ArticleApplied and environmental microbiology1995
A general method for the consecutive integration of single copies of a heterologous gene at multiple locations in the Bacillus subtilis chromosome by replacement recombination.
Article in Applied and environmental microbiology, 1995. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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Who cites it
3 citing papers in PubMed, 11 citations in OpenAlex.
- Multiple integration of the gene ganA into the Bacillus subtilis chromosome for enhanced β-galactosidase production using the CRISPR/Cas9 system.AMB Express · 2019Article
- The spatial architecture of Bacillus subtilis biofilms deciphered using a surface-associated model and in situ imaging.PloS one · 2011Article
- Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli.Nucleic acids research · 2005Article
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
We have devised a two-step procedure by which multiple copies of a heterologous gene can be consecutively integrated into the Bacillus subtilis 168 chromosome without the simultaneous integration of markers (antibiotic resistance). The procedure employs the high level of transformability of B. subtilis 168 strains and makes use of the observation that thymine-auxotrophic mutants of B. subtilis are resistant to the folic acid antagonist trimethoprim (Tmpr), whereas thymine prototrophs are sensitive. First, a thymine-auxotrophic B. subtilis mutant is transformed to prototrophy by integration of a thymidylate synthetase-encoding gene at the desired chromosomal locus. In a second step, the mutant strain is transformed with a DNA fragment carrying the heterologous gene and Tmpr colonies are selected. Approximately 5% of these appear to be thymine auxotrophic and contain a single copy of the heterologous gene at the chromosomal locus previously carrying the thymidylate synthetase-encoding gene. Repetition of the procedure at different locations on the bacterial chromosome allows the isolation of strains carrying multiple copies of the heterologous gene. The method was used to construct B. subtilis strains carrying one, two, and three copies of the Bacillus stearothermophilus branching enzyme gene (glgB) in their genomes.
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