WO2007083137A1

WO2007083137A1 - Whole cell biosensor using the release of a volatile substance as reporter

Info

Publication number: WO2007083137A1
Application number: PCT/GB2007/000171
Authority: WO
Inventors: Stephen Nicklin; Megan Louise Cooper; Natasha Joanna D'souza
Original assignee: The Secretary Of State For Defence
Priority date: 2006-01-18
Filing date: 2007-01-18
Publication date: 2007-07-26
Also published as: GB0600960D0; GB2447396A; GB0812716D0

Abstract

A whole cell biosensor comprising a gene for a recognition protein and a gene fusion of at least one reporter gene and an inducible promoter so as to detect a specific analyte. The recognition protein (14) interacts with the analyte (12) in such a way that it is able to activate transcription from the promoter (18) and the reporter gene (20, 22) encodes a protein which produces a detectable response the reporter gene being located downstream of the inducible promoter. The detection of the analyte is signalled by the production of an odour (26). The whole cell biosensor may also produce a further signal such as light (24).

Description

WHOLE CELL BIOSENSOR USING THE RELEASE OF A VOLATILE SUBSTANCE AS REPORTER

The present invention relates to a biosensor and more specifically to a dual function whole cell biosensor. The present invention also relates to the method of fabricating such a whole cell biosensor.

A biosensor, by definition, consists of a biological material coupled with a transducer to enable the detection of various substances in different media. Whole cell biosensors are intact, living microbial cells that have been genetically engineered to produce a measurable signal in response to a specific chemical or physical agent in the environment. Whole cell biosensors have a number of advantages over other types of biosensor: they avoid lengthy and expensive operations of enzyme purification and they preserve intracellular enzymes in their natural environment which protects them from inactivation by external factors. Whole cells can act as multipurpose catalysts for processes which require more than one enzyme and can be immobilised in proximity to transducers for downstream signal processing.

A whole-cell biosensor has two essential components: (i) a biological recognition element that interacts with the target compound, termed the analyte, in such a way that it is then able to activate transcription from a particular promoter; and (ii) a reporter gene(s) that encodes the proteins that generate a detectable response. The biosensors known in the art usually produce a visible signal. Whole cell biosensors have great potential for signal amplification as a low level of target analyte can induce the expression of many copies of the reporter protein(s).

In order for the whole cell biosensor to signal the presence of an analyte, the combination of the recognition and reporter elements detailed above must be successful. The recognition component of a whole cell biosensor needs to selectively recognise the target analyte and induce transcription from a specific promoter. Many species of micro-organism have adapted to use a diverse range of xenobiotic compounds available in their environment for growth. To do this they produce recognition proteins that bind the analyte of interest. It is therefore possible to engineer bacterial signalling molecules so that they recognise and are activated by the analyte of choice. Whole cell biosensors can be linked to a variety of transducers and can produce a variety of outputs. Several types of reporter gene can be used to create whole-cell biosensors which generate colorimetric, fluorescent, luminescent, chemiluminescent or electrochemical signals. For example, electrochemical biosensors usually have cells immobilised on an electrode and amperometric or potentiometric techniques are used to monitor cellular responses and optical biosensors employ cells immobilised on optical fibres or waveguides and absorbance or luminescence signals are measured. Other measurement devices include oxygen and pH electrodes as well as ion or substrate/product-specific electrodes for detection of reaction products.

The cells used in whole cell biosensors need to be immobilised on the transducer without losing the cells' viability and sensing activity. Immobilisation of cells has many advantages over the use of suspended cell liquid systems for use in biosensors. The chance of accidental spillage is dramatically decreased which is an important consideration when dealing with genetically modified strains. Immobilisation also helps in the stabilisation of the system, thus enhancing its operational and storage stability. Immobilisation increases the mechanical stability and allows the creation of thin films of matrix for rapid diffusion and short analysis times. The biological material can be immobilised via a number of known methods such as adsorption, entrapment, covalent binding, cross-linking or a combination of all of these techniques. Whilst drying cells onto surfaces is known to immobilise cells, it is not considered to be a sufficiently stable system for the biosensor of the invention as there is a potential to lose the cells to the external environment which is undesirable on health and safety grounds.

