Jamboree presentation 11 - 4 - 2006 Edinburgh iGEM 2006 Jamboree presentation 11 - 4 - 2006

Intro to our team www.Macteria.co.uk Team members Engineering: Kim de Mora Bryony Davidson Jen Wilson Biology: Judith Nicholson Farid Bizzari Jelena Aleksic Informatics: Sreemati Lalgudi Seshasayee Patrick Cai Sergii Ivakhno Faculty: Dr Chris French Dr Alistair Elfick Dr Hongwu Ma Laszlo Kozma-Bognar Here is the result we got from the modeling. When there is low concentration of arsenic input, like 5 ppb, the lacZ is produced at a basal level while the Urease is quite high. www.Macteria.co.uk

Why detect arsenic? www.Macteria.co.uk

Bangladesh Another cruel twist of fate… www.Macteria.co.uk

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33-75 million people affected www.Macteria.co.uk

? www.Macteria.co.uk

100 million people worldwide www.Macteria.co.uk

And not just in the developing world either… Here is the result we got from the modeling. When there is low concentration of arsenic input, like 5 ppb, the lacZ is produced at a basal level while the Urease is quite high. www.Macteria.co.uk

Arsenic detection method Current field technology: the Gutzeit Method Expensive and unreliable (max sensitivity of 50 ppb & 33% false negatives) Here is the result we got from the modeling. When there is low concentration of arsenic input, like 5 ppb, the lacZ is produced at a basal level while the Urease is quite high. www.Macteria.co.uk

Our device Arsenic-sensing bacteria with a pH output Very sensitive pH requires an indicator, litmus paper or a simple electrode It is possible to engineer a quantitative response: ureABC urease pH lacZ’ lactose fermentation pH Here is the result we got from the modeling. When there is low concentration of arsenic input, like 5 ppb, the lacZ is produced at a basal level while the Urease is quite high. www.Macteria.co.uk

ArsR mechanism in E. coli Key: Promoter Gene Binding site ArsR sensitive promoter arsR gene Here is the result we got from the modeling. When there is low concentration of arsenic input, like 5 ppb, the lacZ is produced at a basal level while the Urease is quite high. www.Macteria.co.uk

System gene circuit diagram (-) (-) (-) ParsR λ cI ParsD ureABC arsR arsD lacZ’ + Responds to higher As (~50 ppb) + Responds to low As (~10 ppb) Lambda cI / Lac I As As pH Here is the result we got from the modeling. When there is low concentration of arsenic input, like 5 ppb, the lacZ is produced at a basal level while the Urease is quite high. pH 10 ppb As = pH 7 3 step response: 0 ppb As = pH 9 50 ppb As = pH 4 www.Macteria.co.uk

λ cI / LacI hybrid promoter detail λ cI binding site ureABC OR1 OR2 OR3 LacI Here is the result we got from the modeling. When there is low concentration of arsenic input, like 5 ppb, the lacZ is produced at a basal level while the Urease is quite high. www.Macteria.co.uk

The model explained: Urease operon No. Name Equation 1 LacI production promoter3->promoter3+LacI 2 LacI binding to allolactose LacI+allolactose=LacI-allolactose 3 LacI binding to promoter4 LacI+promoter4 = LacI-promoter4 4 Urease production promoter4->promoter4+Urease 5 LacI degradation LacI->null 6 Urease degradation Urease->null

The model explained: λCI operon No. Name Equation 7 ArsR production promoter2->promoter2+ArsR 8 ArsR binding to Arsenic ArsR+2As(III)=ArsR-2As(III) 9 ArsR binding to promoter2 2ArsR+promoter2=2ArsR-promoter2 10 LCI production promoter2->promoter2+LCI 11 LCI binding to promoter 4 LCI+promoter4=LCI-promoter4 12 ArsR degradation ArsR->null 13 LCI degradation LCI->null

The model explained: lacZ’ operon No. Name Equation 14 ArsD production promoter1->promoter1+ArsD 15 ArsD binding to Arsenic ArsD+2As(III)=ArsD-2As(III) 16 lacZ production promoter1->promoter1+lacZ 17 ArsD binding to promoter1 2 ArsD+promoter1=2ArsD-promoter1 18 ArsD degradation ArsD->null 19 lacZ degradation lacZ->null

The model explained: the whole system Operon 1 6 Reactions 10 Parameters Operon 2 6 Reactions 10 Parameters To test our idea of arsenic biosensor, we generated computational model to simulate the response of system. The model is constructed in both COPASI and Symbiology toolbox for Matlab. We use Michaelis-Menten to describe the gene expression, and use Mass action for other process. We break down the whole system into three sub-models: Operon 1 produces LacZ, and it contains 6 reactions and 10 parameters. Operon 2 produces LamdaCI, and it contains 6 reactions and 10 parameters as well. Lamda CI in return represses Operon 3 which produces Urease, and operon 3 contains 7 reactions and 12 parameters. After constructing 3 operons, we assemble them into a whole system. Operon 3 7 Reactions 12 Parameters

Modelling result: low arsenic Here is the result we got from the modeling. When there is low concentration of arsenic input, like 5 ppb, the lacZ is produced at a basal level while the Urease is quite high.

