Abstract
Bacillus subtilis is a well-characterized model bacterium frequently used for a number of biotechnology and synthetic biology applications. Novel strategies combining the advantages of B. subtilis with the DNA assembly and editing tools of Escherichia coli are crucial for B. subtilis engineering efforts. We combined Gibson Assembly and λ red recombineering in E. coli with RecA-mediated homologous recombination in B. subtilis for bacterial artificial chromosome-mediated DNA integration into the well-characterized amyE target locus of the B. subtilis chromosome. The engineered integrative bacterial artificial chromosome iBAC(cav) can accept any DNA fragment for integration into B. subtilis chromosome and allows rapid selection of transformants by B. subtilis-specific antibiotic resistance and the yellow fluorescent protein (mVenus) expression. We used the developed iBAC(cav)-mediated system to integrate 10kb DNA fragment from E. coli K12 MG1655 into B. subtilis chromosome. iBAC(cav)-mediated chromosomal integration approach will facilitate rational design of synthetic biology applications in B. subtilis.
Highlights
The rod-shaped Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Bacillus subtilis are well characterized model bacteria frequently used for metabolic engineering and a number of biotechnology and synthetic biology applications (Schallmey et al, 2004; Juhas et al, 2013a; Juhas, 2015b; Chen et al, 2013; Yim et al, 2011; Ajikumar et al, 2010)
The vast majority of the good DNA assembly and editing tools are in E. coli, B. subtilis is a better host for certain applications
Novel tools combining the advantages of B. subtilis with the efficient and reliable DNA assembly and editing methods available in E. coli are crucial for B. subtilis engineering efforts
Summary
The rod-shaped Gram-negative bacterium Escherichia coli and the Gram-positive bacterium Bacillus subtilis are well characterized model bacteria frequently used for metabolic engineering and a number of biotechnology and synthetic biology applications (Schallmey et al, 2004; Juhas et al, 2013a; Juhas, 2015b; Chen et al, 2013; Yim et al, 2011; Ajikumar et al, 2010). The vast majority of the good DNA assembly and editing tools are in E. coli, B. subtilis is a better host for certain applications. B. subtilis is considered to be a promising host for the construction of the minimal cell factories (Juhas et al, 2014b). Novel tools combining the advantages of B. subtilis with the efficient and reliable DNA assembly and editing methods available in E. coli are crucial for B. subtilis engineering efforts
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