iGEM 2009 Team Newcastle Introduction Environmental project Heavy - - PowerPoint PPT Presentation

iGEM 2009 Team Newcastle

Introduction

• Environmental project
• Heavy metal pollution in soil
• Cadmium accumulation issue
• Use of engineered micro-organisms

Image: http://www.enst.umd.edu/people/Weil/ResearchProjects.cfm

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...

• Specifically target cadmium at an

important stage in the cadmium cycle

• Engineer the life cycle of a bacteria

Objectives of our project

Bacillus subtilis normal life cycle

• Can produce resilient, long-lasting spores
• Naturally lives in soil
• Non pathogenic

Choice of organism: Bacillus subtilis

Vegetative Cell Endospore Spore Cell Division

Our System

Cadmium Sensing Stochastic Switch Metal sequestration Sporulation Tuning Chassis Development

Sub-projects

Population Modelling Stochastic Switch Metal Sensor Sporulation Tuner Chassis Metal Sequestration

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

Biochemical model

Agent

Biochemical model

Agent

Biochemical model

Agent based 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
• verall project

development

Key: Vegetative Cells Normal Spores 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

• ur system which produces a

signal in response

• How do we build our cadmium

sensor BioBrick?

– Use metal sensors CzrA and ArsR

Metal Sensor Metals Sensed ArsR As(III) Ag(I) Cu Cd

Cadmium Sensing: ArsR and CzrA

Metal sensitive promoters can sense more than one metal

Metal Sensor Metals Sensed CzrA Zn Co Ni Cd

• 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

Cadmium Sensing: AND gate

Cadmium ions MntH channel RNA Polymerase ArsR CzrA cadA promoter

Cadmium Sensing: AND gate

Cadmium Sensing: BioBrick Construct

AND gate BioBrick (BBa_K174015)

• In Bacillus subtilis, CadA efflux channels export

cadmium ions

• The CadA promoter is cadmium-sensitive
• The CadA promoter contains CzrA binding site

cadA promoter CzrA binding site ArsR binding site RBS

Cadmium Sensing: Modelling

Cadmium Sensing: Modelling

Time (second) CI (nM)

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

• Tuneable invertible Pveg promoter region
• Controls and tunes key aspects of the Bacillus life cycle
• Hin-Hix system
• Heritable

Pspac PxylA RFP Metal container decision activator/ GFP hixC hixC hin

Pveg Hin recombinase

The Switch

The Switch

Stochastic switch

• Increases rate of Bacillus sporulation
• Activates metal sponge expression
• Upregulates cadmium import
• Downregulates cadmium efflux
• Prevents germination gene complementation

Pspac PxylA RFP

Hin recombinase

Metal container decision activator / GFP

• Aim: To disable germination for the spores containing the

sequestered cadmium, rendering retrieval of the cadmium unnecessary.

Chassis

• 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.

A germination deficient chassis: (1) ∆sleB∆cwlJ spores fail to germinate (2) after treatment for recovery

1 2 1

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 Metal container decision activator / GFP hixC hixC hin

Pveg

Stochastic Modelling

Stochastic modelling could help us choose the strength of promoters to tune the switch.

IPTG Xylose

[RFP] (Arabinose=10000nM)

IPTG

[GFP] (Arabinose=10000nM)

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

Concentration (nM) Concentration (nM) Time Time mRNA Hin mRNA Hin Rfp Hin Gfp

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) Concentration (nM)

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

• 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 Sequestration: What is this sub-project about?

Metallothionein-CotC fusion protein Cadmium

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

Spo0A~P (nM) Time (second)

Increasing IPTG concentrations

• f 0-1000nM

IPTG KinA Spo0A~P Sporulation

Sporulation Tuning: Lab Work and Characterisation

1- Brightfield, IPTG (-) 2- Enhanced GFP, IPTG(-) 3- Brightfield, IPTG(+) 4- Enhanced GFP, IPTG(+) 5- Zoom into 3, spores indicated.

2 3 4 5 1

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 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