mit jamboree 2007. talk outline background system design novel reporter system established modelling...
TRANSCRIPT
![Page 1: MIT Jamboree 2007. Talk Outline Background System design Novel reporter system Established modelling techniques Cutting-edge modelling](https://reader036.vdocuments.mx/reader036/viewer/2022062417/5515c96c55034693758b4a35/html5/thumbnails/1.jpg)
MIT Jamboree 2007
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MIT Jamboree 2007
Talk Outline
• Background
• System design
• Novel reporter system
• Established modelling techniques
• Cutting-edge modelling
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MIT Jamboree 2007
Phenolic compounds
Polycyclic aromatic hydrocarbons (PAH)
BTEX compounds
The Problem
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MIT Jamboree 2007
• 1: Design modular sensor construct
• 2: Create the construct
• 3: Test the system
• 4: Development into a machine
• 5: Model and predict outcomes!
Objectives
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MIT Jamboree 2007
Why a Biosensor?
• Lab-based monitoring
• Skilled workforce
• Expensive!
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MIT Jamboree 2007
operator / promoter reporter gene()
XylR
toluene
What is a Biosensor?• Biosensors include a transcriptional activator
coupled to a reporter
Luciferase gene
luciferase
luciferin luminescence
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MIT Jamboree 2007
Our Construct Design
Responsivepromoter
Doubleterminator
BBa_B0015RBS
BBa_J61101Reporter
gene
Constitutive promoter
Transcriptionalactivator
Doubleterminator
BBa_B0015
RBS
![Page 8: MIT Jamboree 2007. Talk Outline Background System design Novel reporter system Established modelling techniques Cutting-edge modelling](https://reader036.vdocuments.mx/reader036/viewer/2022062417/5515c96c55034693758b4a35/html5/thumbnails/8.jpg)
MIT Jamboree 2007
• 1: Design modular sensor construct – Switch on reporter in presence of pollutants
• 2: Create the construct
• 3: Test the system
• 4: Development into a machine
• 5: Model and predict outcomes!
Objectives
![Page 9: MIT Jamboree 2007. Talk Outline Background System design Novel reporter system Established modelling techniques Cutting-edge modelling](https://reader036.vdocuments.mx/reader036/viewer/2022062417/5515c96c55034693758b4a35/html5/thumbnails/9.jpg)
MIT Jamboree 2007
Phenolic compounds
Polycyclic aromatic hydrocarbons (PAH)
BTEX compounds
Our Solution
DmpR - phenols
DntR - PAHs
XylR - toluene
![Page 10: MIT Jamboree 2007. Talk Outline Background System design Novel reporter system Established modelling techniques Cutting-edge modelling](https://reader036.vdocuments.mx/reader036/viewer/2022062417/5515c96c55034693758b4a35/html5/thumbnails/10.jpg)
MIT Jamboree 2007
Responsivepromoter
RBSBBa_J61101
Doubleterminator
BBa_B0015
Reporter gene
Constitutive promoter
Transcriptional activator
Doubleterminator
BBa_B0015
RBS
LacZ
GFP
Luciferase
DmpR - phenols
DntR - PAHs
XylR - toluene
Our Construct Design
![Page 11: MIT Jamboree 2007. Talk Outline Background System design Novel reporter system Established modelling techniques Cutting-edge modelling](https://reader036.vdocuments.mx/reader036/viewer/2022062417/5515c96c55034693758b4a35/html5/thumbnails/11.jpg)
MIT Jamboree 2007
• 1: Design modular sensor construct – Switch on reporter in presence of pollutants
• 2: Create the construct– Use 3 different sensors to express luciferase or LacZ
• 3: Test the system
• 4: Development into a machine
• 5: Model and predict outcomes!
Objectives
![Page 12: MIT Jamboree 2007. Talk Outline Background System design Novel reporter system Established modelling techniques Cutting-edge modelling](https://reader036.vdocuments.mx/reader036/viewer/2022062417/5515c96c55034693758b4a35/html5/thumbnails/12.jpg)
MIT Jamboree 2007
Testing The SystemXylR - inducible luciferaseDntR - inducible LacZ
[PAH metabolite] (M)
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MIT Jamboree 2007
• 1: Design sensor/reporter construct – Switch on reporter in presence of pollutants
• 2: Create the construct– Use 3 different sensors to express luciferase or LacZ
• 3: Test the system– PAH-metabolite and xylene sensors successful
• 4: Development into a machine
• 5: Model and predict outcomes!