Most current biosensors only produce light as a signal and, as a result, can only be interrogated when there is little or no background light. The biosensor disclosed in US patent no. 5972638 had to be interrogated between dusk and dawn so that the emitted light from bacteria was not obscured by background light. In addition, light emission cannot be detected if the biosensor is to be used underground, which presents difficulties if the biosensor were to be seeded onto a mine field. Electrochemical biosensors produce a signal which can be detected by the user as long as he is near to the device. However, in the case where the sensor is being used for remote detection, sophisticated electronics need to be incorporated into the electrochemical biosensor which greatly increases the cost of the device. The whole cell biosensor of the present invention seeks to overcome this problem by producing a signal which can be detected, irrespective of the amount of background light or area of use.

The biosensor of the present invention is a whole cell biosensor for detecting an analyte and converting cellular responses to said analyte into a detectable signal wherein said biosensor comprises a gene for a recognition protein and a gene fusion of at least one reporter gene and an inducible promoter, said at least one reporter gene being located downstream of the inducible promoter, and wherein the detectable signal comprises an odour.

The use of an odour as a signalling mechanism will facilitate detection of low volatility compounds by providing a secondary vapour trail chosen specifically for ease of detection. The secondary vapour trail can be detected by dogs, commercial off the shelf or bespoke detection equipment or even by the human nose. Examples of commercial off the shelf detection equipment are gas chromatography coupled mass spectroscopy, Fourier transform infra red spectroscopy, Gas Tech tubes and sulphur chemiluminescence. The secondary vapour trail produced will overcome the problems encountered with light emitting biosensors in that it can be used and consulted at any time of the day and in any level of light. The scent also provides an efficient stand-off detection capability. Further, the whole cell biosensor does not require electronic components for the production of a scent which renders the biosensor of the invention cheaper to manufacture than conventional biosensors.

The scent produced by the device must have a higher volatility than the analyte being detected so that it can be readily detected. Selection of the scent to be used is also based on a number of other factors such as a need for precursor agents, biodegradability of the product and the background odours already present in the environment. Examples of detectable scents released by bacterial systems are: acyl esters, hydrogen sulphide and methanethiol. Suitable reporter genes for these odours are, respectively, BEBT, a gene having high homology to the cystalysin gene from Treponema denticola ATCC 35404 and METase.

The preferred gene for scent production in the present biosensor is homologous to the METase gene of Pseudomonas putida sp.ICR3460. METase is a useful reporter gene as it can be easily incorporated in a biosensor and is able to generate large amounts of methanethiol which can be unambiguously detected. A preferred embodiment of the invention is where the biosensor produces a further signal, preferably at the same time as the olfactory signal is produced. In a more preferred embodiment of the invention, the further signal is a visual signal. This visual signal is preferably either fluorescence or luminescence. In this embodiment there will be the need for electronic components such as a transducer, luminometer or fluorometer in order for the light to be detected.

In order to find a suitable recognition protein one can screen a library of biological materials for interactions with analytes. If no interactions are produced as a result of this screening, it is possible to create a mutant strain of the material by using random or site directed mutagenesis to mutate the binding region of the recognition protein so that it will be activated by an analyte of choice. An example of a bacterial recognition protein which can be mutated to detect the presence of analytes other than its natural effectors is XyIR.

A reporter gene will produce the desired signal output of the whole cell biosensor such as odour or light. Examples of preferred reporter genes are: METase for production of methanethiol, an easily detectable scent, green fluorescence protein for a fluorescent signal and bacterial luciferase for a luminescent signal. More than one reporter gene can be incorporated into a whole cell biosensor so that more than one detectable signal can be produced, e.g. a scent and light.

To make a whole cell biosensor the reporter genes must be placed downstream of an appropriate DNA promoter. Transcription from this promoter will be triggered by a recognition protein which recognises and is activated by an analyte. The gene fusion, consisting of the promoter upstream of the reporter gene(s), is cloned into a plasmid or the genome of a host cell along with the recognition protein gene. The genome may be a eukaryotic, prokaryotic or viral host genome.

