ENGINEERING A REPRESSIBLE PROMOTER FROM THE LASR QUORUM Torrey - - PowerPoint PPT Presentation

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Nov 21, 2023 127 likes •326 views

ENGINEERING A REPRESSIBLE PROMOTER FROM THE LASR QUORUM Torrey Pines High School SENSING SYSTEM FOUND IN iGEM 2013 PSEUDOMONAS AERUGINOSA Wild-type LasR Quorum Sensing System in Pseudomonas aeruginosa The Idea Turning the LasR Quorum

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Torrey Pines High School iGEM 2013

ENGINEERING A REPRESSIBLE PROMOTER FROM THE LASR QUORUM SENSING SYSTEM FOUND IN PSEUDOMONAS AERUGINOSA

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Wild-type LasR Quorum Sensing System in Pseudomonas aeruginosa

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The Idea

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Turning the LasR Quorum Sensing System into a Repressor

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Two Plasmid System

LanRFP GFP

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Two Plasmid System

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Promoter Schematics

1 2 3 4 5

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Step 1: Creating The LanRFP Plasmid using CPEC

LanRFP Plasmid

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Before After

Step 2: Add XbaI site to GFP Plasmid

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Normal PCR Inverse PCR

Step 2: Enzymatic Inverse PCR Insertion

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Step 2: Adding the constitutive promoter and LasR gene to our GFP plasmid using CPEC

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Step 3: Creating promoters with overlap extension PCR (Wobble PCR)

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Step 4: Wobble Extension, Digestion, and Ligation

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Step 4: Final Constructs

BTP003 BTP004 BTP005 BTP006 BTP007

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Step 5: Preparing for Measurements

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Final Results

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 Create a successful promoter with strong -35 and -10 sites

Strong sites in the wildtype promoter

Future Direction

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Thanks to:

Mr. Brinn Belyea

UCSD Biodynamics Lab  Spencer Raoul Scott  John De Friel  Daniel Jacobsen New England Biolabs Life Technologies

Acknowledgements

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