A synthetic biology open hardware platform for antimicrobial resistance screening and characterisation in engineered E. coli

Antibiotic resistance is evolving at a pace that is significantly ahead of present-day technology, the drug development pipeline, and pharmaceutical prioritization. Therefore, it falls into the hands of the greater scientific community to address these needs in an efficient and collaborative manner. To address this urgent need, the innovative power of synthetic biology was harness to develop Target Surrogate E. Coli (TESEC) platform as a modular and cost-effective solution for accelerating antibiotic discovery. The TESEC platform leverages engineered E. coli strains to expedite the identification of drug candidates against infectious diseases. By replacing an essential metabolic pathway with a pathogen-derived equivalent, TESEC enables safe, controlled, and accelerated experimental testing. This platform capitalizes on the advantages of E. coli as a safer and faster model organism, with its rapid growth rate and easy quantification using absorbance plate readers. This PhD explores the potential of the TESEC system as an open science platform for high- throughput targeted antibiotic discovery, antibiotic target resistance landscape screening and characterization. This synthetic biology side is complemented by the development of an open hardware cost-effective 96 well microplate absorbance spectrophotometer to further enable accessible dissemination of the TESEC platform. The initial step was to scale up the TESEC protocol from a 96-well to a 384-well format, establishing a robust screening platform for ongoing and future target identification. The subsequently was to employ TESEC to study resistance using the second-line tuberculosis drug D-cycloserine (DCS) and its target, Mycobacterium tuberculosis alanine racemase (Mtb-alr). We optimized the tunable expression of Mtb-alr in E. coli against DCS, enabling solid screening, disk diffusion assays, and liquid culture analysis of resistance. Our TESEC resistance characterization pipeline successfully validated resistance in previously reported DCS-resistant Mtb-alr mutants and identified novel mutants. To validate and characterize these mutants, we analyzed growth characteristics in liquid culture, including final optical density, maximum growth rate, and lag time. Lag time analysis proved most significant, with the majority of mutants exhibiting lower lag time than the wild type, indicating their rapid adaptation to DCS exposure. These findings underscore the importance of considering lag time and the advantages of rapid adaptation in resistance studies. Notably, significant resistance across all analysis methods was observed in only two mutants, reinforcing the efficacy of D-cycloserine as an antibiotic with low resistance development. Furthermore, these results provide insights into future antibiotic target criteria. In addition to resistance characterization, we developed a low-cost tower spectrophotometer for absorbance measurements, a crucial component of the TESEC screening pipeline. Constructed using affordable materials and accessible tools, the prototype presently offers optical density measurements within a specific range of OD from 0.1-1.0. By employing passive light collimation by distance, we were able to avoid expensive optics. The spectrophotometer's open-source design facilitates replication, modification, and customization by other researchers, promoting collaboration and facilitating future enhancements. Overall, this research demonstrates the potential of the TESEC platform for screening and characterizing the resistance landscape of antibiotic targets. By using a range of resistance screening and analysis, the study enhances our understanding of specific adaptive strategies employed by resistant mutants. The development of a low-cost spectrophotometer further empowers researchers with limited resources, fostering collaboration and driving future advancements in antibiotic research.