Development of a robotic directed evolution approach to whole cell biosensor production

Whole cell biosensors are single-celled organisms that have been re-engineered to detect and respond to elements of their environment. Whole cell biosensors can be used to measure the presence of various molecules by responding in a detectable way, for instance by production of fluorescence or a colored pigment. The ability to accurately measure molecular targets is required broadly in fields including medical diagnostics and environmental monitoring.

When compared to existing detection methods, whole cell biosensors are advantageous in that they are low-cost, self-replicating and bio-degradable. These properties would make whole cell biosensors suitable for field-testing and point-of-care diagnostics, including in resource limited locations.

The aim of this research is to create a microfluidics-based platform, coupled to engineered biological biosensor scaffolds, that can be applied for engineering and producing whole cell biosensors that are adaptable to different targets. This will be achieved via three interdisciplinary and complementary research objectives. Our first objective will apply synthetic biological engineering in bacteria to create a modularly designed biosensor in which the sensing element can be 'chopped and changed' for new targets without re-engineering the entire system.

This will leverage past engineering of modular biosensors, which demonstrate it is possible to re-purpose nanobodies as sensing elements in a modular fashion. Our work will similarly leverage physical and computational pipelines for designing and modifying the targets of nanobodies, which are well-established, making nanobodies a particularly promising starting point for creating for re-targetable

sensing domains.

Our second objective will take designed nanobody biosensors and apply state-of-the-art directed evolution technology to optimise their functionality as biosensors, as well as to re-direct them to alternative targets when desired. Our approach will combine a robotic microscope and microfluidics, which make it possible to observe whole cell biosensors for extended periods of time at the level of individual cells, including their response to time varying stimuli.

Via such an approach it is also possible to deliver light signals to individual cells to select the best performing examples out of large libraries of engineered variants. The

process of iteratively delivering 'survive' or 'die' signals to cells will be used to drive large libraries toward desired performance: in this case sensitive and specific detection of important biological targets.

Finally, the third objective will be to apply the modular nanobody designs and microscope based engineering platform to build novel whole cell biosensors, with a particular focus on developing highly specific and reliable biosensors as a low-cost and accessible diagnostics platform. This project falls within the EPSRC 'Healthcare Technologies' and 'Engineering' research areas.

Within Healthcare it directly addresses challenges surrounding optimisation of disease prediction, diagnosis and intervention is one of the four grand challenges. Within Engineering the project's interdisciplinary focus lies at the intersection of the EPSRC Research Areas "Synthetic Biology" and "Robotics"