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

2. 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.

3. Why detect arsenic?

4. Bangladesh
Another cruel twist of fate…

6. 33-75 million people affected

7. ?

8. 100 million people worldwide

9. 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.

10. 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.

11. 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.

12. 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.

13. 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

14. 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.

15. 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

16. 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

17. 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

18. 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

19. 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.

20. 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.

21. 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.

22. 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.

23. 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’

24. 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

25. 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

26. 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

27. 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.

28. 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

29. 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

30. 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

31. 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.

32. 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.

33. 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.

34. 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.

35. 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)