Biochemical Engineering Fundamentals, Part 1 - Bailey J., Ollis D. 1989

Molecular Genetics and Regulatory Systems
Recombinant DNA Technology
Vectors for Escherichia coli

Vectors are DNA molecules that ensure the Amplification of a DNA fragment within a growing Cell population. To meet all the requirements of the cloning process, a vector must possess the following properties:

1. The ability to replicate within the host cell.

2. The capacity to incorporate foreign DNAs of various molecular weights without disrupting Replication ability.

3. Ease of Introduction into the host cell following the incorporation of foreign DNA.

4. The presence of a selectable genetic marker that allows for rapid positive Selection of Cells containing the vector.

5. The presence of only a single site susceptible to Cleavage by one or more Restriction Endonucleases.

Two classes of vectors—Plasmids and Bacteriophages—have been used for DNA Cloning in E. coli. Here, the primary focus will be on Plasmid Vectors.

As mentioned above, the transformation mechanism is used when introducing plasmids into E. coli cells. Since a transformed E. coli cell can take up only a single plasmid, and subsequent isolation, identification, and application of the desired DNA sequences require a clone consisting of A large number of identical plasmid-containing cells, the plasmid must necessarily be capable of replication within the growing bacterial cell. In turn, this requires the plasmid to include an origin of replication—a nucleotide sequence (approximately 600 Base Pairs in the case of ColE-1 type E. coli plasmids) that directs and regulates the replication process so that each cell contains a sufficient number of plasmid copies (typically around 30).

Selectable genetic markers are equally important. For example, if an Antibiotic Resistance Gene is incorporated into the plasmid, rapid positive selection of plasmid-containing cells becomes straightforward. To achieve this, it is sufficient to culture the cells on a medium containing an antibiotic at a concentration that kills all cells lacking the plasmid while permitting the growth of cells that contain the plasmid and, consequently, the antibiotic resistance gene. Another general selection approach involves using a mutant host cell that lacks an enzyme essential for growth in a specific medium, and simultaneously introducing the normal gene corresponding to that enzyme into the recombinant plasmid. In this case, expression of the plasmid gene complements the genetic defect of the host cell.

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FIG. 6.23. Genetic Map of plasmid pBR322, showing gene loci and A number of restriction sites. The sequence of all 4363 base pairs of the plasmid has been deciphered.

In Recombinant DNA technology, selectable markers in plasmids can perform two distinct Functions. First, positive selection Methods, which enable the rapid identification of vector-containing colonies, significantly streamline laboratory research. Second, during the subsequent cultivation of plasmid-containing cells, selective pressure (e.g., introduced by adding an antibiotic to the nutrient medium) minimizes competition from any plasmid-free cells that may arise during population growth.

Plasmid Vectors for Cloning in E. coli have been studied extensively in recent years. One of the most popular is plasmid pBR322 (Fig. 6.23), which contains a series of unique restriction sites, tetracycline and ampicillin resistance genes, and an origin of replication that ensures plasmid amplification within The Cell. Here, amplification (extracopying) refers to a substantial increase in the copy number of plasmids relative to chromosomal DNA. In the case of pBR322 and related plasmids, amplification is made possible by the fact that, unlike the chromosome, plasmid replication in the host cell can proceed even when Protein Synthesis is halted. Therefore, adding a protein synthesis inhibitor, such as chloramphenicol, to the culture medium induces the synthesis of 30 to 1,000 or more copies of the plasmid and, consequently, of the cloned DNA. This yields the latter in quantities sufficient for identification and subsequent use as a reagent in constructing other DNAs. Plasmid pBR322 and related vectors can accommodate foreign DNA segments containing up to 15,000 base pairs.

Bacteriophage λ has also been widely investigated as a cloning vector; it retains its replication capacity and lytic functions following a 25% deletion of the wild-type genome, which allows for the cloning of foreign DNA segments of up to 12,000 base pairs. Significantly larger DNA segments (up to 50,000 base pairs) can be cloned using hybrid plasmid-phage λ vectors known as cosmids, which can be packaged into the HEAD and tail of phage λ. For this reason, bacteriophage vectors are preferable when constructing Genomic Libraries consisting of entire Eukaryotic Genomes or very large fragments thereof. Useful vectors have also been derived from single-stranded DNA phages, such as phage M13. The single-stranded clones obtained using these vectors are particularly well-suited for determining DNA nucleotide sequences via the Sanger method. The following section will cover Methods for determining nucleotide residue sequences and other techniques for identifying cloned DNAs.