Biophysical characterisation and design of optogenetic control elements

Inducible gene 'switches' are a core technology in synthetic biology that enable at-will gene expression and the programming of cellular function. Optogenetics makes use of light-controlled inducers and allows unparalleled temporal and spatial control of either gene expression or repression. A current focus of optogenetics is the development of systems that respond to different wavelengths of light, potentially allowing multiple control elements within a single cell; e.g. individually responding to blue, green and red light. Such 'multichromic control' is particularly desirable to the biological metrology conducted by the industrial partner (National Physical Laboratory; NPL) because targeted (spatiotemporal) stimulation provides greater confidence when measuring networked biological systems. However, until recently, green light inducers were lacking. The discovery and subsequent development of a construct containing coenzyme B12-dependent CarH, which responds to green light, now plugs this gap. However, the in vitro biophysical characterisation of these CarH constructs (they are chimeric fusion proteins) is limited, which limits the improvement and extension of this system in optogenetics. This project will use a range of biophysical techniques to investigate the mechanism of CarH inducers.

The project will make use of (light-activated) time-resolved spectroscopy - both optical and mass spectrometry based - to characterise coenzyme B12 binding and the light-dependence of both the protein oligomeric state and binding to DNA, which are functionally relevant. Mutagenesis and construction of new chimeric proteins will be used to both investigate mechanism and develop systems with improved or altered activity. These new variants will then be tested for optimised optogenetic transcriptional regulation in vivo using green light as previously described. They will then be integrated, into established multichromic optogenetic actuator systems.

The project involves the biophysical characterisation of a recently developed optogenetic control system, based on a natural coenzyme-B12 dependent protein. It falls within the remit of 'technologies and methodological development', 'synthetic biology', and more generally 'world class bioscience' and is firmly embedded at the interface of chemistry, biology and physics, a key driver for BBSRC in the 'Exploiting new ways of working' agenda. It draws on the core bioscience skills of mathematics (data analysis), and provides advanced research training in recombinant protein production and biophysical characterisation using a range of solution spectroscopy and mass spectrometry techniques. Overall, the work will provide a highly interdisciplinary approach to (bio)chemistry/ biophysics-based research, offering highly diverse training opportunities to a PhD student. The Manchester-based supervisors have long-established collaborations with the industrial partner at the National Physical Laboratory (NPL) and the student will have the additional benefit of being able to access the local supervisors' laboratories on a daily basis, as they are co-located within the same building, the Manchester Institute of Biotechnology (MIB).