Improvement of pBBR1MCS plasmids, a very useful series of broad-host-range cloning vectors.

pBBR1MCS vectors are small in size, contain unique cloning sites within the lacZα gene, and are mobilizable and compatible with various plasmid incompatibility groups. We cloned four genes for aminoglycoside resistance methyltransferases from the Arm and Kam families into pBBR1MCS-3 and expressed them in Escherichia coli. The activity of two of these enzymes was impaired because of the fusion with the first 20 amino acids of the β-galactosidase α-peptide derived from the pBBR1MCS-3 vector. In order to overcome this problem, we introduced by site-directed mutagenesis a new NdeI restriction site into pBBR1MCS-3 to generate a start codon directly at the beginning of lacZα gene. We modified the pBBR1MCS-2, 4 and 5 plasmids in the same manner and obtained the enhanced pBBR1MCS_START vector series that retains all the useful features of the previous vectors, but eliminates the unknown effect of the fusion with the β-galactosidase α-peptide.