ArticleNucleic acids research2012
Bacillus subtilis genome editing using ssDNA with short homology regions.
Article in Nucleic acids research, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 31 papers.
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31 citing papers in PubMed, 64 citations in OpenAlex.
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- Engineered bacterial orthogonal DNA replication system for continuous evolution.Nature chemical biology · 2023Article
- A recombineering system forEngineering microbiology · 2023Article
- Barriers to simultaneous multilocus integration in Bacillus subtilis tumble down: development of a straightforward screening method for the colorimetric detection of one-step multiple gene insertion using the CRISPR-Cas9 system.Microbial cell factories · 2023Article
- Recombineering in Non-Model Bacteria.Current protocols · 2022Article
- Positioning Bacillus subtilis as terpenoid cell factory.Journal of applied microbiology · 2021Review
- Simultaneous Multiplex Genome EngineeringFrontiers in microbiology · 2021Article
- Lox'd in translation: contradictions in the nomenclature surrounding common lox-site mutants and their implications in experiments.Microbiology (Reading, England) · 2021Article
- Advances on systems metabolic engineering ofSynthetic and systems biotechnology · 2020Article
- A regulated synthetic operon facilitates stable overexpression of multigene terpenoid pathway in Bacillus subtilis.Journal of industrial microbiology & biotechnology · 2020Article
- Development of a novel strategy for robust synthetic bacterial promoters based on a stepwise evolution targeting the spacer region of the core promoter in Bacillus subtilis.Microbial cell factories · 2019Article
- A rapid and versatile tool for genomic engineering in Lactococcus lactis.Microbial cell factories · 2019Article
- Development and characterization of a CRISPR/Cas9n-based multiplex genome editing system forBiotechnology for biofuels · 2019Article
- Programmed gRNA Removal System for CRISPR-Cas9-Mediated Multi-Round Genome Editing inFrontiers in microbiology · 2019Article
- Highly Efficient Genome Engineering inFrontiers in microbiology · 2019Article
- Deaminase-mediated multiplex genome editing in Escherichia coli.Nature microbiology · 2018Article
Corrections and comments
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Authors and funding
6 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
In this study, we developed a simple and efficient Bacillus subtilis genome editing method in which targeted gene(s) could be inactivated by single-stranded PCR product(s) flanked by short homology regions and in-frame deletion could be achieved by incubating the transformants at 42°C. In this process, homologous recombination (HR) was promoted by the lambda beta protein synthesized under the control of promoter P(RM) in the lambda cI857 P(RM)-P(R) promoter system on a temperature sensitive plasmid pWY121. Promoter P(R) drove the expression of the recombinase gene cre at 42°C for excising the floxed (lox sites flanked) disruption cassette that contained a bleomycin resistance marker and a heat inducible counter-selectable marker (hewl, encoding hen egg white lysozyme). Then, we amplified the single-stranded disruption cassette using the primers that carried 70 nt homology extensions corresponding to the regions flanking the target gene. By transforming the respective PCR products into the B. subtilis that harbored pWY121 and incubating the resultant mutants at 42°C, we knocked out multiple genes in the same genetic background with no marker left. This process is simple and efficient and can be widely applied to large-scale genome analysis of recalcitrant Bacillus species.
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