Genetic engineering can be described as a set of techniques employed to change the genetic make-up of the cells to give rise to an improved and/or new organism. This technology has been widely used to generate transgenic plants with insect and pest resistance along with high nutritional quality of food grains, infection resistant livestock and many more. However for this process to be accomplished one needs to generate recombinant DNA. Recombinant DNA technology emerged as a response to the need for specific DNA segments in amounts sufficient for biochemical analysis. It has provided the means to produce large amounts of highly purified normal and mutant proteins for detailed analysis of their function in the organism.
Genetic engineering employs test tube environment to manipulate DNA segments (from same or different organisms) and this modified DNA be stored in favorable conditions till they are moved or delivered into cellular environment . This technology is all about creating artificial DNA by combining two or more sequences at desired positions,that would not normally occur together in any organism.
Cloning of a DNA fragment or gene is only possible in presence of another DNA segment termed as Vector, which has the ability to self replicate in a cellular host and could be transferred between organisms, an important trait for bacteria, as they use plasmids to transfer genetic information between one another ( horizontal gene transfer ). This trait of plasmid is exploited by the researchers, who use bacterial plasmids as vectors to insert foreign DNA into DNA they are working. The most commonly used vectors for molecular cloning experiments are bacteriophhages, bacteria, viruses and Plasmids.
Using this technique we can isolate and clone a single gene or a DNA fragment of interest into many highly identical copies. This is largely possible because of phages, bacteria and plasmids tend to replicate DNA (or reproduce) in similar fashion even after integration of foreign DNA fragment (insert DNA cloned into cloning vector), so that the newly inserted foreign DNA also replicate normally as part of parent DNA. This blog post is dedicated to Plasmids as cloning vectors.
What is a Plasmid?
Plasmids are small circular arrangements of DNA (containing genes) found in bacteria that can replicate independently of the bacterial DNA found in the nucleoid. The term plasmid was coined by American molecular biologist “Joshua Lederberg in the year 1952. Plasmids can be found in all three major domains: Archea, bacteria, and Eukarya but however they play the most significant biological role in bacteria where they can be passed from one bacterium to another by horizontal gene transfer, usually providing selective advantage, such as antibiotic resistance.
Plasmids contain many important features that made them choice of vectors in all biotechnology and molecular labs for various experiments of gene therapy and cell culture . Some of the important features are outline below:
1) Self replicating
2) Easy to handle because of usually small size
3) Highly stable for many years in native form or in bacteria (glycerol stocks)
4) Functional in many species is the most important feature of plasmids . This allows the researchers to express the gene of interest cloned in a plasmid, in a wide variety of organisms, including plants, worms, mice and even cultured human cells. Although plasmids are commonly used to understand gene function, they can also be used to investigate promoters, small RNAs, or other genetic elements like transposons.