iGEM 2009 Team Newcastle
Introduction • Environmental project • Heavy metal pollution in soil • Cadmium accumulation issue Image: http://www.enst.umd.edu/people/Weil/ResearchProjects.cfm • Use of engineered micro-organisms
What can our project do about it? Aim: Isolate cadmium from the soil environment rendering it bio-unavailable to avoid the damaging effects of accumulation.
Bac-Man Begins...
Bac-Man Begins...
Objectives of our project • Specifically target cadmium at an important stage in the cadmium cycle • Engineer the life cycle of a bacteria
Choice of organism : Bacillus subtilis • Can produce resilient, long-lasting spores • Naturally lives in soil • Non pathogenic Spore Endospore Vegetative Cell Cell Division Bacillus subtilis normal life cycle
Our System Sporulation Cadmium Tuning Sensing Metal Stochastic Chassis sequestration Switch Development
Sub-projects Population Metal Sensor Stochastic Modelling Switch Chassis Metal Sporulation Sequestration Tuner
Sub-project modelling • Modelling was done for each sub-project • Technologies used include: – CellML, SBML, COPASI, COR, Arcadia, Systems Biology Workbench, OpenCell, Java, Jsim, MatLab
Population Modelling
Population Modelling: Aims and Novelty • What is the affect of modifying the bacteria's life cycle? • Independent bacterial cells making decisions in their lives • Each cell runs cellular models, using its own parameters – thus integrating agent-based modelling and biochemical models Agent based model Agent Agent Agent Biochemical Biochemical Biochemical model model model
Population Modelling: How does it work? • Java language – JSim connects to biochemical models • Each bacterial cell runs independently as a thread – Uses a lot of CPU power and RAM • Results fed into the overall project Key: Vegetative Cells Normal Spores development Metallic Spores
Population Modelling: Distributed Computing • The solution: Distributed Computing – Using multiple computers to spread the load • Using Microbase and Networking – University computer clusters – Amazon Elastic Compute Cloud
Cadmium Sensing
Cadmium Sensing: What is this sub-project about? • We need to produce a tightly regulated cadmium sensor in our system which produces a signal in response • How do we build our cadmium sensor BioBrick? – Use metal sensors CzrA and ArsR
Cadmium Sensing: ArsR and CzrA • Both are metal sensitive repressors: • ArsR features in the Arsenic resistance operon • CzrA features in the Cobalt Zinc resistance operon • Why use these metal sensitive promoters Metal Metals Sensed Metal Sensor Metal sensitive promoters can sense more than one metal Metals Sensed Sensor ArsR As(III) Ag(I) Cu Cd CzrA Zn Co Ni Cd
Cadmium Sensing: AND gate Cadmium ions RNA Polymerase CzrA MntH channel ArsR cadA promoter
Cadmium Sensing: AND gate
Cadmium Sensing: BioBrick Construct • In Bacillus subtilis , CadA efflux channels export cadmium ions • The CadA promoter is cadmium-sensitive • The CadA promoter contains CzrA binding site AND gate BioBrick ( BBa_K174015 ) cadA ArsR binding site CzrA binding RBS promoter site
Cadmium Sensing: Modelling
Cadmium Sensing: Modelling CI (nM) Time (second)
Cadmium Sensing: achievements
Stochastic switch
Where in the system? The stochastic switch is central to the re-engineering of the Bacillus life cycle
The switch Hin recombinase Pspac PxylA RFP hin hixC Metal container Pveg hixC decision activator/ GFP • Tuneable invertible Pveg promoter region • Controls and tunes key aspects of the Bacillus life cycle • Hin-Hix system • Heritable
The Switch
The Switch
Stochastic switch Hin recombinase Pspac PxylA RFP Metal container decision activator / GFP • Increases rate of Bacillus sporulation • Activates metal sponge expression • Upregulates cadmium import • Downregulates cadmium efflux • Prevents germination gene complementation
Chassis • Aim: To disable germination for the spores containing the 1 sequestered cadmium, rendering retrieval of the cadmium unnecessary. 1 2 A germination deficient chassis: (1) ∆ sleB ∆ cwlJ spores fail to germinate (2) after treatment for recovery • Objective: To use the non-germination spores, with the inactivated genes, sleB and cwlJ , kindly sent to us by Prof. Anne Moir from Sheffield University. • The knocked out genes can be complemented to recover ‘wild type’ cells.
