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Previous iGEM teams have charaterised an impressive array of inducible promoters, along with other elements of biosensing circuitry... Read More


Parts for a reliable and field ready biosensing platform


Implementation of biosensors in real world situations has been made difficult by the unpredictable and non-quantified outputs of existing solutions, as well as a lack of appropriate storage, distribution and utilization systems. This leaves a large gap between a simple, functional sensing mechanism and a fully realised product that can be used in the field. We aim to bridge this gap at all points by developing a standardised ratiometric luciferase output in a Bacillus chassis. This output can be linked up with prototyped instrumentation and software for obtaining reliable quantified results. Additionally, we have reduced the specialized requirements for the storage and distribution of our bacteria by using Bacillus' sporulation system. To improve the performance of our biosensing platform we have genetically modified Bacillus’ germination speed. Lastly, we demonstrated the robustness of our system by testing it with a new fluoride riboswitch, providing the opportunity to tackle real life problems.

Introductory Video:


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 four parts:

Overview of Systems

1. Ribosense

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.

2. Ratiometrica and use of bacterial luciferase

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. Secondly, 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.

3. Development of a cheap and easy sensing kit - Instrumentation

We are developing a cheap kit for digital quantification of the lux-based construct. Developing cheap, functional bio-electronic equipment is crucial for the development of synthetic biology into an industrialised field. All components are inexpensive and readily available. It is self-contained, yet is is modular, allowing for customisation. It is based on the Arduino microcontroller, and is compatible for use with a PC or an android smartphone, for ease of use in the field.

4. Sporage and Distribution

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.

We hope that, taken together, these constructs will be of considerable use as a reliable and robust reporter chassis both for use by other iGEM teams and more widely.

Previous iGEM teams have characterised an impressive array of inducible promoters, along with other elements of biosensing circuitry. However, at the present time, the output from each is not consistent. Additionally, despite the unifying biobrick standards used, they do not necessarily couple together to make integrated test kits. The Cambridge iGEM 2012 team aims to take the true meaning of biobricks to heart, by creating an open and applied biosensor standard available for use by all subsequent teams. Such a standard could also potentially be used by industry and researchers in the field.

The biosensor aims to be modular in design, allowing the kits to be tailored to an individual's requirements, and to use light as an output to allow computer interfacing. We aim to use two luciferases, one to give a read-out of the input, and the other to act as a standard to allow fluctuations in colony size to be taken into account. Furthermore, we shall be using B. subtilis as our chassis, with the view to making the most of the spore forming capacity of bacteria to send out desiccated kits with long shelf lives.

Finally, we also plan to create a cheap electronic device together with a mechanical chassis, which would be able to automatically read the information provided by the luciferases (light intensity and wavelength) and convert them into calibrated digital information which could then be analysed and manipulated computationally.

Project Details

As stated above, the main aim of the project was to develop a bio-sensing standard to promote a platform for the development of novel biosensors that may work in a wealth of different ways but that can all be characterised and coupled to an output that is predictable, reliable and most importantly meaningful.

Standardised Outputs

Nature's approach to analyzing different chemical parameters. Note the cross talk between the sensory cascades, which renders the system highly unpredictable.

The use of biosensors within synthetic biology has one main advantage over traditional, electronic sensors, which is the diversity of chemical signals to which these finely tuned nanomachines can respond to. The ability of the cells to integrate and process information sensed in this way is powerful, but presently the complexity of the metabolic pathways used by cells for this confounds attempts at synthetic in vivo information processing. Until we have a far more complete picture of the interactions between cellular components that allow information processing (something which may be provided in the future by the field of Systems biology), the use of electronic circuits will remain a far more powerful and simple means of processing such information.

In order to transfer such information to a computer would require a biological - electronic interface. This implies that the bacteria would send a message of some sort, which will be detected by an electronic sensor. Many different cell types, each containing a complement of genes suitable for detecting ]a particular substance, would be used in different spatial locations. Separation of the different genes into different cellular compartments would prevent crosstalk between the different sensory cascades, improving the reliability and predictability of the constructs produced.

Our approach to analyzing different chemical parameters.

In order to best implement these principles, we have decided that every biosensor should be coupled to a standard output with its own response curves, to suit the customer's needs. Decoupling the culture/analyte solution from the detection system (e.g. an electronic one) would be a good idea as otherwise the behaviour of the electrode under different conditions might affect the results. Therefore we chose to use light as the signal transducer. This left us with a choice between biofluorescence and bioluminescence.

Whilst fluorescent proteins have been characterised in far greater detail than luciferases, part of the broader aim of the project is for the kit to be as affordable as possible. And given that the equipment to detect the emission spectra of luciferases is cheaper, we decided that a quantitative measurement of bioluminescence was a better option.

