BioSketch The bacterial sketch pad. Chris Doucette, Thomas Noriega, Hing Eng Yves Wang, Jennifer Gao, Yin Li Group Meeting 2005-08-01.

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BioSketch The bacterial sketch pad. Jennifer Gao, Yin Li, Chris Doucette, Thomas Noriega, Hing Eng, Yves Wang Group Meeting

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BioSketch The bacterial sketch pad. Chris Doucette, Thomas Noriega, Hing Eng Yves Wang, Jennifer Gao, Yin Li Group Meeting

BioBricks Team

CollinsMod Team

BioSketchHarvard iGEM 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 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 Expected Results of IPTG/Heat Treatment Collins switch was always 37C. The switch on pTS241 is (supposed to be) turned 40C. The switch on pTS265 is (supposed to be) turned 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 Reporters are turned OFF by IPTG/heat StrainsUntreated IPTG treated Heat treated IDGenotypeCellsFluor % Fluor CellsFluor CellsFluor 1A Collins switch + GFP % % 1B Collins switch + mCherry % % 2A 40C switch + GFP % % % 2B 40C switch + mCherry % % % 3A 37C switch + GFP % % % 3B 37C switch + mCherry % % % 5AGFP % % % 5BmCherry % % %

BioSketchHarvard iGEM 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 30C. Results Inconclusive: There was no visible difference between UV-treated and untreated cells.

BioSketchHarvard iGEM A FACS Analyzer to Quantify Results A FACS analyzer will permit rapid and high-resolution quantification of results. Experiment planned for FACS Dana Farber Spoke with the flow cytometry 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 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 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 (-ve) Conditions IPTG treatment (0, 2, 5mM) UV treatment (0, 24, 48J/m2)