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

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 Sensing System into a Repressor

Two Plasmid System LanRFP GFP

Two Plasmid System

Promoter Schematics 1 2 3 4 5

Step 1: Creating The LanRFP Plasmid using CPEC LanRFP Plasmid

Step 2: Add XbaI site to GFP Plasmid Before After

Step 2: Enzymatic Inverse PCR Insertion Normal PCR Inverse PCR

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

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

Step 4: Wobble Extension, Digestion, and Ligation

Step 4: Final Constructs BTP003 BTP004 BTP005 BTP006 BTP007

Step 5: Preparing for Measurements

Final Results

Future Direction  Create a successful promoter with strong -35 and -10 sites  Strong sites in the wildtype promoter

Acknowledgements Thanks to: Mr. Brinn Belyea UCSD Biodynamics Lab  Spencer Raoul Scott  John De Friel  Daniel Jacobsen New England Biolabs Life Technologies

Final Results