A number of selection systems have been developed for direct selection of recombinant plasmids in cloning experiments (positive selection). In this study, the commonly used LacZ-based α-complementation plasmid vectors have been used for designing a positive selection system for the selection of recombinants. The basis for the strategy is the phenomenon of galactose sensitivity exhibited by galactose epimerase (galE) mutants of Escherichia coli. It is known that lacZ⁺ galE, but not lacZ^- galE cells are killed upon addition of lactose due to the accumulation of a toxic intermediate, UDP-galactose, by hydrolysis of lactose. Using a galE mutant strain of E. coli that carries the lacZΔM15 allele, various α-complementation plasmids that vary in their copy number were examined for their ability to be killed following addition of lactose. The results show that some plasmids that exhibit relatively high β-galactosidase enzyme activity can be used effectively for positive selection. This selection would be extremely useful during primary cloning experiments such as construction of genomic or cDNA libraries and also in instances involving selection for rare recombinants.

Introduction

Plasmid vectors are commonly used for cloning and propagation of DNA fragments in Escherichia coli. However, identification of plasmids with DNA inserts can be sometimes laborious and time-consuming in gene cloning experiments. To overcome this problem, a variety of positive selection cloning vectors have been developed that allow growth only of bacterial colonies carrying recombinant plasmids. Typically, these plasmids express a gene product that is lethal for certain bacterial hosts; insertion of any DNA fragment into the plasmid inactivates the gene and alleviates the toxicity. There are various conditional lethal genes used in positive selection vectors, and some of them consist of those encoding the bacteriophage λ repressor, EcoRI methylase, EcoRI endonuclease, galactokinase, colicin E3, transcription factor GATA-1, lysis protein of ϕX174, barnase (reviewed in Reference1), SacB protein of Bacillus subtilis (2), RpsL protein of E. coli (3), CcdB protein of F-factor (4), and also the ParD system of the R1 plasmid (5).

α-Complementation plasmids are among the most commonly used vectors for cloning and sequencing the DNA fragments, as they generally have a good multiple cloning site and an efficient blue-white screening system for identification of recombinants in presence of a histochemical dye, 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal), and binding sites for commercially available primers for direct sequencing of cloned fragments (1,6). These vectors express the amino-terminal fragment of the lacZ gene product (β-galactosidase) that is capable of intra-allelic complementation (α-complementation) with the carboxy-terminal fragment encoded by appropriate host strains (7). Insertion of foreign DNA into the polycloning site of the plasmid inactivates the amino-terminal fragment of the β-galactosidase and abolishes α-complementation. Thus, cells bearing the recombinant plasmids (LacZ^-) would be white in presence of X-gal and can be distinguished from the blue parental vector carrying cells (LacZ⁺). However, there is no positive selection available for these vectors to directly select the recombinants. Such a selection would be extremely useful during primary cloning experiments such as construction of genomic or cDNA libraries where parental vector contamination has to be eliminated and also in instances in which rare recombinants need to be identified.

E. coli strains carrying a mutation in galE gene that encodes galactose epimerase are highly sensitive to galactose as they accumulate a toxic intermediate, UDP-galactose, that causes cell lysis (8). Since the β-galactosidase enzyme encoded by lacZ gene cleaves lactose into glucose and galactose moieties, the lacZ⁺ galE strains are killed by the presence of lactose or its analogue, phenyl β-D-galactoside due to the accumulation of UDP-galactose (9). So, essentially, galE strains capable of β-galactosidase synthesis, including those carrying the α-complementation vectors coding for the amino-terminal fragment of β-galactosidase and the chromosomally encoded carboxy-terminal portion, should be killed by addition of lactose, whereas cells having recombinant lacZ^- plasmids would survive the lethal selection and could be selected on lactose-supplemented media. The galE selection has been extensively utilized earlier to obtain lacI or -Z mutations in various mutant isolation procedures (9,10,11,12,13). In the present study, the efficacy of the galE-based positive selection system for direct selection of recombinants in various α-complementation plasmids was examined.

Materials and Methods

Bacterial Strains, Phages, and Plasmids

The E. coli strains and α-complementation plasmids are listed in (Table 1). Strain DH5α (Invitrogen, Carlsbad, CA, USA) has been used for all the manipulations (1,14). Strain PL2 (E. coli Genetic Stock Center) was used as a source of the mutant galE allele (15). Phage P1kc was from the laboratory stock.

Growth Media and Conditions

Luria-Bertani (LB) medium was used for most of the experiments (1). LBOYE is similar to LB, but contained 0.5% tryptone and no yeast extract. Minimal A (MM) media was supplemented with either 0.2% sugar (such as glucose, succinate, or sorbitol) or 0.5% Casamino acids (CAA) as described previously (16). The media components were from Difco (BD, Franklin Lakes, NJ, USA). The test for galactose-sensitivity was performed on MM-CAA plates supplemented with 0.1% d-galactose. Lactose sensitivity was examined by scoring for growth either on MM-CAA or LBOYE plates supplemented with 0.1% d-lactose. For enrichment of lactose-resistant cells in liquid culture, MM-CAA broth with 0.1% lactose was used. The plates for positive selection were made of either LBOYE or MM-CAA agar supplemented with appropriate antibiotics, X-gal, and 0.1% lactose. The growth temperature was 37°C. The antibiotic concentrations used are as follows: 75 µg/mL ampicillin, 50 µg/mL kanamycin, 25 µg/mL chloramphenicol, 50 µg/mL spectinomycin, and 15 µg/mL tetracycline. X-gal and isopropyl β-D-thiogalactopyranoside (IPTG) were used at 25 µg/mL and 0.5 mM, respectively.