A Cell-Free Toolbox to Anticipate, Learn and Counter Antimicrobial Resistance

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Cell-free synthetic biology leverages core biological processes outside of a cell by removing the cell membrane and DNA, providing a cell extract. Bacterial cell-free extracts typically contain around 400 proteins, as well as a plethora of RNA and metabolite species unique to the host cell. Cell-free extracts provide powerful dexterity to explore a range of enzymatic processes, including transcription, translation, energy regeneration, and amino acid metabolism - all potential antimicrobial targets. While E. coli cell-free gene expression (CFE) systems are the dominant tool within cell-free synthetic biology, we, and others, have recently shown that cell-free systems can be used to study almost any non-model microbe. While these new cell-free systems provide new opportunities, they remain relatively uncharacterised.

Specifically, we provide a Klebsiella pneumoniae CFE system that enables the safe study of antimicrobial resistance (AMR) inside a test tube. Since this model system remains uncharacterised, we will first look to quantify the K. pneumoniae CFE system in terms of metabolomics and proteomics, as well as develop a toolbox for mode of action studies (Objective 1). Then, we will uniquely exploit this cell-free toolbox to study a laboratory-evolved resistome (i.e., gentamicin and rifampicin) for K. pneumoniae. Here, the toolbox will show how individual AMR variants alter protein synthesis and intracellular antibiotic resistance (Objective 2). Lastly, we will exploit the cell-free toolbox to explore protein-protein interaction inhibitors (i.e., essential to CFE activity) to study an understudied antibiotic mechanism of action with recognized potential to overcome AMR (Objective 3). Overall, we create a new synergy between the two distinct disciplines of cell-free synthetic biology and infectious diseases to develop a new opportunity and capability that will place UK science at the forefront of international synthetic biology and AMR research.