According to a further aspect, therefore, the invention comprises a method of constructing a whole cell biosensor for detecting an analyte and converting cellular responses to said analyte into a detectable signal comprising an odour, which method comprises cloning a gene fusion including at least one reporter gene which is capable of producing a detectable odorous signal and an inducible promoter into a cell and either previous to or subsequent to said cloning also providing said cell with a gene for a recognition protein selected to recognise said analyte.

The biosensor of this invention can be used in the form of a liquid culture contained in a culture vessel but in a preferred form of the biosensor of this invention, the biological material used is fixed by any suitable means such as an immobilisation matrix. The immobilisation matrix used to fix the biosensor of the present invention must not only allow access of the analyte to the biological material, but also allow emission of a volatile odour as a signal that the analyte has been detected. Immobilisation of cells onto a membrane or direct immobilisation onto a surface (such as beads in a column) would address this issue, but both of these techniques have disadvantages. Immobilisation onto membranes can allow loss of cells unless they are directly adhered to the membrane whereas direct attachment of cells onto a surface requires a specific cross-linker, which poses the risk of viability loss, or the need for cell surface expression of particular proteins such as cellulose binding proteins. Immobilisation of cells into matrices such as agar, agarose and alginates can be easily attempted, while sol-gels are an attractive option due to their optical clarity. Pore size optimisation is crucial if immobilisation matrices are used due to the requirement for substrate entry and odour outlet, as well as acceptable response times. Entrapment onto membranes is relatively easily achieved but requires a secondary barrier to prevent cell loss, for example a 0.2μm filter. The immobilisation matrix must have optical clarity for both the excitation wavelength, if required, and the emission wavelength of light where a visual signal is required to be emitted.

The biosensor according to the present invention is capable of being attached or contained on a solid support incorporating electronics, for example a micro-electro-mechanical system (MEMS) type device which is another preferred embodiment. Also, existing sensor devices can be modified to incorporate the scent detection system into the air outlet valve of the device.

The preferred visual reporter genes used in the biosensor of the invention are green fluorescence protein (GFP), which produces fluorescence, and bacterial luciferase, which produces luminescence, as light production from these proteins does not require addition of external substrates. In the case of GFP, a single gene is involved, but UV light is required for excitation. Bacterial luciferase can be used as a reporter protein by supplying a 5-gene operon and this system has the advantage that it requires no excitation by external light sources. However, the cell has to expend its own energy reserves to luminesce which means that not as much light will be emitted from bacterial luciferase as from GFP containing cells.

The present invention will now be described with reference to the accompanying drawing (Figure 1).

Figure 1 is a schematic of the process which occurs in a preferred whole cell biosensor to produce olfactory and visual signals.

Figure 1 shows an analyte 12 binding to a recognition protein 14. This binding event activates the recognition protein 14 and the complex 16 thus made then binds to a promoter region 18. This binding triggers transcription of the reporter genes 20, 22 for the odorous 26 and visual 24 signals respectively.

A whole cell bioreporter according to the present invention was created containing the wild type xylR. gene and the Pu promoter upstream of the gfp and METase genes. This biosensor produced both a visual signal of fluorescence from gfp and an odorous signal of methanethiol in response to the presence of the analyte toluene.

The use of computer modelling can be used to successfully identify site specific changes to make a protein which would allow recognition of any compound, whether known or novel, which means that any substance could be the potential target of a biosensor.

It is envisaged that the whole cell biosensor of the invention will find application in many fields where analyte detection is required, perhaps where a visual output is not practical, such as where the sensor is placed underground, or where the operator of the device is unable to see a visual output, for example if there is too much background light or if he is blind. It can be used for drug detection, incorporated into masks for hazardous gas detection, transport systems, even to mask smells such as in shoe innersoles or at sewage outlets. However, a preferred use of the biosensor of the invention is for the detection and diagnosis of explosives and other dangerous or illegal substances under all operational conditions. This includes detection of terrorist improvised explosive devices (IED), both conventional and more recently chemical, biological, radioactive and nuclear (CBRN) such as in the remote sensing of airborne bacteria or the detection of pathogens.

