Evaluation of Engineering Potential in Undomesticated Microbes With <scp>VECTOR</scp>

Genetic engineering research has predominantly focused on well-characterised organisms like Escherichia coli and Bacillus subtilis, with methods that often fail to translate to other microorganisms. This limitation presents a significant challenge, particularly given the increasing isolation of large microbial collections through high-throughput culturomics. In response, we developed a scalable, high-throughput pipeline to evaluate the engineerability of diverse microbial community members we named VECTOR (Versatile Engineering and Characterisation of Transferable Origins and Resistance). We utilised a library of vectors with the Bacterial Expression Vector Archive (BEVA) architecture that included combinations of three antibiotic resistance genes and three broad host-range origins of replication (pBBR1, RK2 and RSF1010) or the restricted host-range R6K with an integrative mariner transposon. We tagged each vector with green fluorescent protein and a unique nucleotide barcode. The resulting plasmids were delivered en masse to libraries of undomesticated microbes from plant microbiomes in workflows designed to evaluate their ability to be engineered. Utilising OD₆₀₀ and relative fluorescence measurements, we were able to monitor genetic cargo transfer in real time, indicating successfully engineered strains. Next-generation sequencing of plasmid molecular barcodes allowed us to identify specific vector architectures that worked well in particular bacterial strains from a large community. Modifications to the procedure facilitated isolation of engineered microbes.