Quorum Quenching Cu-Boulder iGEM 2012

Sean Kalra, Jake Sheppard, Max Jacobs Aubrey Jackson, and Simon Greenberg

Presentation Overview 1.  Background of quorum sensing mechanism

2.  Describe the model system used

3.  Discuss our quorum stopping project

4.  Our constructs

5.  Explore future applications

Achievability vs. Coolness

What is Quorum?

Biofilms Require Quorum Sensing

Where is biofilm found? On your teeth, in your shower, on marine drilling

equipment, hot springs...almost anywhere

Typhoid requires quorum sensing

•  Besides biofilm, pathogenic bacteria rely on quorum sensing

•  Up to 70% of infection causing bacteria have some sort of antibiotic resistance

•  example: Salmonella Typhimurium

Quorum Sensing

AHL

Presentation Overview 1.  Background of quorum sensing mechanism

2.  Describe the model system used

3.  Discuss our quorum stopping project

4.  Our constructs

5.  Explore future applications

Characteristics of a good model system

• Safety concerns with pathogenic bacteria

• Biofilms are hard to work with

• Biofilms and pathogenic factors are difficult to quantify

• Need an accurate and consistent baseline

Aliivibrio fischeri quorum sensing system

•  Activation of Lux operon in response to AHLs

•  490nm luminescent output is easily quantifiable

•  Light output served as a surrogate for pathogenic factor activation

Lux operon •  LuxABCDEG are responsible for

luminescent output

•  LuxR/LuxI mediate density dependent control of the lux operon

luxG luxB luxC luxA luxE luxD luxI

luxR

luxR

constitutive Luminescent output

Quorum sensing

Aliivibrio fischeri OD vs luminescence

Presentation Overview 1.  Background of quorum sensing mechanism

2.  Describe the model system used

3.  Discuss our quorum stopping project

4.  Our constructs

5.  Explore future applications

Inhibiting Quorum Sensing

Now we have a good model for quorum activation, but how do we inhibit it?

AHLs

Quorum Quenching A two part system to interfere with quorum

sensing.

Transcription Factor

High Const.

Reporter Inhibitor

Promoter

Detector Responder

uxR

LuxR homologs are more sensitive to AHLs than LuxR

Collins, C. H., Arnold, F. H. and Leadbetter, J. R. (2005), Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones. Molecular Microbiology, 55: 712–723. doi: 10.1111/j.1365-2958.2004.04437.x

LuxR C6 LuxR C8

After we've detected the AHLs how do we inhibit them?

Cleave Ring

Cleave amide

Competitive Inhibitor

Aiia

AiiD

Brominated AHLs from D. Pulchra

Use NucB to destroy extracellular DNA that is transferred in Biofilms

Presentation Overview 1.  Background of quorum sensing mechanism

2.  Describe the model system used

3.  Discuss our quorum stopping project

4.  Our constructs

5.  Explore future applications

Reporter Inhibitor Transcription Factor

Promoter High Const.

•  LuxR C6 •  LuxR C8 •  SdiA

•  LuxpR •  pSrgE

•  RFP •  NucB •  AiiA

Constructing the basic parts we need

Parts Cloned Synthesized Registry BioBricked

LuxR X

SdiA* X X x

LuxR C6 Sensitive*

X

LuxR C8 Sensitive*

X

pSrgE* X

LuxpR X

NucB* X X

Aiia X

RFP X

LuxA* X X X

LuxB* X X X

LuxC* X X X

LuxD* X X X

LuxE* X X X

LuxG* X X X

RFP AiiA

High Const.

RFP Aiia LuxR C8

LuxpR High Const.

RFP Aiia SdiA

pSrgE High Const.

RFP Aiia NucB LuxR C6

LuxpR High Const.

Lux Cassette LuxR

LuxpR High Const.

1)

2)

3)

4)

5)

Our Constructs

Testing construct 1's ability to inhibit luminescence

•  OD measures concentration of both cells •  RFP is directly proportional to OD of E. coli with our construct •  Using RFP, subtract construct 1's concentration to get the effective

concentration of aliivibrio fisheri •  From there we can calculate at what OD the luminescence increased.

Competition Assay

Concentrations of VF:Construct 100:0 90:10 80:20 50:50 0:100

Modelling

What still needs to be done

1. Complete LuxR C6, and C8 constructs

2. Run co-culture experiment with A. fischeri and our constructs

3. Quantify the inhibitory effect of our construct on A. fischeri

4. We expect to see a huge decrease in luminescence at OD .25

Presentation Overview 1.  Background of quorum sensing mechanism

2.  Describe the model system used

3.  Discuss our quorum stopping project

4.  Our constructs

5.  Explore future applications

Probiotic

Prophylactic probiotic to fight internal biofilms and quorum sensing in pathogens

Bathroom Cleaner In households our construct could clean shower heads, shower curtains, and under sinks

http://www.pnas.org/content/106/38/16393.full

Water Mat Destroyer / Consumer Indicator

Stop formation of bacterial mats

Consumer indicator for freshness

Healthier Plants

Take our construct to plants to help them fight biofilm and pathogens

Population Analyzer

Species specific AHL recognition to survey bacterial populations.

Acknowledgements

LuxR G2E, LuxR G2H

Collins, C. H., Arnold, F. H. and Leadbetter, J. R. (2005), Directed evolution of Vibrio fischeri LuxR for increased sensitivity to a broad spectrum of acyl-homoserine lactones. Molecular Microbiology, 55: 712–723. doi: 10.1111/j.1365-2958.2004.04437.x

SdiA