Biology subjects

Liyanagedera, S. B. W.

Publications and source records attributed to Liyanagedera, S. B. W..

4 recordsLinked to original sources

bioRxiv2025

Horseradish peroxidase as an electrochemical reporter protein for cell-free biosensors

Cell-free biosensor systems offer a promising platform for portable diagnostics. However, most employ fluorescent reporter proteins that require complex instrumentation and can be affected by photo-bleaching and auto-fluorescence, limiting translatability. Electrochemical reporters do not suffer from these drawbacks. Here, we evaluate horseradish peroxidase (HRP) as a redox enzyme reporter for cell-free biosensor systems. HRP was synthesized in an E. coli cell-free transcription-translation system supplemented with hemin, calcium acetate, and commercial disulfide bond enhancers. The electrochemical detection of its activity was established by chronoamperometry, with H2O2 as a substrate and tetramethylbenzidine as a redox mediator. Cell-free expressed HRP produced a strong steady state current compared to a catalytically inactive mutant and a no-template control. Kinetic analysis showed a Km for the cell-free expressed HRP close to that of the native enzyme. To explore the potential of HRP as an electrochemical reporter, we placed it under the control of a tetracycline-responsive regulatory promoter and demonstrated a 3-fold current increase in the presence of anhydrotetracycline. These results support HRP as an electrochemical reporter for cell-free biosensors, offering a practical alternative to optical reporters for future use in handheld analytical devices. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=46 SRC="FIGDIR/small/694927v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@1b39294org.highwire.dtl.DTLVardef@5c3578org.highwire.dtl.DTLVardef@1ba827forg.highwire.dtl.DTLVardef@1cd8bd1_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

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

ATP Regeneration from Pyruvate in the PURE System

The Protein synthesis Using Recombinant Elements ( PURE) system is a minimal biochemical system capable of carrying out cell-free protein synthesis using defined enzymatic components. This study extends PURE by integrating an ATP regeneration system based on pyruvate oxidase, acetate kinase, and catalase. The new pathway generates acetyl phosphate from pyruvate, phosphate, and oxygen, which is used to rephosphorylate ATP in situ. Successful ATP regeneration requires a high initial concentration of[~] 10 mM phosphate buffer, which surprisingly does not affect the protein synthesis activity of PURE. The pathway can function independently or in combination with the existing creatine-based system in PURE; the combined system produces up to 233 {micro}g/ml of mCherry, an enhancement of 78% compared to using the creatine system alone. The results are reproducible across multiple batches of homemade PURE, and importantly also generalise to commercial systems such as PURExpress(R) from New England Biolabs. These results demonstrate a rational bottom-up approach to engineering PURE, paving the way for applications in cell-free synthetic biology and synthetic cell construction.

synthetic biology↗

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↗