Low copy number plasmids for regulated low-level expression of cloned genes in Escherichia coli with blue/white insert screening capability

We have constructed pCL1920 and pCL1921, a pair of low copy number plasmids which contain a 580 bp BstVl fragment that carries the lac promoter/operator, multiple cloning sites and lacZ fragment of pUC19 (1) cloned in place of the poly linker region in pGB2 (2), a pSClOl derived plasmid which confers spectinomycin (50 jtg/ml) and streptomycin (100 jig/ml) resistance in Escherichia coli. All multiple cloning sites indicated are unique except for an additional EcoRI site as shown in the figure. pCL1920 and pCL1921 contain the BstVl fragment in opposite orientations with respect to the pGB2 sequences. In the absence of inducer the pCL1920/21 vectors do not produce detectable levels of/3-galactosidase in JM105 (lacP lacZAM\5) (1) cells (less than 2 Miller units) (3). In the presence of 2 mM IPTG (isopropyl-j3-D-thiogalactopyranoside) the /3galactosidase levels of the pCL1920/21 [JM105] transformants rose to 11 units, while the pUC19 [JM105] transformants produced 470 units; a 43 fold increase. These results are consistent with the expected 40 fold difference in plasmid copy number between pCL 1920/21 (5 copies per cell) compared to that of the pUC vectors (200 copies per cell). Thus the pCL1920 and pCL1921 vectors allow