Role of Genetic Transformation in Recombinant DNA Technology and Demonstration Using Escherichia coli strain DH5alpha

Author Name: *Anushree Srivastava
Author Email: anushree.srivastava02@gmail.com

Abstract

In Molecular Biology, genetic transformation occurs when an organism’s character is changed or modified by inserting one or more desired gene(s) in order to utilize them for different purposes, for instance, the betterment of plant species by insertion of genes coding for drought, pest, and frost resistance, transformation of common bacterium E. coli in order to generate mass quantities of the hormone insulin that can be used to treat diabetes, enabling bacteria by genetically transforming them to digest oil spills [1] , and in biological research organisms are genetically engineered to discover the phenotypic expressions and functions of specific genes. So, given the consequential role of genetic transformation in extension of recombinant DNA technology, the present study regarding the demonstration of successful transformation experiment establishes its relevance. In the study the common lab strain Escherichia coli DH5α has been used to demonstrate the transformation experiment and the parameter streptomycin resistance has been used as a marker. First, Escherichia coli strain DH5α were exposed to antibiotic sensitivity test and cells having streptomycin resistant plasmid were used for the plasmid DNA isolation. Agarose gel electrophoresis confirmed the presence of plasmid DNA in the sample. E. coli cells containing genes sensitive to streptomycin were used for the competent cell preparation through CaCl2 treatment. Transformation was performed using heat shock method. Finally, the blue-white lacZ screening method was employed in the identification of successfully transformed white-color colonies of E. coli, which therefore concludes that genetic transformation may avail many commercial and research motives.

Keywords

Escherichia coli, Plasmid DNA, Recombinant DNA Technology, Transformation.

Introduction

The potential and promise of genetic engineering is enormous and the dissemination of the products of Recombinant DNA Technology is limitless and useful to the mankind [2] In recombinant DNA technology, genes from one kind of organism are introduced by lateral gene transfer into the genome of another kind of organism and genetic transformation is one of those few natural means of lateral gene transfer to be artificially manipulated and create recombinant DNA and organism [3] Transformation is an important tool in recombinant DNA technology as the addition of new genes to a recipient cell introduces a heritable modification in the recipient cell’s phenotype [4] Although recombinant DNA technology first emerged in the 1960s and 1970s, the basic principle of recombination had been discovered many years earlier. Indeed, in 1928, Frederick Griffith, an English medical officer studying the bacteria responsible for a pneumonia epidemic in London, first demonstrated what he termed “genetic transformation”; here, living cells took up genetic material released by other cells and became phenotypically “transformed” by the new genetic information. More than a decade later, Oswald Avery repeated Griffith’s work and isolated the transforming molecule, which turned out to be DNA. These experiments showed that DNA can be transferred from one cell to another in the laboratory, thus changing the actual genetic phenotype of an organism [5].
Transformation Mechanism and Competence Factor: The mechanism of transformation involves extraction of DNA from donor organisms by a complex biochemical process that yields hundreds of naked DNA fragments from each bacterial chromosome. (Naked DNA is DNA released from an organism, often after the cell is lysed) When the extracted, naked DNA is placed in a medium with organisms capable of incorporating it, most organisms can take up a maximum of about 10 fragments. Uptake of DNA occurs only at a certain stage in a cell’s growth cycle, in response to high cell density and depletion of nutrients. In this stage, a protein called competence factor is released into the medium and apparently facilitates the entry of DNA. When competence factor from one culture is used to treat a culture that lacks it, cells in the treated culture become competent to receive DNA- they can now take up DNA fragments. Competence in most naturally transformable bacteria is regulated, and special proteins include a membrane-associated DNA-binding protein, a cell wall autolysin, and various nucleases play a role in the uptake and processing of DNA [6] DNA entry depends on factors such as modifications of the cell wall and the formation of specific receptor sites on the plasma membrane that can bind DNA. Once DNA reaches the entry sites, endonucleases cut double-stranded DNA. The strands separate and only one strand enters the cell. Splicing of a DNA strand involves breaking the strand, removing a segment, inserting a new segment, and attaching the ends. Enzymes in the recipient cell excise a portion of the recipient’s DNA and recombine it with the donor DNA, which now becomes a permanent part of the recipient’s chromosome.
Plasmid DNA Vector and Escherichia coli strain DH5α Host cell: Vector is an agent that can carry a DNA fragment into a host cell in which it is capable of replication. Plasmids are autonomously replicating circular, double stranded DNA molecules found in bacteria, where they behave as additional genetic units inherited and replicated independently of the bacterial chromosome [7] The size of plasmids ranges from a few kb to 200 kb [8] Plasmids often contain genes that code for enzymes that can be advantageous to the host cell and may be involved in resistance to, or production of, antibiotics, resistance to toxins found in the environment, or the production of toxins by the bacteria itself. E.coli is a rod shaped Gram negative bacterium [8]. It is the host of choice for cloning experiments due to several reasons. It has a short doubling time (20 min.) and its genetics is well understood and can be easily transformed. The E.coli strain DH5α is used for most common cloning applications. These cells are compatible with lacZ blue/white selection procedure. The lacZΔM15 is a mutation of DH5α that deactivates lacZ activity in the bacteria producing an inactive form of β-galactosidase [9]. Strains with this mutation cannot cleave X-gal and remain colorless on X-gal plates, thus determining the success of transformation experiment [10] Hence, by deliberately introducing the foreign DNA into this bacterium, it is possible to make it produce useful medical products such as insulin or interferon [11].

Conclusion

The transforming principal – DNA is the material of heredity – led the birth of modern genetics [25,26] Transformation is the main route by which plasmid cloning vectors containing DNA inserted in vitro are introduced into bacteria. It is therefore a vital technique in both cloning and reversed genetics/allele replacements [27] procedures. The development of plasmid transformation regimes is thus an important prerequisite for the extension of recombinant DNA technology [28] Conclusively, the observation in the present study that DNA obtained from streptomycin resistant E. coli strain DH5a, coding for streptomycin resistance, is able to transform the sensitive E. coli, which indicates the utility of genetic transformation technique in genetic engineering. By putting a gene of interest into a plasmid, the transformed bacteria will express that gene and that expression can be exploited for the production of a needed protein or for selection purposes. Bacterial transformation is essential to the field of molecular biology in that it allows for the propagation, genetic expression, and isolation of recombinant DNA molecules [29]

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