DNA technology: DNA cloning using an E. coli vector

Researchers attempt to manufacture a protein by inserting a gene of interest, yak-1, into an E. coli cloning vector. The DNA sequence corresponding to the coding region of yak-1 contains 1000 base pairs and has the following structure, where ATG and TAA correspond to the sites of the start and stop codons, respectively: 5'-ATGcttaggcat…976 intervening base pairs…tggcagcccTAA-3'.

A map of the E. coli cloning vector following the insertion of yak-1 is shown in Figure 1. The locations of the origin of replication (ori), ampicillin resistance gene (amp), and restriction enzyme sites are shown. The numbers of base pairs between different loci are indicated; the entire vector, including yak-1, contains 3000 base pairs.

Figure 1 Map of E. coli cloning vector containing yak-1

The restriction sites and cleavage locations of two restriction enzymes, BamHI and SalI, are shown in Figure 2.

Figure 2 BAMHI and SaII restriction sites and cleavage locations

To incorporate the yak-1 gene, researchers employed PCR amplification with primers containing BamHI and SalI restriction sites. Amplified yak-1 and the E. coli vector were digested with BamHI and SalI, and then ligated to form recombinant DNA. This plasmid was introduced into E. coli cells, which were grown on ampicillin plates. Random colonies were selected, and their DNA was re-digested with BamHI and SalI to confirm successful integration, as shown in Table 1.

Table 1 DNA fragment lengths isolated from experimental E. coli colonies following restriction enzyme digestion