Logic-directed evolution of new biosensor molecules in vivo

The ability to rapidly and accurately detect small molecules has widespread use within security and defence contexts such as detection of explosives, biological agents or pathogens. This project will develop a new platform for the development of novel sensors. Sensors require highly specific detection of molecules in very low concentrations. The use of biological approaches for this purpose is attractive since many biological systems have evolved for precisely this purpose. While biological diversity offers a rich source of variety, it is ultimately limited to what can be isolated and characterised. In vitro methods for evolving new biological functionality and diversity have proven useful but are ultimately limited in the range of biological diversity that can be sampled. This project will develop a prototype for the evolution of new biological specificity in vivo, with feedback bio-'logic' circuitry that will enable end-point evolution of biological systems to new specificities. The isolation of new proteins with altered specificity will be essential for the creation of a new generation of biosensors.

This proposal seeks to develop a new platform for the development of new-to-nature protein specificities suitable for use in biosensor devices. We will develop a novel method for the directed evolution of new specificities in signal transduction systems. We have developed an in vivo system in bacteria that is able to direct mutation to specific target genes, thus providing the basis for a mechanism of targeted directed evolution in vivo. This project will create a biological logic circuit that will self-limit the evolution to the point when a new specificity has been created, at this point the evolution will stop and a selectable output will be switched on. We have termed this end-point evolution.

The key problem in creating new biological specificity is identifying it. The sequence space in biological diversity is so vast, that identifying the one mutant that has the desired specificity is difficult. This project aims to tackle this directly by the design, modelling and implementation of genetic control circuits: 'Bio-Logic'. This will be programmed for end-point evolution so that the desired phenotype will switch off further mutation and switch on an output and selectable marker. The ability to threshold the response and increase stringency of selection is important and will come from lowering the concentration of the response molecule for which specificity is being engineered, and/or increasing the stringency of selection.

The heightened terrorist threat has lead to the requirement for rapid and accurate detection of chemical and biological agents, as well as explosives. It must be considered that although the perceived threat from biological agents is low, recent enhancements in technologies such as gene synthesis, means that threat levels should not be treated as non-existent, nor indeed unchanging. The ability to detect biologically infectious agents is a strategic security requirement as well as a medical challenge. The development of cheap and easy to deploy explosives sensors in the environment could have a great impact on reducing the combat threat of expolsive devices, such as landmines and improvised explosive devices, as well as reducing the civilian impact from the millions of landmines that have been left in the ground. We therefore consider that the development of sensors for infectious agents and/or explosives may have the greatest potential for a step-change in technological driven approaches to these key problems.

Imagine a real-time explosives detector that can 'smell' the presence of an IED before it is reached, or identify airline passengers who have been in contact with restricted chemicals. Imagine a pathogen detection system that can easily detect water borne pathogens in remote and inaccessible locations, or identify an infection before it poses a clinical threat.

There is an increasing demand for new fast and accurate sensor technology and these futuristic scenarios are not necessarily far off. Both of the targets identified above require the detection of small molecules at very low concentrations. The exquisite molecular recognition that proteins are capable of provides an ideal platform for the basis of recognition. However, its key limitation is the ability to develop new specificities for molecular recognition. This proposal seeks to tackle this issue directly in a format that is readily adaptable for new molecular targets. This can therefore provide a technological platform that can be applied to the rapid development of new sensors. This has the potential to transform the pathway to the development of new biosensors.