Gibson Assembly-based direct cloning of plasmid DNA in Lactiplantibacillus plantarum WCSF1

Abstract

ABSTRACTLactobacilli are gram-positive bacteria that are growing in importance for the healthcare industry and genetically engineering them as living therapeutics is highly sought after. However, progress in this field is hindered since most strains are difficult to genetically manipulate, partly due since their complex and thick cell walls limit our capability to transform them with exogenous DNA. To overcome this, large amounts of DNA (>1 μg) are normally required to successfully transform these bacteria. An intermediate host, like E. coli, is often used to amplify recombinant DNA to such amounts although this approach poses unwanted drawbacks such as increase in plasmid size, different methylation patterns and limitation of introducing only genes compatible with the intermediate host. In this work, we have developed a direct cloning method based on Gibson assembly and PCR to amplify the DNA to sufficient quantities for successful transformation in L. plantarum WCFS1. This advantage of this method is demonstrated in terms of shorter experimental duration and the possibility to introduce a gene incompatible with E. coli into L. plantarum WCFS1.