*)-mCherry for insertion into the already-made P(trc)-GFP plasmid. pTSGC gfpmut3b P trc mCherry PL*PL*PL*PL*
BioSketchHarvard iGEM 2005 11 This week with CollinsMod Assaying GFP expression with a FACS analyzer Current plan: Stagger several experiments so that the following parameters can be examined in parallel: IPTG treatment (16h and 2 days later) Heat treatment (16h and 2 days later) UV treatment (4h and 16h later) Alternatively: Scale down the parameters for pilot experiment with FACS analyzer Genotypes Collins switch + GFP GFP (+ve) JM 2.300 (-ve) Conditions IPTG treatment (0, 2, 5mM) UV treatment (0, 24, 48J/m2)