Objectives
![Page 14: MIT Jamboree 2007. Talk Outline Background System design Novel reporter system Established modelling techniques Cutting-edge modelling](https://reader036.vdocuments.mx/reader036/viewer/2022062417/5515c96c55034693758b4a35/html5/thumbnails/14.jpg)
MIT Jamboree 2007
Unique Reporter System
• Conventional biosensors use conventional reporter genes
– e.g. LacZ, GFP, luciferase…
• Lengthy and expensive procedures
• Need a novel idea!
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MIT Jamboree 2007
Microbial Fuel Cells
• Clean, renewable & autonomous
• Electrons from metabolism harvested at anode
• Versatile, long-lasting, varied carbon sources
• Advantage over conventional power sources
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MIT Jamboree 2007
Microbial Fuel Cells
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MIT Jamboree 2007
Pyocyanin• From pathogenic Pseudomonas aeruginosa
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MIT Jamboree 2007
• Phz genes – 7 gene operon, pseudomonad specific
• PhzM and PhzS – P. aeruginosa specific
Pyocyanin
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MIT Jamboree 2007
Our Constructs
Constitutive promoter
Inducibletranscription
factor
DoubleterminatorRBS
Targetpromoter
PhzMcoding region
RBSPhzS
coding region
DoubleterminatorRBS
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MIT Jamboree 2007
+-
Pollutant
Microbial Fuel Cell
Electrical Output
xylRRBS Term.Term.Pr Pu
phz genes Term. Term.
PYOCYANIN
RBS
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MIT Jamboree 2007
• 1: Design sensor/reporter construct – Switch on reporter in presence of pollutants
• 2: Create the construct– Use 3 different sensors to express luciferase or LacZ
• 3: Test the system– PAH-metabolite and xylene sensors successful
• 4: Development into a machine– Use Pseudomonas aeruginosa to power a fuel cell
which generates a remote signal sent to base station
• 5: Model and predict outcomes!
Objectives
![Page 22: MIT Jamboree 2007. Talk Outline Background System design Novel reporter system Established modelling techniques Cutting-edge modelling](https://reader036.vdocuments.mx/reader036/viewer/2022062417/5515c96c55034693758b4a35/html5/thumbnails/22.jpg)
MIT Jamboree 2007
Wetlab - Drylab
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MIT Jamboree 2007
Computational Modelling of the Biosensor
Aims• Guide biologists for the better design of
synthetic networks• Use different computational approaches
to model and analyze the systemso Simple biosensor o Positive feedback within the biosensor
• Test and Validate the hypothesis proposed by the biologists
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MIT Jamboree 2007
The Model
24
PhzM PhzS
PCAIntermediate compound
PYO
TF + S TF|S
tf
phzM phzSTF|S
mRNA PhzM mRNA PhzS
mRNA TF• Merge transcription and translation• Merge phzM with phzS (Parsons 2007)
TF: Dntr or Xylr
S: signal
TF|S: complex
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MIT Jamboree 2007
The Model
25
tf
TF + S TF|S
phzMS
PhzMS
PCA PYO
TF|S
PYO
PYO
TF: Dntr or Xylr
S: signal
TF|S: complex
• Merge transcription and translation• Merge phzM with phzS (Parsons 2007)
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MIT Jamboree 2007
Feedback Loop
tf
TF + S TF|S
phzMS
PhzMS
PCA PYO
TF|S
PYO
PYO
TF: Dntr or Xylr
S: signal
TF|S: complex
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MIT Jamboree 2007
Modelling framework
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MIT Jamboree 2007
Modelling framework
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MIT Jamboree 2007
Qualitative Petri-Net Modelling & Analysis
• Graphical representation--Snoopy
• Graphical representation--Snoopy
• Qualitative analysis Charlie– T invariants (cyclic
behavior in pink)
– P invariants
– (constant amount of output)
• Quantitative Analysis by continuous Petri Net– ODE Simulation
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MIT Jamboree 2007
Modelling framework
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MIT Jamboree 2007
Parameters
31
• Literature search• Experts’ knowledge
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MIT Jamboree 2007
Ordinary Differential Equations
32
Available!