One of the greatest problems we seek to overcome in this project is that of consistent readouts. As is always the case with biology, predictability in our biosensing equipment was going to be an issue. To normalise for cell density productivity, it was decided that a ratiometric output would be absolutely necessary if the output from our biosensors was to be meaningful. Drawing on work done by James Brown and the Haseloff lab into reliable, predictable and quantitative ratiometric measurements using fluorescent proteins, we decided to use these principles as the basis of our work with luciferase. We also decided that, as a side experiment and proof of concept, we would attempt to achieve meaningful ratiometric outputs with fluorescent proteins that could be measured with an (all too expensive!) plate reader.

The construct made in the pJS 130 vector for the ratiometric measurement of IPTG concentrations with fluorescent proteins.

On the luciferase side of things, after a fairly deep trawl through the literature, an OFP-luciferase fusion was found where the emission spectra appeared sufficiently distinguishable from that of the normal bacterial luciferase (a fairly distinctive blue) that it could be measured using simple photo-resistors and coloured filter gels. The emission spectra of the OFP/luciferase fusion is shown below:

The expected output spectrum of our MOrange/luciferase fusion - Dachuan Ke and Shiao-Chun Tu (2011) DOI:10.1111/j.1751-1097.2011.01001.x

The construct that we hope to make is shown below:

The construct made in the pJS130 vector for the ratiometric measurement of IPTG concentrations with luciferase.


As the main crux of this project is a standardised output, we aimed to develop several biosensors, employing different mechanisms, to prove the extended functionality of the final product. To date, most of the biosensors in the registry use an inducible promoter to control expression of their reporter protein. We have used some of these as a proof of the compatability of our kit with a theoretical customers' sensors. However, we also explored another mechanism of biosensing in the form of riboswitches. These have the potential to be the detectors of the future providing a more standard way of designing input circuits and hopefully a faster sensing method as the transciption has already occurred (unlike inducible promoters).

The sensors:

Magnesium Riboswitch

Fluoride Riboswitch

Magnesium riboswitch

Magnesium is essential for life, being a vital component of many enzymatic reactions. Of particular interest for synthetic biology is its role in the action of DNA polymerase enzymes such as Taq. and Phusion. However, no teams have really characterized a sensor that can be used to measure its concentration in solution. Such a biological sensor exists in the form of the bacillus Mg2+ riboswitch. As shown in the diagram, we attempted to isolate this component and submit it as a biobrick, characterizing its function by inserting it into a derepressor construct.

The riboswitch acts as a transcriptional attenuator when Mg2+ is bound, causing disengagement of the RNA polymerase before it can access downstream ORFs. Consequently, these proteins are not expressed. The system that we used inserted this riboswitch just upstream of the LacI repressor in plasmid pJS130. The lac operator that LacI acted on was upstream of sfGFP, consequently expression of LacI blocked transcription of GFP.

To characterize this construct, we will use a 96-well plate reader to assay the effects of the different concentrations of Mg2+ and IPTG on the levels of GFP. We would expect the presence of either to allow the expression of GFP, however because transcriptional attenuation by the riboswitch occurs before expression of the repressor protein, it may be expected that Mg2+ will somehow demonstrate a dominant effect.

The construct made in the pJS130 vector for the detection of changes in Mg2+ ion concentrations

Fluoride riboswitch

We also plan on implementing and characterising a Fluoride riboswitch. This, unlike the Magnesium construct, is a positive regulator. The riboswitch, originally isolated from Bacillus cereus, serves as a transcriptional attenuator in the abscence of fluoride. In the presence of fluoride its conformation changes and the repression is lifted. In B. cereus this serves to permit translation of a fluoride efflux pump, which allows the bacteria to cope with the, potentially toxic, elevated fluoride levels in which it finds itself.


We have constructed a mechanical rotary device that is turned by an arduino-controlled motor to 'sense' from 6 different cuvettes that can be placed in the device and then left for automated detection. The arduino is also connected to two light sensors, one supplied with a blue and the other with an orange filter. The ratio of the light intensity at blue and orange frequencies can be measured to determine the strength of output signal from the bacillus.

The hardware is coupled to a graphical user interface (GUI) that was designed using wxpython. Python is particularly useful as the communication with the arduino microcontroller is done using serial programming, for which python has standard libraries. However, before any communication takes place between the user and the device, the arduino is loaded to perform the basic functions which are written in C++. The arduino and python were chosen for the ease of use and open platform. Also, the arduino is cheap and python is free!

Sporulation and Germination

sporulation and storage scheme

Another aspect to this project is the longevity of the product. Strains of bacteria expressing various receptors would be generated and stored as dormant spores. This allows the individual tubes of bacterial 'sensors' to sit in the user's cupboard until needed. When the user requires a specific sensor, the appropriate strain is selected and the bacteria can be germinated by following a simple protocol. They can then be placed into the arduino device, the sample loaded and the concentration profile measured.

Part of the main reason for choosing Bacillus subtilis as our chassis was because of it's abiltiy to form dormant spores. As part of our project, we aim to determine a protocol for inducing sporulation in Bacillus, and determine a very simple protocol for germinating the spores so they can be used as part of the system.

It is essential for the germination procedure to be as straight forward as possible, requiring minimal equipment and expertise, so that it could in theory be performed in the field, in a situation where the biosensor might be used.