Arsenic Biosensor
From 2006.igem.org
This is our first formalised project idea. The details are as follows:
Contents |
Aim
Develop a bacterial biosensor that responds to a range of arsenic concentrations and produces a change in pH that can be calibrated in relation with the arsenic concentration.
Background
This project is motivated by the needs of parts of the developing world. For details, see http://2006.igem.org/Arsenic_Biosensor_Background Background
Possible mechanism 1 - the bare minimum
INPUT [Arsenate/Arsenite] -> Detector -> Reporter -> OUTPUT [H+]
The arsR gene codes for a repressor which binds to the arsenic promoter in the absence of arsenate or arsenite. These two genes can be linked to the lacZ gene, in order to place lactose metabolism under the control of an arsenic activated promoter.
The ability to metabolise lactose should result in the acidification of the medium, and an output of H+ ions, giving a pH response to the input of arsenate/arsenite molecules.
It should be possible to obtain a curve for pH vs. arsenic concentration, where a range of pH values representing an unsafe level, a safe level, or an intermediate level of arsenic is present can be elucidated after calibrating the biosensor with known concentrations of arsenic.
Parts needed: Arsenic binding repressor protein Arsenic repressor protein responsive promoter lacZ protein coding gene
Possible mechanism 2 - more ambitious
After discussion, it was decided that it would be unfeasible to try and engineer a sensitive enough analogue output just using the pH - instead, a set of a couple of defined 'stepped' outputs would be preferable.
In order to increase the range that is indicated by the biosensor, the following mechanism might be worth considering.
Sample: Water sample + Lactose + Bacterial culture
The mechanism involves 3 'devices' inside the bacteria.
Device 1: The lac operon regulatory region is connected to an enzyme which, if activated, catalyses a reaction that produces an excess of OH-, thus making the surrounding solution alkali.
Device 2: A promoter that's regulated by a repressor very sensitive to arsenic. Thus anything above 5 ppb of arsenic would bind to the repressor and enable gene expression. This promoter is connected to a gene that expresses a molecule that represses the lac operon in device 1, thus keeping the pH at neutral. The usual lac repressor molecule wouldn't work since there's lactose in the solution to bind to it, so either we would have to make a lac operon regulatory region that also happens to respond to another repressor, or we would have to come up with a different solution for the problem (like somehow utilising an anti-switch or something?)
Device 3: A promoter regulated by a repressor that's somewhat less sensitive to arsenic, so only switches on at, say, 20 ppb. Connected to lacZ, as detailed above in Possible mechanism 1.
So, if all goes according to plan, we would get the following results:
If no arsenic is present: The lac operon in Device 1 detects the presence of lactose that's been put in, activates the expression of the genes behind it, and the enzyme produced catalyses a reaction that raises the pH of the surrounding solution. So the pH with no arsenic present is around 9-10.
If 5 ppb arsenic is present: The arsenic activates Device 2, which produces a repressor that deactivates Device 1. No enzyme is produced, and the pH remains neutral. 5ppb arsenic = pH 7
If 20 ppb arsenic is present: The arsenic activates Device 3, which produces lacZ, which digests the lactose in the solution and turns it acidic. Device 2 is still switched on and Device 1 is still switched off accordingly, so there are no problems with the acid and teh alkali neutralising one another. 20ppb arsenic = pH 4.5
The figures for the concentrations and the pH are basically currently just made up, it would take weeks of calibration in the lab to tune everything properly and see what's feasible. Whether the E. coli survives the given range of pH or not is basically irrelevant, as each infusion of bacteria would be single use, and neither the alkali nor the acid will be produced until the bacteria are already sitting in the test sample.
Possible mechanism 3 - ultra ambitious
This mechanism relies on the existence of a number of different ars operon style promoters with varying sensitivities to arsenic. I don't know if such things actually exist. If they don't, it's not actually worth it reading any further. However, if they do:
This mechanism is an elaboration on mechanism 2, combining pH as an indicator with fluorescence.
It also requires promoters that need 2 activator molecules to switch them on. As far as I know, such things exist, with the activator molecule binding to the specific gene sequence, and the co-activator binding to the activator and increasing the gene expression. The same coactivator can act on a number of different promoters, since it is only the activator part that's sequence specific.
Anyway.
We leave Device 1 as it is. We leave Device 2 as it is, except that along with a repressor, it also expresses a coactivator molecule (lets call it CA1) We leave Device 3 as it is.
And then we add another couple of devices:
Device 4: An ars operon that's activated at 10 ppb, that produces an activator molecule A1
Device 5: promoter activated by A1+CA1, with a fluorescent gene attached, eg. GFP.
Now, if we have ars promoters that can bind two repressors, we can really play around with this:
Device 6: an ars operon that's activated at 15 ppb, that produces activator molecule A2, as well as repressor molecule R1 which shuts down Device 4.
Device 7: promoter activated by A2+CA1, with a fluorescent gene attached, eg. YFP.
Device 8: activated at 20 ppb, produces A3 and R2, R2 shuts down Device 6
Device 9: A3+CA1 make colour
etc. potentially ad infinitum, with each colour repressing the previous colour.
Failing that: Without the promoters that bind 2 repressors, it's still possible to incorporate the colours, it's just that the number of colours we can incorporate will be limited. We can do this by stopping at Device 5, then making Device 3 produce a different coactivator CA2, and having
Device 6: ars operon that's activated at, say, 30 ppb, produces A2
Device 7:A2+CA2 produce a colour, eg. RFP
In this case, either another mechanism should be used to deactivate Device4+5, or Device 7 should produce a colour that's darker than that of Device 5, and thus still clearly visible even when both are active at the same time.
So what we get with the latter, simpler version is a display as follows:
Arsenic conc. (ppb) pH Colony colour
0 9.5 none
5 7 none
10 7 green
20 4.5 green
30 4.5 red
The results for the more complex version above are pretty similar, except with a lot more different concentrations, and a proper rainbow spectrum of colours.
Advantage: once a colony of bacteria is engineered, no matter how complex the genetic engineering was, it's still very simple to grow. So provided this version was made in real life, it would still have all the advantages of the pH system we discussed - it would be cheap and easy to use in the field, only requiring a bit of lactose and indicator. However, this version would have the added advantage that it would also be possible to determine the concentration more precisely by consulting the above table if a fluorescence microscope was used. It could be a high throughput reasonably detailed technique used in labs as well as a quick easy test used in the field. A sort of all-in-one.
Problems
The arsenic promoter shows some background activity even when arsenic is not present. It may be possible to reduce this noise by adding an extra control method to repress the lacZ gene parallel to the action of the arsR product, with for example an antiswitch.
It will not be possible to get a linear relationship between pH and the arsenic concentration because the relationship between gene expression and the cofactor concentration is not linear.
The arsenic concentration has to be the limiting factor in the reaction to result in the steady state obtained being the correct pH in relation to arsenic concentration. If the cell number is the limiting factor the pH may not drop low enough to indicate the arsenic level present in the time taken before measuring.
Experiments required
A lot of this project will be calibrating the sensor, finding the time to reach a steady state, finding out which amount of cells is ideal for reaching the steady state which gives distinct results for each pH range, and finding out which pH range represents which concentration of arsenic. It would be ideal to have the threshold for the legal limit of arsenic at the point of the curve which gives the most accurate result.
