iGEM 2010 Presentation2010.igem.org/files/presentation/St_Andrews.pdf · sensing system To...

CholeraBiology

ModellingCharacterisationHuman Practices

iGEM 2010 Presentation

University of St Andrews iGEM 2010 Team

November 6, 2010

University of St Andrews iGEM 2010 Team iGEM 2010 Presentation

CholeraBiology


Team

� 9 Undergraduates:� Biologists, Medics, Physicists, a Computer Scientist and a Chemist

� 5 Advisors:� 2 Postgraduates� 3 Faculty


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Our Project: Cholera and Quorum Sensing


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IntroductionCqsABistable Switch

OutlineCholera

BiologyIntroductionCqsABistable Switch

ModellingIntroductionModelsParameter Testing

Characterisation

Human PracticesMethodResults


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Cholera and Quorum Sensing

� Vibrio cholerae� Acute infection which is

often fatal� Leads to dehydration and

loss of electrolyte balance� Transmitted through

contaminated food andwater

� Outbreaks often follownatural disasters strikingvulnerable communities


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Low Cell Density


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High Cell Density


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Hence. . .

If we can trick V. cholerae into thinking it is at highcell density it will become avirulent and safe


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Expressing CqsA in E.coliThe Cholera Autoinducer: CAI-1

K356000B0034J23100 B0010 B0012

CqsA

CqsA


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New Functionality From Existing PartsRe-Engineering the Lux Operon

� Well understood quorum sensing system in Vibrio fischeri

� In the registry and commonly used by iGEM teams


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Wild-Type Expression Pattern


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Bistable Switch


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Bistable Expression Pattern


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IntroductionModelsParameter Testing

OutlineCholera



Characterisation



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Model Design

� Based on ODEs

� Solved computationallyusing RK4

� Models written in C++

� Custom written solvers andmodels


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Modelling Goals

� To create a mathematical representation of the E.coli Lux quorumsensing system

� To understand the bistable switch mechanism of our syntheticE.coli

� To predict how the CAI-1 production mechanism will be activatedand deactivated under the control of the bistable switch


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LuxR Quorum Sensing Circuit


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Our Model

� Cell growth phase: doubling time of 20 minutes

� Cell death phase: halving time of 20 minutes

� Diffusion of HSL throughout the system is an instantaneous process

� HSL diffuses evenly across the entire system


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GFP: High/Low Cell Density


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Parameter TestingWhat Parameters Affect Bistability?

� Association and dissociation

� HSL production and degradation

� LuxR degradation

� Promoter binding site strengths


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Measuring Bistability


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Find the parameter space inwhich the system switches on

Investigate ∆ Cell densityvarying one parameter

Investigate ∆ Cell densityvarying the other parameter


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Tuning Our SwitchIn the lab we can control:� Promoter Strength (PoPs)� Ribosome Binding Site Efficient

Use these parameters to control the operating point of our switch


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OutlineCholera



Characterisation



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Ribosome Binding Site

� Sequence on mRNA

� Required in bacteria for translation


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Determining RBS Efficiency

� Translation initiation determines rate of protein translation.� Translation initiation rate depends on:

� Downstream of RBS� Sequence of RBS� Upstream of RBS


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Determining the Optimal RBSThe thermodynamic model quantifies the strength of the molecularinteraction between an mRNA and the 30 S ribosome complex byGibbs free energy1.

� RBS calculator uses this thermodynamic model(http://voigtlab.ucsf.edu/software/).1H. M. Salis, E. A. Mirsky and C. A. Voigt. Automated design of synthetic

ribosome binding sites to control protein expression. Nature Biotechnology2009, 27(10):946-50.


http://voigtlab.ucsf.edu/software/

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RBS in vitro

� Ordered the sequence as primers

� Ligated them into I13401

� Measured the level of GFP expression by flow cytometry


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MethodResults

OutlineCholera



Characterisation



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MethodResults

Human PracticesIntroduction

Two views of human practices:

Local Data collected in sunny St Andrews

� Not everyone has an Internet connection� Gets data from those not involved in the subject at

hand� Different groups of people than trend setters

Global Data collected from the World Wide Web

� Poll thousands of users easily� Collect repeat data quickly� Huge repository of data: Facebook, Twitter, Blogs,

News etc


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MethodResults

Crawler Design

Facebook

Twitter

BBC News

NY Times

CNN

Blog

Parse Pages and Extract Links

Journal

Blog Link

News Link

Initial Input

Queue of Links

A new discovery in synthetic biology….New field of synthetic biology requires regulation….Disaster ahead with synthetic biology …Synthetic biology brings hope to millions…

Paper Link

“SyntheticBiology”

“Synbio”

Positive Negative

Opinion Finder

Synthetic biology brings hope to millions

Disaster ahead with synthetic biology


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MethodResults

Popularity of Synthetic BiologyLocal


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MethodResults

Popularity of Synthetic BiologyGlobal

1 2 3 4 5 6 7 8 9 10

0

10

20

30

40

50

60

70

80

Change of Opinion Over Time

synthetic biology

Genetic Engineering

Week

Opi

nion


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MethodResults

Popularity of SciencesGlobal


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MethodResults

Amount of Discussion of SciencesGlobal


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MethodResults

Popularity of Different Subjects

1 2 3 4 5 6 7 8 9 10

0

10

20

30

40

50

60

70

80

90

Change of Opinion Over Time

synthetic biology

politics

football

lady gaga

Week

Opi

nion


Conclusions

� Constructed a biobrick to generate the cholera autoinducer CAI-1

� Created a system of equations to describe the Lux quorum sensingnetwork

� Predicted the RBS strength by the RBS calculator program andmeasured it in vitro

� Introduced two new RBS sequences with measured strength

� Introduced new approaches to human practices were introducedwith the automated collection of large amounts of readily availableweb data pertaining to current opinion and trend

Sponsors

Thanks

Dr V. Anne Smith for forming the team and kindly donating her labspace

Mr Choon Tan for aiding us in the lab and putting up with his labbeing invaded

Professor Jeff Graves for helping us with sequencing

Dr Elaine Campbell for helping us with the flow cytometry

The St Andrews Ice Cream Van which drives past the lab

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