Further potential applications of the biosensor of this invention include environmental applications, for example the detection of pesticides and other contaminants of surface waters such as streams, rivers or ponds or use in connection with conventional munitions disposal (CMD), force protection or mine detection. It could also be used for on-line monitoring of buildings and transport systems by incorporation into a handheld biosensor for pollutants and other analytes of interest.

There is in particular a requirement for chemical and biological detection devices with reduced cost and size that yet provide increased stand-off detection capability. The biosensor described herein can provide these desired capabilities by virtue of its vapour signalling output, preferably in conjunction with a visual output. Also, due to the nature of the biosensor, it will be capable of working in seawater and hence may find future application in the field of underwater detection.

Claims

Claii

1. A whole cell biosensor for detecting an analyte and converting cellular responses to said analyte into a detectable signal, wherein said biosensor comprises a gene for a recognition protein and a gene fusion of at least one reporter gene and an inducible promoter, said at least one reporter gene being located downstream of the inducible promoter, and wherein the detectable signal comprises an odour.

2. A whole cell biosensor according to claim 1 wherein the odour produced is selected from one of acyl esters, hydrogen sulphide and methanethiol.

3. A whole cell biosensor according to claim 2 wherein the reporter genes selected are, respectively, BEBT, a gene with high homology to the cystalysin gene from Treponema denticola ATCC 35404 and METase.

4. A whole cell biosensor according to any of claims 1 to 3 wherein the biosensor produces a further detectable signal in response to the analyte.

5. A whole cell biosensor according to claim 4 wherein the further signal is produced at the same time as the odour.

6. A whole cell biosensor according to claim 4 or claim 5 wherein the further detectable signal is visual.

7. A whole cell biosensor according to claim 6 wherein the visual signal is fluorescence or luminescence.

8. A whole cell biosensor according to claim 7 wherein the fluorescence is produced by green fluorescence protein.

9. A whole cell biosensor according to claim 7 wherein the luminescence is produced by bacterial luciferase.

10. A whole cell biosensor according to any of claims 1 to 9 which is in a liquid culture contained in a culture vessel.

11. A whole cell biosensor according to any of claims 1 to 9 which is immobilised into a matrix comprising agar, agarose or a solgel.

12. A whole cell biosensor according to any of claims 1 to 9 which is immobilised on a solid support.

13. A whole cell biosensor according to claim 12 wherein the solid support comprises a transducer.

14. A whole cell biosensor according to claim 12 incorporated into a micro-mechanical system (MEMS) type device.

15. A whole cell biosensor according to claim 13 or claim 14 which is immobilised by adsorption, entrapment, covalent binding, cross-linking, or a combination of any of these.

16. A whole cell biosensor according to any preceding claim in operational combination with means for detecting the odour produced.

17. A whole cell biosensor according to claim 16 wherein the said means comprises gas chromatography coupled mass spectroscopy, Fourier transform infra red spectroscopy, Gas Tech tubes or sulphur chemiluminescence.

18. A method of constructing a whole cell biosensor for detecting an analyte and converting cellular responses to said analyte into a detectable signal comprising an odour, which method comprises cloning a gene fusion including at least one reporter gene which is capable of producing a detectable odorous signal and an inducible promoter into a cell and either previous to or subsequent to said cloning, also providing said cell with a gene for a recognition protein selected to recognise said analyte.

19. A method according to claim 18 wherein the gene fusion includes a further reporter gene downstream of the promoter element, said further reporter gene being capable of producing a second detectable response to an analyte to be detected.

20. A method according to claim 19 wherein the further reporter gene is a visual reporter gene which produces a detectable visible signal.

21. A method according to one of claims 18 to 20 and comprising the further step of immobilising the cell onto a solid support by adsorption, entrapment, covalent binding, cross-linking or a combination of any of these.

22. A method according to any one of claims 18 to 21 wherein the cloning takes place into a plasmid.

23. A method according to any one of claims 18 to 21 wherein the cloning takes place into a eukaryotic, prokaryotic or viral host genome.