igem 2009 Team Newcastle

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1 igem 2009 Team Newcastle

2 Introduction Environmental project Heavy metal pollution in soil Cadmium accumulation issue Image: Use of engineered micro-organisms

3 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.

4 Bac-Man Begins...

5 Bac-Man Begins...

6 Objectives of our project Specifically target cadmium at an important stage in the cadmium cycle Engineer the life cycle of a bacteria

7 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

8 Our System Cadmium Sensing Sporulation Tuning Chassis Development Stochastic Switch Metal sequestration

9 Sub-projects Population Modelling Metal Sensor Stochastic Switch Chassis Metal Sequestration Sporulation Tuner

10 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

11 Population Modelling

12 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 Biochemical model Agent Biochemical model Agent Biochemical model

13 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 Key: Vegetative Cells Normal Spores Metallic Spores Results fed into the overall project development

computers to spread the load Using Microbase and

14 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

15 Cadmium Sensing

16 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

17 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 Sensor Metal Metals Sensed Sensor ArsR As(III) Ag(I) Cu Cd CzrA Zn Co Ni Cd Metal sensitive promoters can sense more than one metal

18 Cadmium Sensing: AND gate Cadmium ions RNA Polymerase MntH channel CzrA ArsR cada promoter

19 Cadmium Sensing: AND gate

20 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) ArsR binding site cada promoter CzrA binding site RBS

21 Cadmium Sensing: Modelling

22 CI (nm) Cadmium Sensing: Modelling Time (second)

23 Cadmium Sensing: achievements

24 Stochastic switch

25 Where in the system? The stochastic switch is central to the re-engineering of the Bacillus life cycle

26 The switch Hin recombinase Pspac PxylA RFP hixc Pveg hin hixc Metal container decision activator/ GFP Tuneable invertible Pveg promoter region Controls and tunes key aspects of the Bacillus life cycle Hin-Hix system Heritable

27 The Switch

28 The Switch

29 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

1 2 A germination deficient chassis: (1) sleb cwlj spores fail to germinate (2) after treatment for recovery

30 Chassis Aim: To disable germination for the spores containing the sequestered cadmium, rendering retrieval 1 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.

31 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 hixc Pveg hin hixc Metal container decision activator / GFP

32 Xylose Stochastic Modelling [RFP] (Arabinose=10000nM) [GFP] (Arabinose=10000nM) IPTG IPTG Stochastic modelling could help us choose the strength of promoters to tune the switch.

33 Concentration (nm) Concentration (nm) 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 mrna Hin Rfp Hin Gfp Time Time

34 Concentration (nm) 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. Arabinose SspB Hin degradation Time (second)

35 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

36 Cadmium Sequestration

37 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

38 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

39 Metal Sequestration: achievements

40 Sporulation Tuning

vegetative cells Spo0A Governs sporulation pathway Activated by the

41 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

42 Sporulation Tuning Objective: To use kina to gradually increase the concentration of Spo0A~P

43 Spo0A~P (nm) Sporulation Tuning Sporulation signal concentration of 3000nM, and varied IPTG concentrations of 0 to 1000nM IPTG KinA Spo0A~P Sporulation Time (second) Increasing IPTG concentrations of nM

44 Sporulation Tuning: Lab Work and Characterisation Brightfield, IPTG (-) 2- Enhanced GFP, IPTG(-) 3- Brightfield, IPTG(+) 4- Enhanced GFP, IPTG(+) 5- Zoom into 3, spores indicated

45 Bac-Man: Achievements Summary

46 Bac-Man: Achievements summary Achievement Designed and shared our ideas on the igem wiki: 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 Complete

47 Acknowledgements Our instructors and advisors: Prof. Anil Wipat Dr. Jennifer Hallinan Dr. Daniel Swan Morgan Taschuk Dr. Matthew Pocock Dr. Mike Cooling With help from: Prof. Anne Moir, Sheffield University Prof. Nigel Robinson Dr. Jan-Willem Veening Keith Flannagan

A New Chassis for Synthetic Biology: Bacteria Without a Cell Wall. L-forms

A New Chassis for Synthetic Biology: Bacteria Without a Cell Wall L-forms Pros & Cons of Cell Wall Cell wall Cell membrane Cell membrane DNA ribosomes RNA metabolites Bacterium with cell wall Bacterium