Molecular Biotechnology. Bernard R. Glick

2.3C). T4 DNA ligase cannot join the ends of the dephosphorylated linear plasmid DNA. However, the target DNA is not treated with alkaline phosphatase and therefore provides phosphate groups to form two phosphodiester bonds with the alkaline phosphatase-treated vector DNA (Fig. 2.6). Following treatment with T4 DNA ligase, the two phosphodiester bonds are sufficient to hold the circularized molecules together, despite the presence of two nicks (Fig. 2.6). After introduction into a host bacterium, these nicks are sealed by the host cell DNA ligase system.

      Figure 2.6 Cloning target DNA into pUC19. The restriction endonuclease BamHI cleaves pUC19 at a unique sequence in the multiple-cloning site (MCS) and at sequences flanking the target DNA. The cleaved vector is treated with alkaline phosphatase to remove 5′ phosphate groups to prevent vector recircularization. Digested target DNA and pUC19 are mixed to join the two molecules via complementary single-stranded extensions and treated with T4 DNA ligase to form a phosphodiester bond between the joined molecules. Several ligation products are possible. In addition to pUC19 inserted with target DNA, undesirable circularized target DNA molecules and recircularized pUC19 that escaped treatment with alkaline phosphatase are produced.

Transformation and Selection of Cloned DNA in a Bacterial Host

      After ligation, the next step in a cloning experiment is to introduce the vector–target DNA construct into a suitable host cell. A wide range of prokaryotic and eukaryotic cells can be used as cloning hosts; however, routine cloning procedures are often carried out using a well-studied bacterial host, usually E. coli. The process of taking up DNA into a bacterial cell is called transformation, and a cell that is capable of taking up DNA is said to be competent. Competence occurs naturally in many bacteria, usually when cells are stressed in high-density populations or in nutrient-poor environments, and enables bacteria to acquire new sequences that may enhance survival. Although competence and transformation are not intrinsic properties of E. coli, competence can be induced by various treatments.

      One method to induce the uptake of plasmid DNA by a bacterial host such as E. coli is by treating mid-log phase cells with ice-cold calcium chloride (CaCl2) and then exposing them for two minutes to a high temperature (42°C). This treatment creates transient openings in the cell wall that enable DNA molecules to enter the cytoplasm. Alternatively, uptake of free DNA can be induced by subjecting bacteria to a high-voltage electric field in a procedure known as electroporation. The experimental protocols for electroporation are different for various bacterial species. For E. coli, the cells (∼50 microliters [μL]) and DNA are placed in a chamber fitted with electrodes (Fig. 2.7A), and a single pulse of approximately 25 microfarads, 2.5 kilovolts, and 200 ohms is administered for about 4.6 milliseconds (ms). Although the precise mechanism of DNA uptake during electroporation is not known, it has been deduced that transient pores are formed in the cell wall as a result of the electroshock and that, after contact with the lipid bilayer of the cell membrane, the DNA is taken into the cell (Fig. 2.7B). Generally, transformation is an inefficient process, and therefore, most of the cells will not have acquired a plasmid; at best, about 1 cell in 1,000 E. coli host cells is transformed. The integrity of the introduced DNA constructs is also more likely to be maintained in host cells that are unable to carry out exchanges between DNA molecules because the gene encoding recombination enzyme RecA has been deleted from the host chromosome.

      Figure 2.7 Electroporation. (A) Electroporation cuvette with a cell suspension between two electrodes. (B) (1) Cells (yellow) and DNA (red) in suspension in an electroporation cuvette prior to the administration of high-voltage electric field (HVEF) pulses. (2) HVEF pulses induce transient openings in the cells (dashed lines) that allow entry of DNA into the cells. (3) After HVEF pulsing, some cells acquire exogenous DNA.

      Cells transformed with vectors that carry a gene encoding resistance to an antibiotic can be selected by plating on medium containing the antibiotic. For example, cells carrying the plasmid vector pUC19, which contains the bla gene encoding β-lactamase, can be selected on medium containing ampicillin (Fig. 2.8A). Nontransformed cells or cells transformed with circularized target DNA cannot grow in the presence of ampicillin. However, cells transformed with the pUC19–target DNA construct and cells transformed with recircularized pUC19 that escaped dephosphorylation by alkaline phosphatase are both resistant to ampicillin. To differentiate cells carrying the desired vector–target DNA construct from those carrying the recircularized plasmid, loss of β-galactosidase activity that results from insertion of target DNA into the lacZ′ gene is determined. Recall that the multiple-cloning site in pUC19 lies within the lacZ′ gene (Fig. 2.5). An E. coli host is used that can synthesize the part of β-galactosidase (LacZω fragment) that combines with the product of the lacZ′ gene (LacZα fragment) encoded on pUC19 to form a functional enzyme. When cells carrying recircularized pUC19 are grown in the presence of isopropyl-β-D-thiogalactopyranoside (IPTG), which is an inducer of the lac operon, the protein product of the lacI gene (the LacI repressor) is prevented from binding to the promoter–operator region of the lacZ′ gene, so the lacZ′ gene in the plasmid is transcribed and translated (Fig. 2.8B). The LacZα fragment combines with a host LacZω fragment to form an active hybrid β-galactosidase. If the substrate 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) is present in the medium, it is hydrolyzed by the hybrid β-galactosidase to form a blue product (5,5′-dibromo-4,4′-dichloro-indigo). Under these conditions, colonies containing recircularized pUC19 appear blue (Fig. 2.8A). In contrast, host cells that carry a plasmid-cloned DNA construct produce white colonies on the same medium. The reason for this is that target DNA inserted into a restriction endonuclease site within the multiple-cloning site usually disrupts the correct sequence of DNA codons (reading frame) of the lacZ′ gene and prevents the production of a functional LacZα fragment, so no active hybrid β-galactosidase is produced (Fig. 2.8B). In the absence of β-galactosidase activity, the X-Gal in the medium is not converted to the blue compound, so these colonies remain white (Fig. 2.8A). The white (positive) colonies subsequently must be confirmed to carry a specific target DNA sequence.

      Figure 2.8 (A) Strategy for selecting host cells that have been transformed with pUC19 carrying cloned target DNA. E. coli host cells transformed with the products of pUC19–target DNA ligation are selected on medium containing ampicillin, X-Gal, and IPTG. Nontransformed cells and cells transformed with circularized target DNA do not have a gene conferring resistance to ampicillin and therefore do not grow on medium containing ampicillin (recircularized target DNA also does not carry an origin of replication, and therefore, the plasmids are not propagated in host cells even in the absence of ampicillin). However, E. coli cells transformed with recircularized pUC19 or pUC19 carrying cloned target DNA are resistant to ampicillin and therefore form colonies on the selection medium. The two types of ampicillin-resistant transformants are differentiated by the production of functional β-galactosidase. (B) IPTG in the medium induces expression of the lacZ′ gene by binding to the LacI repressor and preventing LacI from binding to the lacO operator sequence. This results in production of the β-galactosidase LacZα fragment in cells transformed with recircularized pUC19. The LacZα fragment combines with the LacZω fragment of β-galactosidase encoded in the host E. coli chromosome to form a functional hybrid β-galactosidase. β-Galactosidase