Biology subjects

Sagona, A. P.

Publications and source records attributed to Sagona, A. P..

3 recordsLinked to original sources

bioRxiv2025

High-yield bioproduction of virus-free P4-EKORhE multi-lysin transducing particles as an antimicrobial gene therapeutic

A description of the construction of the bioengineered P4-EKORhE and a comprehensive method for producing very high yields (up to 1012 particles per millilitre) enable the use of virus-like particles to transduce genetically encoded antimicrobials through a combination of synthetic biology and optimised upstream and downstream processing. The final product, a gene-delivered antimicrobial in the form of the multi-lysins cassette, is fully functional before and after packaging within P4-EKORhE particles. The antimicrobial activity of the multilysins cassette, characterized by its lysis proteins, was tested in vivo in both pure bacterial Escherichia coli (E. coli) cultures and in a model of infection using A549 immortalised human epithelial tissue cell cultures. This work exemplifies several bioproduction methods and demonstrates how the virology of the P4 and P2 phages can be harnessed to establish a bioprocess for producing transducing particles at very high yields, avoiding contamination by the natural virus while maintaining the antimicrobial effectiveness of the final product.

bioengineering↗

bioRxiv2024

Directional Immobilisation of SpyTag Bacteriophage on PDMS surfaces for Phage based Microfluidics

The increasing incidence of bacterial infections caused by antibiotic-resistant pathogens worldwide underlines the need to develop novel diagnostic tools enabling the early initiation of targeted antimicrobial therapy. One promising possibility is to unite the high specificity and sensitivity of phage-based applications with the speed and sensitivity provided by microfluidic devices. As a prerequisite of developing such systems, we aimed at the directional immobilization of phages on the surface of Polydimethylsiloxane (PDMS), a material commonly used for building such devices. Our work utilised the covalent interaction between two proteins: SpyTag, genetically encoded on the capsid of the phage, and BslA-SpyCatcher fusion protein, purified and surface displayed on PDMS. We demonstrate a simple methodology for the directional tail up immobilisation of SpyTagged Phage on to user defined locations on the surface of a PDMS device and subsequent on chip capture and infection of a cognate host. Our technique serves to illustrate a generally applicable solution to develop the next generation of phage based bio-sensors.

bioengineering↗

bioRxiv2024

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.

synthetic biology↗