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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
ModellingCharacterisationHuman Practices
Team
� 9 Undergraduates:� Biologists, Medics, Physicists, a Computer Scientist and a Chemist
� 5 Advisors:� 2 Postgraduates� 3 Faculty
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
Our Project: Cholera and Quorum Sensing
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionCqsABistable Switch
OutlineCholera
BiologyIntroductionCqsABistable Switch
ModellingIntroductionModelsParameter Testing
Characterisation
Human PracticesMethodResults
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionCqsABistable Switch
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
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionCqsABistable Switch
Low Cell Density
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionCqsABistable Switch
High Cell Density
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionCqsABistable Switch
Hence. . .
If we can trick V. cholerae into thinking it is at highcell density it will become avirulent and safe
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionCqsABistable Switch
Expressing CqsA in E.coliThe Cholera Autoinducer: CAI-1
K356000B0034J23100 B0010 B0012
CqsA
CqsA
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionCqsABistable Switch
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
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionCqsABistable Switch
Wild-Type Expression Pattern
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionCqsABistable Switch
Bistable Switch
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionCqsABistable Switch
Bistable Expression Pattern
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionModelsParameter Testing
OutlineCholera
BiologyIntroductionCqsABistable Switch
ModellingIntroductionModelsParameter Testing
Characterisation
Human PracticesMethodResults
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionModelsParameter Testing
Model Design
� Based on ODEs
� Solved computationallyusing RK4
� Models written in C++
� Custom written solvers andmodels
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionModelsParameter Testing
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
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionModelsParameter Testing
LuxR Quorum Sensing Circuit
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionModelsParameter Testing
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
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionModelsParameter Testing
GFP: High/Low Cell Density
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionModelsParameter Testing
Parameter TestingWhat Parameters Affect Bistability?
� Association and dissociation
� HSL production and degradation
� LuxR degradation
� Promoter binding site strengths
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionModelsParameter Testing
Measuring Bistability
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionModelsParameter Testing
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionModelsParameter Testing
Find the parameter space inwhich the system switches on
Investigate ∆ Cell densityvarying one parameter
Investigate ∆ Cell densityvarying the other parameter
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
IntroductionModelsParameter Testing
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
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
OutlineCholera
BiologyIntroductionCqsABistable Switch
ModellingIntroductionModelsParameter Testing
Characterisation
Human PracticesMethodResults
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
Ribosome Binding Site
� Sequence on mRNA
� Required in bacteria for translation
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
Determining RBS Efficiency
� Translation initiation determines rate of protein translation.� Translation initiation rate depends on:
� Downstream of RBS� Sequence of RBS� Upstream of RBS
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
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.
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
RBS in vitro
� Ordered the sequence as primers
� Ligated them into I13401
� Measured the level of GFP expression by flow cytometry
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
MethodResults
OutlineCholera
BiologyIntroductionCqsABistable Switch
ModellingIntroductionModelsParameter Testing
Characterisation
Human PracticesMethodResults
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
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
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
MethodResults
Crawler Design
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
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
MethodResults
Popularity of Synthetic BiologyLocal
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
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
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
MethodResults
Popularity of SciencesGlobal
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
MethodResults
Amount of Discussion of SciencesGlobal
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
CholeraBiology
ModellingCharacterisationHuman Practices
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
University of St Andrews iGEM 2010 Team iGEM 2010 Presentation
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