From carbon nanotubes to zinc porphyrins: engineering proteins to interface with non- biological molecular systems.

Bacterial resistance to antibiotics is one of the most significant crises in modern healthcare. The most widely utilised class of antibiotics (and therapeutics in general)are the beta-lactams, which include ampicillin, amoxicillin and methicillin. By far the main mechanism bacteria use to confer beta-lactam resistance is the production of beta-lactamases that hydrolyses the pseudo-peptide bond in the beta-lactam ring critical for its antibacterial activity. The aim of this project is to generate a high-information content miniaturised sensor with the capacity to rapidly detect the presence and type of beta-lactamase to allow more effective clinical use of current beta-lactams. To achieve this engineered beta-lactamase inhibitory protein (BLIP) will be selectively interfaced with a graphene field-effect transistor so that protein-protein interactions are transduced into a change in resistance of graphene-bridged microelectrodes through both bulk binding effects and electrostatic surface interactions. This system has the potential to sense events connected to single protein molecules. BLIP is the ideal sensor as it binds a wide variety of clinically relevant beta-lactamases with very distinct surface-facing electrostatics and interaction properties, which will potentially offer discrete electronic signatures. A key requirement is defined and directed interfacing of BLIP with graphene to maximise communication between the two components. This will be achieved using a synthetic biology approach to design in silico residue positions for the recombinant incorporation of non-native chemical handles (e.g. phenyl azide chemistry) that will allow high precision and optimal interfacing of protein with graphene. Detailed single molecule analysis (AFM/STM) of the protein-graphene interface will lead to the microfabrication of the protein-graphene-electrode biosensing platform and begin monitoring and assessing binding events through changes in conductivity.

First rotation project (Jones, Cardiff). The student will initiate the first round of BLIP variant design in silico, introduce the selected mutations, and using a reprogrammed genetic code approach incorporate the new reactive handles using non-natural amino acid at the defined positions. The student will produce and functionally test the selected BLIP variants to ascertain the effect of the mutations.

Second rotation project (Cardiff and Exeter Physics). The student will begin optimising approaches for directly assembling the BLIP variants on graphene. Two approaches will be taken: (1) direct covalent linkage through the photogeneration of reactive nitrene radicals; (2) Click addition of ?-stacking adducts to the BLIP. The student will analyse the BLIP-graphene interface using single molecule imaging approaches (AFM-STM). Functional studies will also begin through imaging the formation of BLIP-beta-lactamase complexes on grapheme.