TXTL-Powered K1F Internal Capsid Protein Engineering for Specific, Orthogonal and Rapid Phage-based Pathogen Detection

The internal capsid proteins that reside within phage of the Podoviridae family hold high potential for being used as sensitive and reliable diagnostic tools. The concealed nature of the capsid interior ensures that any encapsulated signal or signal generating enzyme, e.g., fused to an internal capsid protein, is suppressed whilst the phage is unaccompanied by its host. Furthermore, the only naturally occurring mechanism for releasing the internal capsid proteins, and therefore exposing their amalgamated signal/enzyme, is for them to be passed through the tail and subsequently ejected out of the phage, a post-adsorption phenomenon which occurs when the host is present, thus presenting a precise model for signal/enzyme release only upon pathogen presence. Here, a small N terminal subunit of the NanoLuc luciferase is fused and incorporated into the K1F internal capsid structure using a simple, non-genomic method. This internalised subunit is exposed to the test solution containing its C terminal counterpart (natural complementation immediately forms the full NanoLuc enzyme) and substrate furimazine in an inducible manner which mimics the presence of the K1F host, E. coli K1 thereby presenting a novel method for rapidly detecting this disease causing pathogen. Finally, it is expected that by building upon this internal capsid protein engineering approach, which completely bypasses the time-inducing processes of intracellular nucleic acid transcription and translation, an unprecedentedly rapid detection device can be developed for an array of bacterial pathogens. ### Competing Interest Statement The authors have declared no competing interest. * AMR : Antimicrobial resistance ELISA : Enzyme linked immunosorbent assay AuNP : Gold nanoparticle PCR : Polymerase chain reaction NASBA : Nucleic acid sequence-based amplification LAMP : Loop-mediated isothermal amplification RPA : Recombinase polymerase amplification GP : General practice LFT : Lateral flow test TXTL : transcription-translation ICP : Internal capsid protein CtNL : C-terminal sub-unit of NanoLuc NtNL : N-terminal sub-unit of NanoLuc Wt : Wild-type PFU : Plaque forming units RLU : Relative light units GFP : Green fluorescent protein LoD : Limit of detection SoD : Speed of detection