title: A versatile mini-mazF-cassette for marker-free targeted genetic modification in Bacillus subtilis.
abstract: There are some drawbacks for MazF-cassette constructed in previous reports for marker-free genetic manipulation in Bacillus subtilis, including cloning-dependent methodology and non-strictly controlled expression system. In our study, the modifications on mazF-cassette are carried out, such as using mini Zeocin resistance gene as positive-selectable marker and strictly controlled xyl promoter from the B. subtilis to replace non-strictly controlled IPTG-inducible Pspac or xyl promoter from Bacillus megaterium. Then the mini-mazF-cassette was successfully applied to knock-out the amyE gene, to delete a 90-kb gene cluster, and to knock-in a green fluorescent protein expression cassette employing a cloning-independent methodology, without introducing undesirable redundant sequences at the modified locus in the B. subtilis 1A751. Besides, the mini-mazF-cassette could be used repeatedly to delete multiple genes or gene clusters with only a 2- to 2.5-kb PCR-fused fragment, which largely reduced the frequency of nucleic acid mutations generated by PCR compared to previous reports. We further demonstrated that the frequency of spontaneous mazF-resistant mutants was lower, and the frequency of generating desired clones was nearly 100%. The entire procedure for marker-free genetic manipulation using the mini-mazF-cassette can be finished in about 3days. This modified cassette has remarkable improvement compared to existing approaches and is applicable for available manipulating Bacillus species chromosomes.
authors.affiliation: Key Laboratory of Molecular Animal Nutrition of Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, People's Republic of China.
authors.forename: Zhiwei
authors.initials: Z
authors.lastname: Lin
authors.forename: Bin
authors.initials: B
authors.lastname: Deng
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Jiao
authors.forename: Bingbing
authors.initials: B
authors.lastname: Wu
authors.forename: Xin
authors.initials: X
authors.lastname: Xu
authors.forename: Dongyou
authors.initials: D
authors.lastname: Yu
authors.forename: Weifen
authors.initials: W
authors.lastname: Li
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics*; D001761:Bleomycin / Q000494:pharmacology; D024881:Drug Resistance, Bacterial / Q000235:genetics; D017353:Gene Deletion; D055879:Gene Knock-In Techniques / Q000379:methods; D055786:Gene Knockout Techniques / Q000379:methods; D005818:Genetic Engineering / Q000379:methods*; D005819:Genetic Markers; D005822:Genetic Vectors; D016254:Mutagenesis, Insertional / Q000379:methods; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
chemical_list: D005819:Genetic Markers; D001761:Bleomycin; C105427:Zeocin
keywords: Bacillus subtilis; Fusion PCR; Marker-free; Mini-mazF-cassette; mazF