New pSC101-derivative cloning vectors with elevated copy numbers

Abstract

Mutations that increase the copy number of the pSC101 replicon have been used for construction of new cloning vectors. Replacement of glutamate at position 93 in RepA yields plasmids that replicate at medium (27 copies/cell) and high (∼240 copies/cell) copy numbers. Based on the crystal structure of RepE, a structurally similar replication initiator protein from the F factor, the pSC101 repA mutants are predicted to be defective in dimerization. The cloning vectors permit increased expression of gene products along with the advantages of pSC101-derivative plasmids, including stable maintenance and compatibility with ColE1 plasmids. The plasmids also allow blue/white screening for DNA inserts and impart resistance to ampicillin, chloramphenicol and kanamycin. The vectors were used in a genetic assay to suppress temperature-sensitive mutants of ffh, encoding the protein component of the E. coli signal recognition particle, by overproduction of 4.5S RNA. While expression of 4.5S RNA from a wild type pSC101-derivative plasmid was not sufficient for suppression, use of the new vectors did suppress the temperature-sensitive phenotype.

Engineering orthogonal dual transcription factors for multi-input synthetic promoters

“…The weak M13 packaging signal (PS), gene III and gene VI were removed from the M13KO7 helper phage (HP-ΔPS-ΔgIII-ΔgVI). The pJPC12 vector was obtained from Peterson and Phillips43 and the M13 packaging signal was deleted from the vector backbone. The higher copy number version pJPC13-ΔPS was obtained by site-directed mutagenesis according to Peterson and Phillips43.…”

“…The pJPC12 vector was obtained from Peterson and Phillips43 and the M13 packaging signal was deleted from the vector backbone. The higher copy number version pJPC13-ΔPS was obtained by site-directed mutagenesis according to Peterson and Phillips43. For the construction of gene circuits, a pLITMUS derivative with a lower copy number was cloned by substituting the origin of replication pUC to p15A.…”

Isolation of Highly Persistent Mutants of Salmonella enterica Serovar Typhimurium Reveals a New Toxin-Antitoxin Module

“…A DNA fragment representing lacI-P trc -shpB was then inserted into the pSC101-derived cloning vector pJPC12 (46), generating pJPC-Trc-shpB (Table 1). Gene expression was induced with isopropyl-␤-D-thiogalactopyranoside (IPTG) at a final concentration of 0.1 mM.…”

Vectors for ligation-independent construction of lacZ gene fusions and cloning of PCR products using a nicking endonuclease

“…pKanCOs1 was constructed by modifying pJPK12 (Peterson and Phillips, 2008) in a similar manner. Plasmid DNA was first digested with Eag I and religated to eliminate a unique Bsp QI site.…”