Modelling result: high arsenic When we increase the concentration of arsenic to higher level, say 20ppb, we can see that the Urease is repressed, and lacZ increases to high level. And this results in the decrease of pH value.

Parameters sensitivity analysis Also, we analysis the sensitivity of each parameter in the model to identified their relative importance. Roughly speaking, we calculate the parameter sensitivity in this way: the percentage of system output changed divided by the percentage of parameter changed. For example, here we regard lacZ as the system output, and the parameter affects it most is K3, the ArsD degradation rate. This finding makes sense because the ArsD occupies the free binding site for producing lacZ. So, when we increase the value of K3, it will result in positive effect on lacZ, that means the concentration of lacZ will increase. With the help the parameter sensitivity analysis, we can refine out model more easily.

Parameter scanning After finding out the most sensitive parameters, we scan parameter from 1/10 to 10 times the normal value, and observe the system response. We hope the computational modeling can shed light on the wet-lab experimental design, and provide the biologists with the theoretical support.

Building the biosensor To prove our concept and provide a starting point for the larger device, we built a simple construct To test arsenic sensitivity and magnitude of pH change (if any!) Pars ArsR LacZ’ www.Macteria.co.uk

Biobricks Putting new parts in the registry one of our project aims Created ArsR and Ars promoter parts from E. coli, with B. subtilis for comparison Also built new lacZ’ Hybrid promoter built and urease planned (site-specific mutagenesis on urease) cI repressor binding site lacI binding site www.Macteria.co.uk

Urease part ideas Secondary idea - 3D Structure Builder Links to biosensor model by using the urease part Tissue engineering, bioremediation, etc. pH Urea + H2O CO2 + NH3 = = Calcium carbonate www.Macteria.co.uk

Outcomes Result: Simple construct built and ready to be characterised with pH experiments Several parts sequenced for registry Future: Test hybrid promoter and urease device, work towards full device www.Macteria.co.uk

Characterization procedure Now we have the engineered constructs, we need to test them Aims: to measure the scale of the pH response To test the sensitivity of the B. subtilis and E. coli Arsenic promoters Here is the result we got from the modeling. When there is low concentration of arsenic input, like 5 ppb, the lacZ is produced at a basal level while the Urease is quite high. www.Macteria.co.uk

Urease characterization The Wildtype urease contains 2 forbidden restriction sites The strain with the restriction sites removed failed to change the pH (i.e. the mutagenesis failed) Only the wild type Urease operon produced a change in pH Here is the result we got from the modeling. When there is low concentration of arsenic input, like 5 ppb, the lacZ is produced at a basal level while the Urease is quite high. Note: only 1 response from wildtype urease www.Macteria.co.uk

E. coli biosensor characterization Only testing a yes/no response here arsR promoter begins to function after about 300 min Difference of 0.6 pH units between control and average readings Here is the result we got from the modeling. When there is low concentration of arsenic input, like 5 ppb, the lacZ is produced at a basal level while the Urease is quite high. Note: successful response at all concentrations of arsenic www.Macteria.co.uk

E. coli biosensor characterization Non-optimised growth medium conditions (response could be faster) Average overnight difference of 0.81 pH units Despite slower response, 5 ppb within sensitivity of promoter Can detect WHO guideline levels of Arsenic (arsenate) Here is the result we got from the modeling. When there is low concentration of arsenic input, like 5 ppb, the lacZ is produced at a basal level while the Urease is quite high. Note: successful response at all concentrations of arsenic www.Macteria.co.uk

The desired end result Cheap, reliable, robust field test device Foolproof to operate and get accurate results Can be produced for less than 1$ in mass volumes Here is the result we got from the modeling. When there is low concentration of arsenic input, like 5 ppb, the lacZ is produced at a basal level while the Urease is quite high. www.Macteria.co.uk

The field test device A test tube could contain all the necessary components: Freeze dried bacteria, growth medium, indicator powder, Ampicillin salt, etc… These tubes could then be given to local villagers to monitor their own water quality themselves A good alternative to the widely used Gutzeit method Here is the result we got from the modeling. When there is low concentration of arsenic input, like 5 ppb, the lacZ is produced at a basal level while the Urease is quite high. www.Macteria.co.uk

Conclusions Successfully designed and modelled complex device Proved a measurable pH response could be obtained with As concentration to WHO standards Successfully biobricked a variety of parts to deposit in the registry With further work, our device could potentially help millions worldwide Here is the result we got from the modeling. When there is low concentration of arsenic input, like 5 ppb, the lacZ is produced at a basal level while the Urease is quite high. www.Macteria.co.uk

We would like to thank our sponsors Funding assistance: Sysbio 2.0 toolbox licenses: SYNBIOCOMM Here is the result we got from the modeling. When there is low concentration of arsenic input, like 5 ppb, the lacZ is produced at a basal level while the Urease is quite high. www.Macteria.co.uk

Questions? www.Macteria.co.uk Here is the result we got from the modeling. When there is low concentration of arsenic input, like 5 ppb, the lacZ is produced at a basal level while the Urease is quite high. Edinburgh Castle (on the only day this year it wasn’t raining) www.Macteria.co.uk