Novel plasmid marker rescue transformation system for molecular cloning in Bacillus subtilis enabling direct selection of recombinants.
A versatile plasmid marker rescue transformation system was developed for homology-facilitated cloning in Bacillus subtilis. It is based on the highly efficient host-vector system 6GM15-pHPS9, which allows the direct selection of recombinants by means of beta-galactosidase alpha-complementation. The system offers several advantages over previously described cloning systems: (1) the convenient direct selection of recombinants; (2) the ability to effectively transform B. subtilis competent cells with plasmid monomers, which allows the forced cloning of DNA fragments with high efficiency; (3) the availability of 6 unique target sites, which can be used for direct clone selection, SphI, NdeI, NheI, BamHI, SmaI and EcoRI; and (4) the rapid segregational loss of the helper plasmid from the transformed cells.
Haima
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Department of Microbiology, Center of Biological Sciences, Haren Groningen, The Netherlands.
Bron
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Venema
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D001412:Bacillus subtilis / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D004274:DNA, Recombinant / Q000032:analysis*; D005816:Genetic Complementation Test; D005819:Genetic Markers; D010957:Plasmids; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D004274:DNA, Recombinant; D005819:Genetic Markers