Molecular Biology Topics

PLASMIDS

Written by MicroDok

Plasmids are extrachromosomal DNA molecules found in both eukaryotic and prokaryotic microorganisms that have the ability to replicate independently. In prokaryotes, plasmids are found in both Gram positive bacteria and Gram negative bacteria; and they usually range from 1.5 kb to 300 kb in size. In terms of the strands of their DNA molecule, most bacterial plasmids are dsDNA molecules while others are ssDNA molecules. Plasmids are circular piece of autonomously replicating DNA that may express antibiotic resistance gene and can also be engineered to express a particular protein of interest. A plasmid controls its own replication by using the biosynthetic machinery of its host cell to make other plasmid-encoded protein molecules or materials that are needed for a particular function.

Plasmids are different from the chromosome of the organism even though they may be found in the same organism. In a microorganism, there are a number of genetic materials that are found besides the organism’s chromosome. These genetic materials include plasmids, transposons, integrons, and other genomes which in addition to the chromosome help in the survival of the organism. Plasmids which originally evolved from bacterial cells are extrachromosomal DNA molecule of bacteria that can replicate independently of the organisms’ own chromosome (Figure 1). Though they naturally occur in bacterial cells, plasmids can also be found in eukaryotic cells. Many plasmids encode products or functions which modify the phenotype of the host cell.

For example, a given bacterial cell may harbour plasmid that confers antibiotic resistance traits or property on the organism. Certain experiments such as gel electrophoresis or plasmid curing experiments (using mutagens such as acridine orange dye) may be used to determine whether a particular bacterial cell harbour a plasmid or not. The presence of a plasmid in a bacterial cell may often be inferred by the host cell’s phenotype (for example: resistance to a particular antibiotic) since certain properties in an organism such as antibiotic resistance are commonly plasmid-specified or plasmid-mediated. The loss of this particular phenotypic property or function in the organism after treating the host cell with chemical agents that specifically kills its plasmid – will help the researcher to know confirm the presence of plasmid in the organism.

Plasmids are not necessary required for the growth of bacterial cell but rather possess some unique features (e.g. antibiotic resistance ability) that give them some selective advantage over other materials in a bacterial cell. Plasmids have no extracellular form and they are normally smaller in size than the bacterial chromosome. They are known to be generally circular and double-stranded DNA molecules, though some linear plasmids also exist. Plasmids are different from an organism’s chromosome in that the chromosome contains genes that are necessary for cellular functions while plasmids rarely contain such important genes required for the growth of an organism. They are found in different numbers in an organism, and this ranges from one plasmid per cell to hundreds of plasmids per cell (a phenomenon called copy number). Plasmids are indispensible tools in recombinant DNA technology (genetic engineering) because of their inherent antibiotic resistance marker, small molecular size and high copy number during replication. The plasmids that are used in recombinant DNA technology to transfer genes from one organism to another are generally classified as vectors.

Bacterial plasmids have one or more DNA sequence which serve as the origin of replication or ori (Figure 1). Ori is the starting point for plasmid DNA replication in the organism. It also help the bacterial plasmid DNA to be duplicated autonomously (i.e. on its own) from the host’s chromosome. In addition to the ori, plasmids also have sites for the expression of antibiotic resistance genes such as the beta-lactamase or bla genes. The site for the expression of beta-lactamase genes or (bla genes) could also serve as important location for the insertion of other gene of interest into a recipient bacterial cell. Plasmids are veritable tools in the hands of molecular biologists for bacterial transformation. Plasmids are the easiest cloning vectors to work with; and a piece of plasmid can be used to clone a mixture of DNA fragments. Plasmids are known to possess sites for antibiotic resistance genes, and this allows for easy detection of recombinant plasmids after a transformation experiment in the laboratory.

Figure 1: Diagrammatic representation of a plasmid. Ori = origin of replication. bla = site for antibiotic resistance gene (e.g. beta-lactamase or bla genes).

Plasmids are important tools for gene cloning techniques, and they are also applied in a wide variety of recombinant DNA technology applications. Plasmid vectors usually contain three sites which makes them unique and widely used for molecular biology manipulations. Restriction site or cloning site, drug resistance gene or marker and a replication origin (ORI) are the main constituents of a plasmid vector. Restriction or cloning site is the site or region on the plasmid vector where the foreign or exogenous DNA fragment could be inserted. Drug resistant gene or marker is the region that harbours gene that destroys antibiotics (e.g. ampicillin); and this particular region allows for the selective growth and isolation of the plasmid vector in a host cell or in vitro after the gene cloning process. Ori is the region that allows the plasmid vector to replicate the gene of interest it is carrying within its host organism.

The insertion of a foreign piece of DNA into the ampicillin resistance gene of a plasmid makes that particular plasmid to be susceptible to the antibiotic ampicillin i.e. the recombinant plasmid will no longer confer resistance to ampicillin. Bacterial transformants lacking such ability to confer resistance to a particular antibiotic (in this case: ampicillin) because of transformation are said to contain a chimeric plasmid. Chimera is a recombinant plasmid that contains a foreign DNA. Plasmids are the first cloning vectors, and they are easier to isolate and purify after a cloning experiment, thus aiding detection of transformed plasmids. Other examples of cloning vectors include: cosmids, yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), viral and phage vectors.

         CLASSIFICATION OF PLASMIDS

Plasmids can be classified based on their ability to transfer genetic material and based on the function that they perform in their host organism.

  • Their ability to transfer their genetic material or gene to other bacteria. Such plasmids are called conjugative plasmids; and they contain or carry a set of genes called tra (for transfer) genes which coordinates the process of conjugation in plasmids (i.e. the sexual transfer of plasmid to susceptible bacterium). Only plasmids with the tra region (i.e. region that contains the tra genes) can successfully carry out conjugation because this region on the plasmid contains genes that encodes proteins that functions in bacterial mating process, DNA replication and DNA transfer. Some conjugative plasmids have a wide variety of microorganisms that they can transfer their genes to. This ability is significant because such bacterial plasmids can transfer their genes (e.g. those for antibiotic resistance) to related and non-related organisms, consequently affecting a broad range of both Gram positive and Gram negative bacteria.
  • Their specific function in their host organism. For example, some plasmids can confer on an organism the exceptional ability to build resistance against an antimicrobial agent (such plasmids are called R plasmid), some are virulent (i.e. they can turn an organism into becoming a pathogen) and others are degradative (i.e. they can breakdown substances).

REFERENCES

Alberts B, Bray D, Johnson A, Lewis J, Raff M, Roberts K and Walter P (1998). Essential Cell Biology: An Introduction to the Molecular Biology of the Cell. Third edition. Garland Publishing Inc., New York.

Alberts B.M, Johnson A, Lewis J, Raff M.C, Roberts K and Walter P (2002). Molecular Biology of the Cell (4th Edn). Garland, New York. A good general reference volume.

Dale J (2003). Molecular genetics of bacteria. Jeremy W. Dale and Simon Park (4th eds.). John Wiley & Sons Ltd, West Sussex, UK.

Tamarin Robert H (2002). Principles of Genetics. Seventh edition. Tata McGraw-Hill Publishing Co Ltd, Delhi.

Twyman R.M (1998). Advanced Molecular Biology: A Concise Reference. Bios Scientific Publishers. Oxford, UK.

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