Cling-E.coli: Quorum Sensing George, Perry, and Stephanie Presented by Stephanie 16 July 2007

Overview

OHHL

Some new constructs in use S03608 S03623 I13507 F2620-E0240

Colony PCRs B0015 F20-E40 F20-I07 F2620 F2621 F622 I13273 I5311 I6042 J06702 J-T S08-B S08-E40 S08-I07 S23-B S23-E0240 S23-I07 T02 J37015 I15030 Worked Did not work

Correct Sequencing Results F2620-E0240 I13273 J23039-T9002 S03608-I13507 S03623-E0240 S03623

Flow Cytometry: Overview Constitutive GFPConstitutive YFP 45.5% 26.5% 97.3% 99.9%

Flow Cytometry Results T02 noninduced v. induced with 100nM OHHL 83.5% Leaky fluorescence 99.0% 56.5% Nearly all cells expressed GFP as expected Slight read from RFP filter noninducedinduced

Flow Cytometry Results As expected, nearly all cells fluoresced in GFP Nearly identical results (counts, percent fluorescence) as T9002 induced with OHHL J39-T %

What we’d like to see J T9002

J-T alone J-T induced with 100nM OHHL

J-T OD and Fluorescence OD Fluorescence

Constitutive GFP: I13522 OD Fluorescence

Improvement: Tet-controllable luxI and luxR P0140 P0340 J23039-T9002 Constitutive promoters to create tetracycline, which inhibits J-T Induce expression with anhydrotetracycline

Pickup of YFP by RFP filter I73 I73 noninduced and induced with 100nM OHHL Flow Cytometry Results 99.2% 68.4% 99.0% Leaky fluorescence noninducedinduced

Flow Cytometry Results About 32.5% of cells were “senders” and 67.5% were “receivers”. GFP fluorescence was about 2/3 total amount compared to I73 induced with OHHL Overlap of YFP and RFP reading S08-I07I73 Combination of S08-I07 and I % 67.5%

Plate Reader Results: Combination of S08-I07 and I73/FE Tested different sender and receiver ratios for peak fluorescence readings Suggestion that slightly more (2x-4x) sender versus receiver is optimal S08-I07 I73 FE

Another Concern : luxI expression / RFP OD and fluorescence readings of saturated liquid cultures indicates weak RFP expression from some of our constructs J04450 (constitutive): 1.695, S23I07: 1.807, S08I07: 1.690, J04450

Improvements Tet-controllable luxI for J-T construct RFP as a population tracker Constitutive positive RFP at flow cytometry appointments Continue to explore optimal ratio of luxI versus luxR constructs Plate reader: preventing dry-out over long assays