1 / 16

BioSketch The bacterial sketch pad.

BioSketch The bacterial sketch pad. Jennifer Gao, Yin Li, Chris Doucette, Thomas Noriega, Hing Eng, Yves Wang Group Meeting 2005-07-18. BioBricks Team. Ligations. Pairwise Assembly Successful Ligations P l + QPI LacIts(241) P l + QPI LacIts(265) P l + RBS-mCherry-T

ananda
Download Presentation

BioSketch The bacterial sketch pad.

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

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

  2. BioBricks Team

  3. Ligations • Pairwise Assembly • Successful Ligations • Pl+ QPI LacIts(241) • Pl+ QPI LacIts(265) • Pl + RBS-mCherry-T • PlacI + RBS-mCherry-T • PlacI-Hyb + RBS-mCherry-T • P434 + RBS-mCherry-T • PlacI-Hyb + RBS-Venus-T • Pl + RBS-Venus-T • PlacI-Hyb +RBS-lcI • Unsuccessful Ligations • PLacIQPIl • PlQPI • QPIlRBS-mCherry-T • QPI434+RBS-mCherry-T • PlacI(hyb) + RBS-lambda • Pl + RBS-434 • Pl + QPILacI • PlacI + RBS-Venus-T • P434 + RBS-Venus-T

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

  5. This week with Biobricks • Pairwise Assembly • Terminator + P434-mCherry • Terminator + P434-Venus • Terminator + Pl-mCherry • Terminator + Pl-Venus • Repeat all unsuccessful ligations from last week

  6. CollinsMod Team

  7. 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. PL* mCherry pWCh 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.

  9. Ptrc gfpmut3b pEG 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

  10. pWG PL* gfpmut3b pTS Ptrc l cI lacI PL* 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/m2 UV • Assay time • next day

  11. 384J/m2 UV kills most cells

  12. Circuit Response to UV (I) 0 J/m2 reporter-only (pWG) 0 J/m2 12 J/m2 24 J/m2 48 J/m2 on plate MC4100 background 10x objective FITC 96 J/m2 192 J/m2 384 J/m2 reporter (pWG) + toggle switch (pTS)

  13. 12 J/m2 24 J/m2 48 J/m2 0 J/m2 96 J/m2 192 J/m2 384 J/m2 Consitutive GFP Expression Low in solution pWG in MC4100 100x, oil-immersion FITC & DIA-DLL

  14. 12 J/m2 24 J/m2 48 J/m2 0 J/m2 96 J/m2 192 J/m2 384 J/m2 Circuit Response to UV (I) in solution pWG+pTS in MC4100 100x, oil-immersion FITC & DIA-DLL

  15. pWG PL* mCherry pWCh PL* gfpmut3b pTS Ptrc l cI lacI PL* Replicating Kobayashi et al.'s Work Exactly • Strain: JM 2.300 • 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

  16. 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!

More Related