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BioSketch The bacterial sketch pad. Jennifer Gao, Yin Li, Chris Doucette, Thomas Noriega, Hing Eng, Yves Wang Group Meeting 2005-07-18.

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Presentation on theme: "BioSketch The bacterial sketch pad. Jennifer Gao, Yin Li, Chris Doucette, Thomas Noriega, Hing Eng, Yves Wang Group Meeting 2005-07-18."— Presentation transcript:

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

2 BioBricks Team

3 BioSketchHarvard iGEM Ligations Pairwise Assembly Successful Ligations P + QPI LacIts(241) P + QPI LacIts(265) P + RBS-mCherry-T P lacI + RBS-mCherry-T P lacI-Hyb + RBS-mCherry-T P RBS-mCherry-T P lacI-Hyb + RBS-Venus-T P + RBS-Venus-T P lacI-Hyb +RBS- cI Unsuccessful Ligations P LacI  QPI P  QPI  QPI  RBS-mCherry-T QPI 434 +RBS-mCherry-T P lacI(hyb) + RBS-lambda P + RBS-434 P + QPI LacI P lacI + RBS-Venus-T P RBS-Venus-T

4 BioSketchHarvard iGEM Lambda cI mutations Made two variants of temperature sensitive Lambda cI, they will be sequenced this week and incorporated into the ligation schedule.

5 BioSketchHarvard iGEM This week with Biobricks Pairwise Assembly Terminator + P 434 -mCherry Terminator + P 434 -Venus Terminator + P -mCherry Terminator + P -Venus Repeat all unsuccessful ligations from last week

6 CollinsMod Team

7 BioSketchHarvard iGEM What has been done: CollinsMod Cloning mCherry and GFP reporter constructs, as well as the empty vector pTV Testing the Collins Circuit Replicating the Kobayashi work exactly

8 BioSketchHarvard iGEM Cloning CI-Repressed mCherry (pWCh) Bacteria-optimized mCherry cloned successfully, verified by: Analytical digests Cells spun down are visibly pink/red/violet Cells (on plate & in-solution) fluorescece when excited with ~600nm (bleaches very quickly) Sequences pending Transformants that were streaked out were not visibly "cherry" until at least two days later, and even then not as darkly colored as expected/desired. mCherry PL*PL*PL*PL* pWCh

9 BioSketchHarvard iGEM Other Cloning Experiments Cloning pTV, the backbone of the toggle-switch vector lacking lacI or cI Analytical digests indicate correct fragments Co-transformation with the reporter constructors will indicate whether the presence of the vector affects fluorescence Cloning of pEG, the LacI-repressed GFP reporter Analytical digests indicate correct fragments gfpmut3bpEG P trc

10 BioSketchHarvard iGEM Testing the Collins Circuit Parental strain MC4100 (lacI-; from The Registry) Introduced constructs cI-repressed GFP reporter (pWG), alone reporter (pWG) + toggle-switch (pTS) Test conditions 0mM IPTG 0, 12, 24, 48, 96, 192, 284J/m 2 UV Assay time next day gfpmut3b PL*PL*PL*PL* pWG pTS cI cI P trc PL*PL*PL*PL*lacI

11 BioSketchHarvard iGEM J/m 2 UV kills most cells

12 BioSketchHarvard iGEM Circuit Response to UV (I) reporter-only (pWG) reporter (pWG) + toggle switch (pTS) 0 J/m2 12 J/m224 J/m248 J/m2 96 J/m2192 J/m2384 J/m2 on plate MC4100 background 10x objective FITC

13 BioSketchHarvard iGEM Consitutive GFP Expression Low 0 J/m2 12 J/m2 24 J/m2 48 J/m2 192 J/m2 96 J/m2 384 J/m2 in solution pWG in MC x, oil-immersion FITC & DIA-DLL

14 BioSketchHarvard iGEM Circuit Response to UV (I) 0 J/m2 12 J/m2 24 J/m2 48 J/m2 192 J/m2 96 J/m2 384 J/m2 in solution pWG+pTS in MC x, oil-immersion FITC & DIA-DLL

15 BioSketchHarvard iGEM Replicating Kobayashi et al.'s Work Exactly Strain: JM Used by Kobayashi et al. (2004) Has been made competent Introduced Constructs CI-repressed GFP reporter (pWG)-only CI-repressed mCherry reporter (pWCh)-only GFP reporter (pWG) + toggle switch (pTS) mCherry reporter (pWCh) + toggle switch (pTS) GFP repoter (pWG) + empty vector (pTV) mCherry repoter (pWCh) + empty vector (pTV) Test Conditions 0, 2mM IPTG 0, 6, 12, 24, 48 (96, 192, 384?) J/m2 UV Assay Conditions 4h and 16h after UV irradiation Cells will also be examined before and after the addition of IPTG gfpmut3b PL*PL*PL*PL* pWG pTS cI cI P trc PL*PL*PL*PL*lacI mCherry PL*PL*PL*PL* pWCh

16 BioSketchHarvard iGEM CollinsMod: A Summary Cloning experiments (success!) mCherry reporter, regulated by CI (pWCh) GFP reporter, regulated by LacI (pEG) empty toggle-switch vector (pTV) Testing the Collins circuit UV kills readily at 384J/m2, and even at 192J/m2. On-plate assay indicates upregulation of GFP expression following UV treatment Not clearly corroborated by in-solution, single-cell examination No IPTG was used Constitutive GFP expression much lower than expected Replicating Kobayashi's work exactly On our way!


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