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Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
Published on: January 8, 2015
New shuttle vector for cloning in Bacillus stearothermophilus
E De Rossi1, P Brigidi, N E Welker
1Department of Genetics and Microbiology A. Buzzati Traverso, University of Pavia, Italy.
Abstract:
Cloning vector plasmid pRP9 was constructed on the basis of the broad host-range plasmid pLM6. pRP9 was a small plasmid (2.9 kb), possessed a convenient polyrestriction site sequence and efficiently transformed Bacillus subtilis, Bacillus stearothermophilus and Escherichia coli. Furthermore, pRP9 presented a very high segregational stability in Bacillus hosts. Also, the structural stability in Bacillus strains, grown under selective pressure, of pRP9 carrying a 3-kb fragment, was high. No single-stranded and high-molecular weight pRP9 DNA was found in B. stearothermophilus. The host/vector systems described possessed all the properties required for efficient gene cloning.

