Caro Astorga, J.; Malci, K.; Ellis, T.; James, P.; Debenedictis, E.; Ming, H.; Rogan, M.

Building DNA constructs of increasing complexity is key to synthetic biology. Golden Gate methods led to the creation of cloning toolkits - collections of modular standardized DNA parts hosted on hierarchic plasmids, developed for yeast, plants, Gram-negative bacteria, and human cells. However, Gram-positive bacteria have been neglected. Bacillus subtilis is a Gram-positive model organism and a workhorse in the bioindustry. Here, we present the SubtiToolKit, a high-efficiency cloning toolkit for B. subtilis and Gram-positive bacteria. Its design permits DNA constructs for transcriptional units, operons, knock-in and knock-out applications. It contains libraries of promoters, RBSs, fluorescent proteins, protein tags, terminators, genome integration parts, a no-leakage genetic device to control the expression of toxic products during E. coli assembly, and a toolbox for industrially relevant strains of Geobacillus and Parageobacillus as an example of SubtiToolKit versatility for other Gram-positive bacteria and its future perspective as a reference toolkit.