The pRH030 plasmid was developed mainly for use with phages. It ensures consistent and reliable expression of the cas9 gene in B. subtilis and enables easy cloning of up to 5 gRNA simultaneously. It is available from the AddGene repository.
Otte K, Kühne NM, Furrer AD, Baena Lozada LP, Lutz VT, Schilling T, Hertel R (2020) A CRISPR‐Cas9 tool to explore the genetics of Bacillus subtilis phages. Lett Appl Microbiol 71(6):588-595 (DOI:10.1111/lam.13349).
Thank you for generously providing the TS01 Bacillus strain and the pRH030 CRISPR plasmid. I am having good success in introducing point mutations into the phi29 phage genome. Ninety to 100% of the sequenced plaques contain the desired mutation. I am currently designing an insert for the pRH030 plasmid to estimate the span and frequency of crossover events with the thought of trying deep mutagenesis in the future. Thanks again!
Diagnostic and Biological Sciences
University of Minnesota
pV2 and pV3-vectors
The pV2 vector is a small and straightforward shuttle vector replication in E. coli and Bacillus. It can be individually modified in E. coli and transferred to B. subtilis by natural competence or via conjugation to many other Bacillus species. The plasmids pV3lacZ and pV3SDlacZ are vectors for the analysis of promoter regions.
Hertel R, Volland S, Liesegang H. (2015) Conjugative reporter system for the use in Bacillus licheniformis and closely related Bacilli. Lett Appl Microbiol 60:162-167 (DOI:10.1111/lam.12352)