Size unlimited markerless deletions by a transconjugative plasmid-system in Bacillus licheniformis |
| |
Authors: | Michael Rachinger Melanie Bauch Axel Strittmatter Johannes Bongaerts Stefan Evers Karl-Heinz Maurer Rolf Daniel Wolfgang Liebl Heiko Liesegang Armin Ehrenreich |
| |
Affiliation: | 1. Labor für Genomanalyse, Institut für Mikrobiologie und Genetik, Georg-August Universität Göttingen, Germany;2. Henkel AG & Co KGaA, Global R&D Chemistry Laundry and Home Care, Düsseldorf, Germany;3. AB Enzymes GmbH, Darmstadt, Germany;4. Lehrstuhl für Mikrobiologie, TU-München, Freising, Germany |
| |
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 939 bp in-frame deletion of the yqfD gene and the deletion of a 31 kbp genomic segment carrying a PBSX-like prophage. |
| |
Keywords: | Markerless deletion Transconjugation Temperature sensitive replication B. licheniformis PBSX prophage |
本文献已被 ScienceDirect 等数据库收录! |
|