Recombinant DNA and cloning

In an experiment, a scientist attempts 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 (ATG and TAA correspond to the sites of the start and stop codons, respectively):

Restriction enzyme sites for BamHI and SalI are provided below:

A map of the E. coli cloning vector, following the insertion of yak-1, is provided below. Ori denotes the origin of replication, amp denotes the ampicillin resistance gene. HindIII, BamHI, SalI, and NdeI designate restriction enzyme sites. There are no other restriction enzyme sites found on this vector. The numerals denote the number of base pairs between different locations on the plasmid.

Figure 1 E. coli cloning vector map for yak-1

First, yak-1 is amplified from the genomic DNA via PCR, using primers designed to incorporate the BamHI and SalI restriction sites to ensure that the amplification product would be able to be digested by those restriction enzymes. Following amplification, yak-1 and the E. coli vector are both cut by BamHI and SalI. Given that the same restriction enzyme sites are used, yak-1 (1000 base pairs) can be integrated into the E. coli vector with DNA ligase to form recombinant DNA (3000 base pairs). The resulting recombinant plasmid is introduced into E. coli via a laboratory technique. No additional vectors are used.

The altered E. coli are grown on ampicillin plates, and several random colonies are selected from the plate. The DNA of these colonies are then extracted, and the isolated genetic product cut again via BamHI and SalI to verify success of ligation. The results of this digestion are presented in Table 1.

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