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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

BioSketchThe bacterial sketch pad.

Chris Doucette, Thomas Noriega, Hing Eng

Yves Wang, Jennifer Gao, Yin Li

Group Meeting

2005-08-01

what has been done collinsmod
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
testing the circuit

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).
expected results of iptg heat treatment
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.
uv treatment should turn on switches
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.
a facs analyzer to quantify results
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.
cloning the double reporter

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.
this week with collinsmod
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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