Cloning vectors based on E.coli plasmids

In this article, I briefly describe the cloning vectors based on E.coli plasmids.

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Cloning vectors

A small piece of DNA into which a foreign DNA fragment is inserted for cloning purposes is known as a cloning vector. The cloning vector may be the plasmid from a bacterium, a higher organism’s cell, or DNA taken from a virus. The vector contains restriction sites that help in the convenient insertion and removal of a DNA fragment. The cloning vector and the foreign DNA are treated with a restriction enzyme that creates the same overhang, then ligating the fragments together. Genetically engineered plasmids and bacteriophages (such as phage λ) are perhaps most commonly used. Other types of cloning vectors are also used, which include bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs).

Cloning vectors based on E.coli plasmids

Plasmids are self-replicating circular extra-chromosomal DNA, the most commonly used cloning vectors. The simplest cloning vectors based on small bacterial plasmids, e.g., E.coli plasmids are the most extensive in gene cloning experiments. A large number of different plasmid vectors are available for use with E.coli. These vectors combine ease of purification with desirable properties such as high transformation efficiency, convenient selectable markers for transformants and recombinants, and the ability to clone reasonably large pieces of DNA. One of the most popular vectors used in gene cloning is pBR322, and its nomenclature is:

p- A plasmid
BR- The laboratory in which the vector was originally constructed (Bolivar and Rodriguez, the two researchers who developed pBR322).
322- This number distinguishes the plasmid from other plasmids developed in the same laboratory.

Properties of pBR322 plasmid

The plasmid pBR322 is 4363bp in size, which helps its easy purification and easy construction of recombinant molecules with it.
pBR322 carries two sets of antibiotic-resistance genes (figure 1a). These are ampicillin and tetracycline resistance genes. Either of them can be used as a selectable marker for cells containing the plasmid. Each marker gene includes unique restriction sites that can be used in cloning experiments. A great variety of restriction sites (PstI, PvuI, ScaI, BamHI, HindIII) can be used. pBR322 can be used to clone DNA fragments (figure 1b) with any of several kinds of sticky ends.
The plasmid pBR322 has a reasonably high copy number. Normally, there are about 15 molecules present in a transformed E.coli cell. However, the number can be increased up to 1000-3000 by the process of plasmid amplification in the presence of a protein synthesis inhibitor(chloramphenicol).

pBR322 derived plasmid cloning vectors

pBR327 plasmid

It is a higher copy number plasmid. It was constructed by removing a 1089 bp segment from pBR322. This deletion did not bring any change in the amp^Rtet^Rgenes but changed the replicative and conjugative abilities of the resulting plasmid.

When compared to pBR322, it has a higher copy number (30-45 molecules), which makes this vector more befitting. The deletion of a segment destroys the conjugative ability of the pBR322 plasmid, making pBR327 a non-conjugative plasmid. Thus, it can’t direct its transfer to other E.coli cells.

pUC8 plasmid

It is derived from pBR322 by doing some minor modifications. The origin of replication and the ampicillin resistance gene remains the same as the pBR322 plasmid. All the cloning sites are clustered into a short segment of the lacZ’ gene carried by pUC8 (figure 2). The manipulations are done to contain the lacZ’ gene instead of the tetracycline resistance gene and a cluster of recognition sites in the lacZ’ gene.

Properties of pUC8 plasmid with its advantages

The size of the pUC8 plasmid is 2750 bp in length. The small size of the plasmid allows the easy insertion of the foreign DNA into it.
The plasmid has a high copy number. The manipulations involved in the construction of pUC8 were accompanied by a chance mutation within the origin of replication that results in the plasmid having a copy number of 500-700 even before amplification. The high copy number has a marked effect on the yield of cloned DNA from E.coli cells transformed with recombinant pUC8 plasmids (figure 3).

pUC vectors carry various combinations of restriction sites and show more flexibility in the types of DNA fragments that can be cloned. A DNA fragment with two different sticky ends can be cloned without involving the linker attachment. This can be done due to the clustering of the restriction sites. DNA cloned into a member of the pUC series can be transferred directly to its M13mp counterpart (figure 4) because of the same restriction site clusters. It can be analyzed by DNA sequencing or in vitro mutagenesis.

**Figure 4: Transfer of a DNA fragment from pUC8 to M13mp8**

pGEM3Z plasmid

The plasmid pGEM3Z contains the amp^Rgenes and lacZ ‘genes containing a cluster of restriction sites, similar to a pUC vector (figure 5). The pGEM3Z plasmid has two additional, short pieces of DNA. Each of which acts as the recognition site for the attachment of an RNA polymerase enzyme. These two promoter sequences lie on either side of the cluster of restriction sites used for the introduction of new DNA into the pGEM3Z molecule.

Conclusion

A cloning vector is a small piece of DNA into which a foreign DNA fragment is inserted for cloning purposes. The cloning vector may be the plasmid from a bacterium, the cell of a higher organism, or maybe DNA taken from a virus. The simplest cloning vectors based on small bacterial plasmids, e.g., E.coli plasmids are the most extensive in gene cloning experiments.

The plasmid pBR322 is 4363bp in size, which helps its easy purification and construction of recombinant molecules with it. It contains two sets of antibiotic-resistance genes with reasonably high copy number.

The pUC8 plasmid and pBR327 plasmid are derived from the pBR322 plasmid by doing some modifications. pUC vectors carry various combinations of restriction sites. Thus, shows more flexibility in the types of DNA fragments that can be cloned.

The pGEM3Z plasmid contains the amp^Rgenes and lacZ’ genes containing a cluster of restriction sites, similar to a pUC vector. However, it contains two additional, short pieces of DNA, each of which acts as the recognition site for the attachment of an RNA polymerase enzyme.