(530a) A Toolbox of Characterized Genetic Parts for Staphylococcus Aureus

Staphylococcus aureus is a prominent organism of study as the source of many diseases in humans, including serious antibiotic resistant infections. However, synthetic biology approaches have not been implemented in S. aureus due to limited numbers of characterized genetic parts, and previous efforts to genetically manipulate this organism have employed inefficient cloning techniques. Here, we report the first toolbox of standardized genetic parts for S. aureus, including 24 constitutive promoters, 13 RBSs, 5 terminators, and 3 plasmid replicons used to precisely control gene expression. To standardize measurements of the genetic parts, we established a relative expression unit for S. aureus using a single fluorescent plasmid as reference, then used this standard unit to assay genetic parts in strain ATCC 12600 via flow cytometry. We designed a Type IIS DNA assembly strategy for the efficient construction of multi-part synthetic DNA constructs and created compatible promoter and terminator part plasmids. Twenty-four constitutive promoters were assayed in S. aureus and Bacillus subtilis and demonstrated dynamic ranges of 380-fold and 122-fold, respectively, and illustrated transferability of our promoters between these gram-positive bacteria (Pearson’s coefficient = 0.946). Subsets of these promoters showed different sequence enrichment patterns depending on their strengths. The characterized RBSs exhibited 87.7-fold dynamic range, and each plasmid replicon presented different characteristics. The Rho-independent terminators showed up to 95.3% reduction in gene expression in S. aureus and correlate with termination strength as previously measured in Escherichia coli (Spearman’s coefficient = 1). We applied our toolbox to design and rationally engineer tetracycline and IPTG sensors in S. aureus through an iterative design-build-test-learn cycle, with the final tetracycline sensor demonstrating 25.8-fold dynamic range. This toolbox extends the number of genetic parts available for gram-positive bacteria and will allow researchers to apply techniques used in synthetic biology to examine and engineer biological processes in S. aureus.