Biosketch the bacterial sketch pad
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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


Biobricks team

BioBricks Team


Collinsmod team

CollinsMod Team


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.


Reporters are turned off by iptg heat

Reporters are turned OFF by IPTG/heat


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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