Introduction

From our work with the Environment Agency and Human Practices team it was clear that there was great demand for a cheaply manufactured and effective biosensor to detect chlorinated solvents. Take a look at our Policy & Practices pages to find out more about our research in this area.
To develop a cheap and user friendly way of detecting chlorinated solvents (focusing specifically on DCM) the engineering design team worked very closely with the biochemistry team to characterise a previously unknown genetic circuit and then adapt it to respond to DCM in our DCMation kit. We modelled and optimised the parameters that we could control to get the fastest visible response. Our biosensor, based on the M. extorquens DM4 degradation pathway for DCM is designed to give a detectable fluorescent output that when integrated into an electronic circuit signals when there is very little to no DCM left in our DCMation kit.

See below for links to the wet lab work, modelling and physical realisation of our product!

What is a biosensor?

Biological systems are very good at sensing the huge range of chemical and physical inputs in the world around them, often at very low levels. They need to do this in order to respond to and survive the constant changes in their environment. In many cases, this sensing results in a change at the transcriptional level in the organism. For example, Methylobacterium extorquens DM4 increases expression of DCM dehalogenase in the presence of DCM in order to exploit this carbon source. This means we can use these natural sensing systems to engineer novel genetic circuits that will respond to specific inputs with detectable outputs; in other words, to create a biosensor.

Optimising our Design

The ideal performance criteria we want in our biosensor are:

- A fast response to DCM.
- High amplitude of output signal.
- A robust system can cope with slight perturbations and still retain original behaviour.
- High sensitivity to input.

However, it is not possible to fulfil all these criteria in one system. The parameters we can alter biologically are limited; furthermore altering one parameter in the system impacts multiple criteria. Working with these restrictions, the challenge was to design a biosensor with properties as close to ideal as possible without sacrificing any one criterion entirely.
We therefore modelled the effect of changing our controllable parameters (see design optimisation page) and used this to guide our initial design decisions.

Characterisation, Construction and Realisation

The promoter of dcmA (DCM dehalogenase gene) is placed upstream of sfGFP; therefore we will get a fluorescent output instead of dcmA expression as in the native bacterium. Repression or activation of the dcmA promoter relies on the regulatory protein DcmR which responds to the [DCM]; in our genetic circuit, the dcmR gene is constitutively expressed.

Click on the characterisation link below to find out more about the genetic regulatory network we are characterising and engineering to produce our biosensor!

By having an electronic circuit, we can quickly adapt our DCMation kit to give the same user-friendly output (a green LED comes on on top of the bench-top kit to indicate the contents can be poured down the sink) depending on the result of our characterisation of the action of DcmR. This design meant that we could develop both the genetic circuit and the physical realisation of our product at the same time rather than sequentially - saving us time!

Click on the realisation link below to see more about our fluorescence detecting circuit!

References

[1] Shaun Rowson BA (Hons) MSc CIWEM CWEM (Team Leader - Groundwater & Contaminated Land,Lincolnshire and Northamptonshire)