Properties and uses of plasmids
Describe the properties and uses of plasmids in G.M. experimentation.
Plasmids are extrachromosomal genetic elements found in a variety of bacterial species. They are double stranded; autonomously replicating, supercoiled, covalently closed circular (ccc) DNA molecules that range in size from 1 kb to greater than 200 kb. Often, plasmids contain genes coding for enzymes that, under certain circumstances, are advantageous to the bacterial host (Table 1).
Table 1. Some of the phenotypes conferred by different plasmids that are beneficial to the host cell.
(Taken and adapted from Old and Primrose 1999)
Why plasmids as vectors?
Plasmids are frequently used as they are the easiest class of vector to work with from the point of view of experimental manipulation, that is isolated from bacteria, modification with enzymes, ligation and introduction into their hosts.
Those plasmids used as cloning vectors in genetic experimentation are specifically designed for the purpose of gene cloning and have certain minimal requirements since most naturally occurring plasmids do not combine all of the elements necessary for gene cloning, thus, vectors had to be developed using recombinant DNA technology (Radnedge and Richards 1999). One of the earliest specifically designed vectors was the E. coli plasmid pBR322, which was named after the scientists Bolivar and Rodriguez, who were involved in engineering this plasmid (Brown 1995), and for the purpose of this paper, reference will be given to the plasmid pBR322 as it is still a popular cloning tool, which fits many of the usual requirements of a cloning vector (Fig. 1).
Plasmids, which are successfully used as cloning vectors, must possess the following properties:
Replicate autonomously
The first important feature of pBR322 and any cloning vector is the presence of an origin of replication (ori). pBR322 is based on the ori element of the natural occurring E. coli Col E1 plasmid
Plasmids are extrachromosomal genetic elements found in a variety of bacterial species. They are double stranded; autonomously replicating, supercoiled, covalently closed circular (ccc) DNA molecules that range in size from 1 kb to greater than 200 kb. Often, plasmids contain genes coding for enzymes that, under certain circumstances, are advantageous to the bacterial host (Table 1).
Table 1. Some of the phenotypes conferred by different plasmids that are beneficial to the host cell.
(Taken and adapted from Old and Primrose 1999)
Why plasmids as vectors?
Plasmids are frequently used as they are the easiest class of vector to work with from the point of view of experimental manipulation, that is isolated from bacteria, modification with enzymes, ligation and introduction into their hosts.
Those plasmids used as cloning vectors in genetic experimentation are specifically designed for the purpose of gene cloning and have certain minimal requirements since most naturally occurring plasmids do not combine all of the elements necessary for gene cloning, thus, vectors had to be developed using recombinant DNA technology (Radnedge and Richards 1999). One of the earliest specifically designed vectors was the E. coli plasmid pBR322, which was named after the scientists Bolivar and Rodriguez, who were involved in engineering this plasmid (Brown 1995), and for the purpose of this paper, reference will be given to the plasmid pBR322 as it is still a popular cloning tool, which fits many of the usual requirements of a cloning vector (Fig. 1).
Plasmids, which are successfully used as cloning vectors, must possess the following properties:
Replicate autonomously
The first important feature of pBR322 and any cloning vector is the presence of an origin of replication (ori). pBR322 is based on the ori element of the natural occurring E. coli Col E1 plasmid
References: Balbás, P., Soberon. X., Merino, E., Zurita, M., Lomeli, H., Valle, F., Flores, N., and Bolivar, F. (1986). Plasmid vector pBR322 and its special purpose derivatives – a review. Gene 50:3-40. Brock, T.D. and Madigan, M.T. (1981). Biology of Microorganisms. 6th Edition. Prentice Hall. USA. Brown, T.A. (1995). Gene Cloning – an introduction. 3rd Edition. Chapman and Hall. London. Dawson, M.T., Powell, R. and Gannon, F. (1996). Gene Technology – Introduction to Biotehniques. BIOS Scientific Publishers Ltd. Oxford. Dillon, J.R., Nasim, A. and Nestmann, E.R. (1985). Molecular cloning in Prokaryotes: in Recombinant DNA Methodology. John Wiley and Sons Inc Glazer, A.N Greenway, D. (1999). University of Central Lancashire student notes: Overhead 52 and 52a. Lehninger, A.L., Nelson, D.L. and Cox, M.M. (1993). Principles of Biochemistry. 2nd Edition. Worth Publishers. Lewin, B. (1995) Genes V. Oxford University Press Old, R.W Radnedge, L. and Richards. H. (1999). The Development of Plasmid Vectors. pp 51-96. In: Methods in Microbiology – Genetic Methods for Diverse Prokaryotes. Vol. 29. (Eds.) Smith, M.C.M. and Sockett, E. Academic Press. London. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1984). Molecular Cloning: A Laboratory Manual. Cold Harbor Laboratory Press, Cold Spring Harbor, New york. Talaro, K. and Talaro, A. (1993) Foundations in Microbiology. WCB Weaver, R.F