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Revision as of 22:33, 21 September 2012



Parts for a reliable, cost-effective, biosensing standard

Introductory Video:


The fundamental aim of this project is to develop parts towards a standard for biosensors that allows high accuracy and reliability, whilst being cost-effective, practical and straightforward to use, even for users with no biological background. We hope that our work will be particularly useful to environmental scientists or health workers.


Electronic circuits excel at logic and speed of computation, but aren't particularly flexible when it comes to sensing the wide-range of small molecules present in the world. The use of biosensors as an approach to determining concentrations of analytes in samples for a huge variety of applications (medical, doping, ground water contamination - See human Practices) has great potential to be revolutionary technology.

Currently, however, whilst plenty of biosensors exist and have been characterised to a greater or lesser degree, they are in no way unified or standardised. Not only that, they are almost exclusively either non-quantitative or only usable in the lab, read using expensive and delicate laboratory equipment. They are also limited, as is often the case in synthetic biology, by the stochasticity of life, often giving unreliable or poorly reproducible results. Furthermore, at present very little thought has gone into the storage and distribution on biosensors for use in the field where shelf life, cost of transportation, storage conditions and biocontainment all become important factors.

We aim to develop a new standard for biosensors that tackles all these issues. The standard is to be back-compatible, so not only will new biosensors developed with the standard have these properties, but so will pre-existing sensors after minimal adaptation.

In order to develop our final system we went through a comprehensive Design Process.

The final result of our standard design is outlined below:

The standard consists of five parts:

1. Use of a ratiometric reporter system

Biosensors may give unreliable outputs. This is due to differences in the number and state of the cells from test to test. By including an internal control signal, to which another inducible signal may be normalised, the reliability and reproducibility of a sensor may be significantly improved. We are currently working on two such two-signal systems. Firstly, a construct that uses an inducible eCFP and a constitutively expressed eYFP. All components, save the vector, are existing biobricks. This will serve as a proof of concept and a way of testing old and new sensors. However, this will require a platereader to use. Our second construct will not, as described below.

2. Use of bacterial luciferase

Luciferase light emission is visible to the naked eye, and can therefore be sensed and quantified using inexpensive, off-the-shelf electronic components, giving it an advantage over fluorescent proteins in this context

Bacterial luciferase has a major advantage over other luciferases in this context in that the substrate regeneration enzymes are known and included in the operon. Other luciferases require addition of exogenous luciferin, which is expensive and unstable at room temperature for any significant length of time. The major problem is the lack of colour change variants of bacterial luciferase for the second signal. We are investigating two possibilities for the colour variant. Firstly, a fusion of the luxA subunit with mOrange, a fluorescent protein. This has recently been shown (Dachuan Ke1 and Shiao-Chun Tu, 2011) to result in an additional peak on the emission spectrum at 560 nm, compared to the natural peak at 490 nm. Secondly, a natural accessory YFP for the Vibrio fischeri luciferase, isolated from a yellow bioluminescent strain, which shifts the peak to longer wavelengths and increases the intensity.

In either case, an unaltered lux operon would be expressed constitutively. Either the accessory protein or the mOrange fusion would be expressed inducibly, and a ratio taken between the two peak intensities.

3. Development of a cheap and easy sensing kit

We are developing a cheap kit for quantification of the lux-based ratiometric construct. All components are inexpensive and readily available. It is self-contained, based on arduino, and will be compatible for use with a PC or an android smartphone, for ease of use in the field.

4. Development of new biosensors and adaptation of old biosensors

It is important that this standard is compatible with existing biobrick sensors. Therefore, we have selected a few biosensors to adapt to and characterise with our constructs. Additionally, we are investigating new and novel biosensors. We have decided to use riboswitches, as they are not represented in the registry and may become more widely used in the future. We have obtained fluoride and magnesium riboswitches to characterise and adapt.

5. Development and use of a custom quick-germination strain of Bacillus subtilis

Bacillus subitilis forms extremely hardy spores, which can be kept at room temperature on desiccated medium. Compared to E.coli, which must be kept in a freezer or freeze-dried for transport, the practical benefits of using subtilis are self-evident. We have isolated genes in subtilis that can be overexpressed to considerably shorten germination time.