High-copy-number and low-copy-number plasmid vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection

Three types of alpha-complementation plasmid vectors were constructed which contain a chloramphenicol- or kanamycin-resistance (CmR or KmR) gene and polylinker cloning sites within the coding region of lacZ'. These vectors are essentially based on high- or low-copy-number replicons. The low-copy-number vectors, 3.61 kb in size, confer CmR and contain the pSC101 replicon and pUC8-/pUC9-type polylinker. On the other hand, the high-copy-number vectors, 2.21 to 2.68 kb in size, confer either CmR or KmR, and contain the pBR322 replicon and pUC18-/pUC19-type or other modified polylinkers. All cloning sites except HindIII and SmaI sites in the KmR vectors are unique in each plasmid. Since almost all frequently used plasmid vectors confer ampicillin resistance, these vectors may be useful to simplify the subcloning/DNA joining experiments due to unnecessity of radioisotope labelling, size fractionation and purification of foreign DNA segments.     

Shuttle vectors containing a multiple cloning site and a lacZ alpha gene for conjugal transfer of DNA from Escherichia coli to gram-positive bacteria

The mobilizable shuttle cloning vectors, pAT18 and pAT19, are composed of: (i) the replication origins of pUC and of the broad-host-range enterococcal plasmid pAM beta 1; (ii) an erythromycin-resistance-encoding gene expressed in Gram- and Gram+ bacteria; (iii) the transfer origin of the IncP plasmid RK2; and (iv) the multiple cloning site and the lacZ alpha reporter gene of pUC18 (pAT18) and pUC19 (pAT19). These 6.6-kb plasmids contain ten unique cloning sites that allow screening of derivatives containing DNA inserts by alpha-complementation in Escherichia coli carrying the lacZ delta M15 deletion, and can be efficiently mobilized by self-transferable IncP plasmids co-resident in the E. coli donors. Plasmids pAT18, pAT19 and recombinant derivatives have been successfully transferred by conjugation from E. coli to Bacillus subtilis, Bacillus thuringiensis, Listeria monocytogenes, Enterococcus faecalis, Lactococcus lactis, and Staphylococcus aureus at frequencies ranging from 10(-6) to 10(-9). The presence of a restriction system in the recipient dramatically affects (by three orders of magnitude) the efficiency of conjugal transfer of these vectors from E. coli to Gram+ bacteria.     

pACYC184-derived cloning vectors containing the multiple cloning site and lacZ alpha reporter gene of pUC8/9 and pUC18/19 plasmids

A new series of vectors, pSU2716, pSU2717, pSU2718, and pSU2719, has been constructed. The plasmids contain (i) the P15A replicon, (ii) the chloramphenicol acetyl transferase (CAT)-coding gene from Tn9, and (iii) the HaeII fragment which carries the multiple cloning site and the lacZ alpha reporter gene of pUC8, pUC9, pUC18 and pUC19, respectively. These vectors allow rapid and simple transfer of inserts from pUC plasmids, have an intermediate copy number (which allows regulated expression from the lac promoter), and are compatible with ColE1-derived vectors (and, therefore, can be used in studies requiring the joint expression of two genes, for example, in genetic complementation analysis). Furthermore, the accumulation of CAT instead of beta-lactamase, allows an easy visualization in sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of proteins of 28-35 kDa, which can otherwise be obscured by the beta-lactamase.     

Construction of Plasmid Vectors for Screening Replicons from Gram-Positive Bacteria and Their Use as Shuttle Cloning Vectors

Plasmids play a central role in engineering recombinant bacteria because they are the primary vehicles used to manipulate targeted sequences. In some cases, bacteria of interest are poorly provided with suitable tools for these molecular or genetic manipulations. In this context, we constructed from two shuttle cloning vectors, pUCB2871 and pUCB2872, the basic vectors pUCB30 and pUCB31, which could represent suitable tools to isolate replicons from Gram-positive bacteria. These plasmid vectors are characterized by the following after-features: (a) the pUC origin of replication is unable to replicate in Gram-positive bacteria; (b) an erythromycin-resistance encoding gene that is functional in both Gram-negative and -positive bacteria; (c) the pUC19 multiple cloning site (MCS) within the lacZα reporter gene; and (4) an additional multiple cloning site (MCS). Cloning replicons from Gram-positive bacteria in this additional MCS would allow the derivative vectors to function directly as shuttle cloning vectors.     

Vectors for generating nested deletions and facilitating subcloning G+C-rich DNA between Escherichia coli and Streptomyces sp.

New multiple cloning sites (MCS), which facilitate the subcloning of G+C-rich DNA, were added to pUC18, M13mp18, pVE616 (a pBR322-derived insertion vector), and the low-copy-number Streptomyces vector, pIJ922. The MCS in these vectors contain sites found infrequently in Streptomyces DNA, facilitating the exchange of subclones between the vectors. The MCS added to M13mp18 and pUC18 was also designed to generate nested deletions within subcloned fragments.     

pHG165: A pBR322 copy number derivative of pUC8 for cloning and expression

During the construction of the Messing pUC plasmid series, the rop(rom) gene of pBR322 which mediates the activity of RNAI was deleted. This has resulted in an elevated copy number for the pUC plasmids which makes the expression of beta-galactosidase activity constitutive in a host containing the Iqtss lac repressor. We describe the construction of a new series of vectors which retain the pUC multiple cloning site (MCS) but in which copy number control has been recovered. In addition, the lac alpha/lac promoter expression region has been inserted into a HpaI cassette. This facilitates the movement of recombinant DNA clones within the MCS. It also increases the complementation activity of the lac alpha peptide by an order of magnitude, allowing selection of recombinants by their Lac- phenotype on MacConkey agar.