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BioSketch The bacterial sketch pad.

BioSketch The bacterial sketch pad. Chris Doucette, Thomas Noriega, Hing Eng Yves Wang, Jennifer Gao, Yin Li Group Meeting 2005-08-01. BioBricks Team. CollinsMod Team. What has been done: CollinsMod. Testing the circuits Collins switch & thermo-sensitive circuits

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BioSketch The bacterial sketch pad.

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

  2. BioBricks Team

  3. CollinsMod Team

  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

  5. UV PL* gfpmut3b l cI Ptrc PL* lacIts Heat Testing the Circuit • The (Final) Circuit: • Treatments: • IPTG or heat treatment (expected to reduce reporter expression). • UV treatment (expected to increase reporter expression).

  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.

  7. Reporters are turned OFF by IPTG/heat

  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.

  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 107 cells/ml • Filter: 0.22um Millipore Millex-GV membrane • Tentative appointment made for Aug 9.

  10. pTSGC gfpmut3b Ptrc PL* mCherry 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.

  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)

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