Fundamentals of Molecular Biology. Part 2: Molecular Genetic Mechanisms - A. N. Ogurtsov 2011

DNA Cloning
E. coli Plasmid Vectors

Plasmids are double-stranded DNA molecules that exist within a Cell independently of chromosomal DNA. Found in Bacteria and lower Eukaryotic Cells (such as Yeast), these extrachromosomal DNA elements act as cellular parasites or symbionts.

Like chromosomal DNA, plasmids replicate prior to each Cell Division. During division, plasmid copies are distributed between the daughter cells, ensuring their propagation across subsequent generations.

In Recombinant DNA technology, researchers typically utilize plasmids that replicate in E. coli. These plasmids are engineered to optimize their performance as vectors for DNA Cloning.

For instance, removing "unnecessary" regions from "natural" E. coli plasmids yields a plasmid vector approximately ~1,2-3 kb in length, containing three essential elements for cloning (Figure 86):

1) an origin of Replication,

2) a selectable marker that enables vector Selection (typically a drug-resistance Gene),

3) a site where exogenous DNA—the DNA fragment destined for replication—can be inserted.

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Figure 86 – Main Components of a plasmid cloning vector

Figure 86 illustrates a schematic diagram of a plasmid vector containing the ampr gene, which provides selection capability for the plasmid because it encodes the enzyme ß-lactamase, conferring resistance to the antibiotic ampicillin.

Exogenous DNA can be inserted into the site indicated in the figure without impairing the plasmid's ability to replicate or express the ampr gene.

Incorporating an artificially synthesized polylinker containing restriction sites for several different restriction Enzymes expands the versatility of the vector.

A polylinker is an artificially synthesized nucleotide sequence containing a single copy of several different restriction sites that do not occur anywhere else in the plasmid backbone.

Cellular enzymes initiate plasmid replication at THE ORIGIN OF replication (ORI), a specific DNA sequence 50–100 Base Pairs long. Once initiated at the origin, replication proceeds along the entire circular DNA, regardless of whether The nucleotide sequences are "native" plasmid components or artificially "inserted" fragments.

Thus, any DNA sequence inserted into the plasmid will be replicated along with the rest of the plasmid.

Figure 87 shows a schematic representation of DNA fragment cloning using an E. coli plasmid vector.

When E. coli cells are mixed with recombinant vector DNA under specific conditions, transformation may occur—the uptake of Plasmid Vectors by certain cells. Typically, one cell out of 10,000 (0.01%) takes up a single plasmid DNA molecule, thereby becoming transformed.

Transformed cells can be selected from untransformed ones. For instance, if the plasmid carries a gene conferring ampicillin resistance, only transformed cells will survive in an ampicillin-containing medium. These cells subsequently multiply, forming an ampicillin-resistant colony.

Figure 87 – DNA cloning using a plasmid vector

DNA fragments ranging from a few base pairs up to ~20 kb are typically accommodated in plasmid vectors. With special precautions to avoid mechanical shearing of the DNA, even longer fragments can be inserted.

When a recombinant plasmid with an inserted fragment transforms an E. coli cell, all daughter cells will contain copies of this plasmid and exhibit Antibiotic Resistance.

The integrated DNA fragment replicates alongside the plasmid and is passed down from generation to generation as The Cell colony grows. In this manner, the original DNA fragment is replicated within the cell colony into a vast number of identical copies.

Because the cells in the colony descend from a single transformed parental cell, they form a cell clone, and the original DNA fragment inserted into the parental cell is referred to as cloned DNA or a DNA clone.

An E. coli plasmid vector can be made more versatile by incorporating a polylinker (Figure 86). When such a vector is treated with a restriction enzyme that recognizes a site within the polylinker, the vector is cleaved precisely once at that single site.

Following this, any DNA fragment of the appropriate size generated by the same restriction enzyme can be inserted into the cut plasmid using DNA ligase.

Plasmids containing a polylinker allow researchers to clone DNA fragments generated by different restriction enzymes using a single plasmid vector, which undoubtedly simplifies experimental Procedures.

Real-world plasmids used in recombinant DNA technology are much more complex than the schematic plasmid shown in Figure 86.

As an example, Figure 88 shows a map of the pBR322 plasmid vector, which is frequently used in biotechnology with Escherichia coli cells.

The figure illustrates a plasmid map containing 4,361 base pairs of DNA. The plasmid contains an origin of replication (ori) and two genes encoding Proteins that confer antibiotic resistance—one to ampicillin (amp^R) and the other to tetracycline (tet^R). The locations of restriction sites targeted by various restriction enzymes, along with their respective designations, are indicated around the perimeter of the plasmid.