Systematic part transfer by extending a modular toolkit to diverse bacteria

Abstract

ABSTRACTIt is impractical to develop a new parts collection for every potential host organism. It is well-established that gene expression parts, like genes, are qualitatively transferable, but there is little quantitative information defining transferability. Here, we systematically quantified the behavior of a parts set across multiple hosts. To do this, we developed a broad host range (BHR) plasmid system compatible with the large, modular CIDAR parts collection forE. coli. This enabled testing of a library of DNA constructs across the Pseudomonadota –Escherichia coli, Pseudomonas putida, Cupriavidus necator, andKomagataeibacter nataicola. Part performance was evaluated with a standardized characterization procedure that quantified expression in terms of molecules of equivalent fluorescein (MEFL), an objective unit of measure. The results showed that the CIDAR parts enable graded gene expression across all organisms – meaning that the same parts can be used to programE. coli, P. putida, C. necator, andK. nataicola. Most parts had a similar expression trend across hosts, although each organism had a different average gene expression level. The variability is enough that to achieve the same MEFL in a different organism, a lookup table is required to translate a design from one host to another. To identify truly divergent parts, we applied linear regression to a combinatorial set of promoters and ribosome binding sites, finding that the promoter J23100 behaves very differently inK. nataicolathan in the other hosts. Thus, it is now possible to evaluate any CIDAR compatible part in three other hosts of interest, and the diversity of these hosts implies that the collection will also be compatible with many other Proteobacteria (Pseudomonadota). Furthermore, this work defines an approach to generalize modular synthetic biology parts sets beyond a single host, making it possible to create a small number of parts sets that can span the tree of life. This will accelerate current efforts to engineer diverse species for environmental, biotechnological, and health applications.