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bioRxiv · 10.1101/2024.06.24.600474

SubtiToolKit - a bioengineering kit for Bacillus subtilis and Gram-positive bacteria.

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

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.

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Caro Astorga, J., Malci, K., Ellis, T., James, P., Debenedictis, E., Ming, H., Rogan, M.. 2024-06-25. SubtiToolKit - a bioengineering kit for Bacillus subtilis and Gram-positive bacteria.. https://doi.org/10.1101/2024.06.24.600474

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