Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
6491769 | Journal of Biotechnology | 2013 | 5 Pages |
Abstract
Conjugative shuttle vectors of the pKVM series, based on an IncP transfer origin and the pMAD vector with a temperature sensitive replication were constructed to establish a markerless gene deletion protocol for Bacilli without natural competence such as the exoenzyme producer Bacillus licheniformis. The pKVM plasmids can be conjugated to strains of B. licheniformis and B. subtilis. For chromosomal gene deletion, regions flanking the target gene are fused and cloned in a pKVM vector prior to conjugative transfer from Escherichia coli to B. licheniformis. Appropriate markers on the vector backbone allow for the identification of the integration at the target locus and thereafter the vector excision, both events taking place via homologous recombination. The functionality of the deletion system was demonstrated with B. licheniformis by a markerless 939Â bp in-frame deletion of the yqfD gene and the deletion of a 31Â kbp genomic segment carrying a PBSX-like prophage.
Keywords
Related Topics
Physical Sciences and Engineering
Chemical Engineering
Bioengineering
Authors
Michael Rachinger, Melanie Bauch, Axel Strittmatter, Johannes Bongaerts, Stefan Evers, Karl-Heinz Maurer, Rolf Daniel, Wolfgang Liebl, Heiko Liesegang, Armin Ehrenreich,