Created in
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MIT Jamboree 2007
Parameters
33
• Literature search• Experts’ knowledge
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MIT Jamboree 2007
Model Parameter Refinement
34
• Modified MPSA
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MIT Jamboree 2007
Modelling framework
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MIT Jamboree 2007
Advantages and disadvantages of stochastic modelling
• Living systems are intrinsically stochastic due to low numbers of molecules that participate in reactions
• Gives a better prediction of the model on a cellular level
• Allows random variation in one or more inputs over time
• Slow simulation time
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MIT Jamboree 2007
Chemical Master EquationsA set of linear, autonomous ODE’s, one ODE for each
possible state of the system. The system may be written:
• Ф → TF - production of TF• TF → Ф - degradation of TF• TF+S → TFS - association of TFS• TFS → TF+S - dissociation of TFS• TFS → Ф - degradation of TFS• Ф → PhzMS - production of PhzMS• PhzMS → Ф - degradation of PhzMS• PhzMS → PYO - production of pyocyanin• PYO → Ф - degradation of pyocyanin
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MIT Jamboree 2007
Propensity Functions
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MIT Jamboree 2007
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MIT Jamboree 2007
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MIT Jamboree 2007
Simulink Modelling Environment
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MIT Jamboree 2007
In the end…Our Contributions:
– standard SBML models of the systems– new biobricks with mathematical description – Practical comparison of modelling apporaches –
qualitative, continuous, stochastic, based on sound theoretical framework
– Tools to support synthetic biology (Code available) : • Minicap: multi-parametric sensitivity analysis of dynamic
systems• Simulink environment
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MIT Jamboree 2007
• 1: Design sensor/reporter construct – Switch on reporter in presence of pollutants
• 2: Create the construct– Use 3 different sensors to express luciferase or LacZ
• 3: Test the system– PAH-metabolite and xylene sensors successful
• 4: Development into a machine– Use Pseudomonas aeruginosa to power a fuel cell
which generates a remote signal sent to base station
• 5: Model and predict outcomes!
Objectives
![Page 44: MIT Jamboree 2007. Talk Outline Background System design Novel reporter system Established modelling techniques Cutting-edge modelling](https://reader036.vdocuments.mx/reader036/viewer/2022062417/5515c96c55034693758b4a35/html5/thumbnails/44.jpg)
MIT Jamboree 2007
Our Constructs So Far…
Native promoter
XylRDouble
TerminatorBBa_B0015
NativeRBS
XylR responsivepromoter
DoubleTerminatorBBa_B0015
RBSBBa_J61101
Renilla Luciferase
BBa_J52008
IRES XylR
XylR responsivepromoter RBS
Renilla Luciferase
BBa_J52008
DoubleTerminatorBBa_B0015IRES XylR
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MIT Jamboree 2007
Number BioBrick Number Description 1 BBa_I723032 Xylene-sensitive promoter 2 BBa_I723029 Xylene-sensitive promoter plus RBS
3 BBa_I723023 Xylene-inducible luciferase 4 BBa_I723031 Inducible luciferase 5 BBa_I723024 PhzM 6 BBa_I723025 PhzS 7 BBa_I723026 PhzM plus terminator
8 BBa_I723027 PhzS plus terminator 9 Bba_I723030 Salicylate-inducible transcription factor
10 BBa_I723020 Salicylate-sensitive promoter
Registry Contributions
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MIT Jamboree 2007
Instructors
• David Forehand
• David Gilbert
• Gary Gray
• Xu Gu
• Raya Khanin
• David Leader
• Susan Rosser
• Emma Travis
• Gabriela Kalna
Students
• Toby Friend • Rachael Fulton• Christine Harkness• Mai-Britt Jensen • Karolis Kidykas • Martina Marbà • Lynsey McLeay • Christine Merrick • Maija Paakkunainen • Scott Ramsay
• Maciej Trybiło
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MIT Jamboree 2007
Thank You!