Insertion of Unmarked DNA Sequences in Multiple Loci of theBacillus subtilis168 Genome: an Efficient Selection Method

Abstract

The construction process of Bacillus subtilis strain with multiple mutations is accelerated by simultaneous use of a positive selection method. All the strains selected by neomycin acquired two simultaneous mutations at different genomic loci. The extremely low rate of false positives was accounted for by employing the improved method.

Combining two genomes in one cell: Stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome

“…3). To minimize the imbalance introduced by megacloning, we prepared two more GpBR loci at leuB (19) and ytqB (29) on the left side of the B. subtilis genome. Megacloning of regions [II], [III], and [IV] in GpBR at proB, leuB, and ytqB yielded BEST7383, BEST8648, and BEST8817, respectively (Fig.…”

Direct cloning of full-length mouse mitochondrial DNA using a Bacillus subtilis genome vector

“…1A, vii-ix). Screening for spectinomycin sensitivity eliminated the false-positives frequently associated with this counter-selection protocol (Itaya, 1999b;Itaya et al, 2001;Uotsu-Tomita et al, 2005), and finally yielded one clone, BEST7688, as a candidate recombinant (Fig. 1A, ix).…”

Recombinant Genomes: Novel Resources for Systems Biology and Synthetic Biology

“…Absence of the cI gene restores the Pr promoter activity and makes the strain resistant to neomycin and vise versa. This small transcriptional genecircuit worked nicely as a counter selection system known as cI-Pr [30,31] and has proven useful for cloning any DNA lacking an appropriate selection marker in the BGM vector [32][33][34][35]. More importantly, the reusable cI-Pr system allows repeated integration of DNA segments in the same BGM vector.…”

“…Replacement of the cI gene by foreign DNA confers resistance to neomycin (bottom left) and permits another cI-Pr selection. Details are described in Section 5.4.1 and[30,31,36].…”