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BioSketch The bacterial sketch pad.

BioSketch The bacterial sketch pad. Yin Li, Chris Doucette, Thomas Noriega, Hing Eng, Yves Wang, Jennifer Gao Group Meeting 2005-07-25. BioBricks Team. Ligations. Pairwise Assembly Successful Ligations QPI l  RBS-mCherry-T QPI 434 +RBS-mCherry-T RBS + l cI 857 P 434 + RBS-Venus-T

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BioSketch The bacterial sketch pad.

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  1. BioSketchThe bacterial sketch pad. Yin Li, Chris Doucette, Thomas Noriega, Hing Eng, Yves Wang, Jennifer Gao Group Meeting 2005-07-25

  2. BioBricks Team

  3. Ligations • Pairwise Assembly • Successful Ligations • QPIlRBS-mCherry-T • QPI434+RBS-mCherry-T • RBS + l cI 857 • P434 + RBS-Venus-T • Pl- QPI LacIts(265) + RBS-mCherry-T • Unsuccessful Ligations • Pl-QPI LacIts(241) + RBS-mCherry-T • PlQPI4 • RBS + l cI JP • PlacI + RBS-Venus-T

  4. Lambda cI mutations • Lambda cI 857 (canonical ts mutant) and cI JP (Japanese patent) verified by sequencing and HindIII digestion of mutated site

  5. Bacterial mCherry BioBrick • mCherry and mCherry-LVA verified by sequencing • Ready to be sent back to Registry • Construction of full reporter constructs (RBS-mCherry-T) begun

  6. Microscopy w/ mCherry • Examined RBS-mCherry-T and RBS-Venus-T behind PlacI, Plh, P434, Pl • No fluorescence • Fragment sizes of mCherry, Venus inserts larger than expected, so we suspect a problem with these ligations.

  7. Sequencing mCherry/Venus • Plh-RBS-mCherry-T and Plh-RBS-Venus-T were sequenced with primers flanking the insert region of Biobrick vector pSB1A2 • Sequences are identical and do not align with mCherry or Venus • BLAST identifies sequence as part of various bacterial vectors, but the sequence does not align with pSB1A2 • All intermediate parts including original Biobrick should be sequenced to determine where this random vector fragment was substituted for the real inserts

  8. This week with Biobricks • Pairwise Assembly • RBS + mCherry (bacterial) • RBS + mCherry-LVA (bacterial) • Pl-QPI LacIts(241) + QPIl-RBS-mCherry-T • Pl-QPI LacIts(265) + QPIl-RBS-mCherry-T • The complete circuit! (but uncertain about mCherry) • RBS-l cI 857 + T • Repeat failed ligations • Microscopy

  9. CollinsMod Team

  10. What has been done: CollinsMod • Establishing the UV killing curve. • Replicating the work in Kobayashi et al. • Testing the thermo-sensitive circuits.

  11. The UV Killing Curve(s)

  12. UV PL* gfpmut3b l cI Ptrc PL* lacIts Heat Testing the Circuit • The (Final) Circuit: • Expected Results: • IPTG or heat treatment should reduce GFP expression. • UV treatment should should increase GFP expression.

  13. PL* gfpmut3b l cI l cI Ptrc Ptrc PL* PL* lacI lacI(ts)241 pWG pTS pTS241 The Experiments • Background strain • JM 2.300 used by Kobayashi et al. • Experimental strains • 40C TS switch (pTS241) + GFP reporter (pWG) in JM 2.300 • Collins switch (pTS) + GFP reporter (pWG) in JM 2.300 • GFP reporter (pWG) in JM 2.300 (ctrl) • JM 2.300 (ctrl) • Planned but not done • 37C TS switch (pTS265) • mCherry reporter (pWCh) • Conditions • 0, 2mM IPTG • 0, 6, 12, 24, 48J/m2 UV • 30, 37, 40C

  14. A Typical Experiment • Toggle-switch UV- and thermo-sensitive toggle-switch (pTS241) + CI-repressed GFP reporter in JM 2.300

  15. PL* gfpmut3b l cI l cI Ptrc Ptrc PL* PL* lacI lacI(ts)241 pWG pTS pTS241 Expected IPTG/Heat Treatment Results Note: The Collins switch (pTS + pWG) was tested at 37C.

  16. Collins switch Reporter JM 2.300 TS switch Observed IPTG Treatment Results 0mM IPTG 30/37C 2mM IPTG 30/37C

  17. Reporter JM 2.300 TS switch Observed Heat Treatment Results 0mM IPTG 30C 0mM IPTG 40C

  18. UV-induction of GFP Expression Is Modest Note: Data for Reporter @ 12J/m2 was not analyzed due to poor image quality.

  19. pTSCG gfpmut3b PL* Ptrc mCherry This week with CollinsMod • Examine if there are differences with the mCherry reporter. • Consider cloning the pTSCG reporter • GFP is repressed by CI • mCherry is repressed by LacI • IPTG/heat treatment: GFP-/mCherry+ • UV treatment: GFP+/mCherry- • Use BioBrick parts? • Assaying GFP expression with a FACS analyzer • Ira helped to signup a machine for use on 2005-08-03 1-2pm @ the Dana-Farber. • High-resolution, quantitative profile of GFP expression. • A "FACSCaliber flow cytometer" was used in Kobayashi et al.

  20. And, introducing …

  21. The UV Pen! • FlashUV2 from MaxMax.com • Ultra-High Brightness UV LED pen • 375nm • 10 Degree Lens • 2,000uW • Questions • Will it induce SOS response? • How reliable is it?

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