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Advances in Bacterial BioBrick Assembly and UV-Induced Gene Expression Modulation

This document outlines the progress and challenges encountered by the BioBricks team in 2005 regarding bacterial ligations, focusing on the assembly of various constructs such as RBS-mCherry and RBS-Venus. Successful and unsuccessful ligations are discussed, including analysis via sequencing and microscopy. Additionally, the CollinsMod team's experiments on UV-killing curves and thermo-sensitive circuits are detailed, highlighting expected outcomes like GFP expression modulation under different conditions. The report also addresses experimental designs and planned future experiments involving reporter constructs.

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Advances in Bacterial BioBrick Assembly and UV-Induced Gene Expression Modulation

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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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