Gene Cloning in Plasmid Vector | By - Dr. Suresh Kaushik
Gene cloning is a technique which is commonly used in biotechnology. In this technique a section of DNA is put into a vector that acts as a vehicle to transfer the DNA to a host cell e.g. a bacterium. The plamid vector multiplies in the bacterial cell. Thus, many copies of the original section of DNA are produced. Ultimately, a colony of bacteria forms, each containing one or several copies of the section of DNA contained in the vector DNA. The colony is termed a clone and the molecule of DNA contained in the vectors has been cloned. If the original section of DNA represented a gene, the process is known as gene cloning.
A DNA fragment to be cloned is obtained through the application of restriction endonucleases. Most of restriction enzymes cleave duplex DNA at specific palindromic sequences. An inherent advantage of this method is the ability to precisely excise the desired fragment through its restriction sites. The complimentary ends of the two DNAs specifically are covalently joined through action of the enzyme DNA ligase to form recombinant plasmid.
How can one select only those host organisms that contain a properly constructed vector? In the case of plasmid transformation, this is usually done through the use of antibiotics and/or chromogenic substrates. Addition of an antibiotic such as ampicillin will eliminate any colonies that did not take up the plasmid. The plasmids intact gene contains a gene that confers antibiotic resistance. In case of blue/white colony selection, E. coli transformed with a plasmid containing a foreign DNA insert in its polylinker region that lacks β-galactosidase activity because the insert interrupts the protein-encoding sequence of the lacZ' gene. Thus, when grown in the presence of blue dye X-gal bacterial colonies that have an insert in their polylinker region form colorless colonies, whereas bacteria containing only plasmids that lack an insert form blue colonies.
A cloned structural gene must be inserted into an expression vector, a plasmid that contains the properly positioned transcriptional and translational control sequences for the protein's expression. With the use of a relaxed control plasmid and an efficient promoter, the production of protein of interest may reach thirty percent of the host's total cellular protein. The ability to synthesize a given protein in large quantities is already having enormous medical, agricultural, and industrial impact.
Gene cloning has contributed hugely to agricultural and medical research. It has mainly been applied in agriculture to develop genetically modified crops or transgenic crops resistance to pests and diseases and improve nutritional quality of crops. It has allowed genes and their functions to be studied in much greater detail than was previously possible. In the future, there will be further possibilities for understanding gene function, protein formation and metabolism using gene cloning techniques.
About Author / Additional Info:
Dr. Suresh Kaushik
A Biotechnogical Professional and author from India