Tuneable? We think of the stochastic switch as a biased heads or tails: • Two differing strength promoters • Inducible degradation of the protein responsible for the switching We modelled our stochastic switch using inducible promoters Pspac and PxylA. Pspac PxylA RFP hin hixC Metal container Pveg hixC decision activator / GFP
Stochastic Modelling [RFP] (Arabinose=10000nM) [GFP] (Arabinose=10000nM) Xylose IPTG IPTG Stochastic modelling could help us choose the strength of promoters to tune the switch.
Tuning? The device had to be modelled due to the many variables that contribute to the stochastic decision: • Pulse lengths of Hin • Net number of flips mRNA Hin Rfp mRNA Hin Hin Concentration (nM) Concentration (nM) Gfp Time Time
Degradation controller • Hin recombinase expressed with a degradation tag. • Degradation induced by expression of chaperone SspB which recognises this tag. • SspB expression controlled by an arabinose inducible promoter. Concentration (nM) Arabinose SspB Hin degradation Time (second)
Stochastic switch: achievements • Successfully designed a tuneable stochastic switch device that controls cellular differentiation and sent the DNA to the parts registry • Completed a stochastic model for this switch, from which parameters can be estimated • Designed and cloned a degradation controller BioBrick and submitted the DNA to the Parts Registry
Cadmium Sequestration
Cadmium Sequestration: What is this sub-project about? • Aim: To render cadmium bio-unavailable by mopping it up using a metallothionein and moving it into spores • By wrapping a spore coat protein around cadmium ions, the ions become isolated from the environment (and humans) and no longer have harmful effects. • Novelty: Moving cadmium into resilient spores have not been accomplished before. Cadmium Metallothionein-CotC fusion protein
Cadmium Sequestration: BioBrick Construct • SmtA is translationally fused with CotC and Gfp − SmtA, Metallothionein − CotC, Spore coat protein − Gfp, reporter protein smtA metallothionein BioBrick Construct
Metal Sequestration: achievements
Sporulation Tuning
Sporulation Tuning • Aim: To control sporulation, deciding how much of the population becomes spores, and how much continue as vegetative cells • Spo0A − Governs sporulation pathway − Activated by the phosphorelay • Used the expression of kinA, a major histidine to activate Spo0A
Sporulation Tuning • Objective: To use kinA to gradually increase the concentration of Spo0A~P
Sporulation Tuning Sporulation signal concentration of 3000nM, and varied IPTG concentrations of 0 to 1000nM IPTG Spo0A~P (nM) KinA Spo0A~P Increasing IPTG Sporulation concentrations of 0-1000nM Time (second)
Sporulation Tuning: Lab Work and Characterisation 1 2 1- Brightfield, IPTG (-) 2- Enhanced GFP, IPTG(-) 3- Brightfield, IPTG(+) 4- Enhanced GFP, IPTG(+) 5- Zoom into 3, spores indicated. 4 5 3
Bac-Man: Achievements Summary
Bac-Man: Achievements summary Achievement Complete Designed and shared our ideas on the iGEM wiki: √ • http://2009.igem.org/Team:Newcastle Register and submit DNA for new BioBrick Parts and Devices √ to the Parts Registry: • 19 parts • Sent DNA for 10 parts √ Characterise a BioBrick: • IPTG inducible KinA sporulation trigger (BBa_K174011) • Works as expected Improve an existing BioBrick part: √ • BioBrick ‘ Pspac promoter’ (BBa_K174004) Help another iGEM team: √ • Mercury sensing model for UQ
Acknowledgements Our instructors and With help from: advisors: Prof. Anne Moir, Sheffield University Prof. Anil Wipat Prof. Nigel Robinson Dr. Jennifer Hallinan Dr. Jan-Willem Veening Dr. Daniel Swan Keith Flannagan Morgan Taschuk Dr. Matthew Pocock Dr. Mike Cooling
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