Transformation of Penicillin Resistance Plasmid from Penicillin Resistant soil Bacteria to E.coli DH5α

Author Name: *Ahmed Waleed Majeed ,Sara Pakseresht
Author Email: am.waleed86@gmail.com

Abstract

Transformation, an active uptake of free DNA by bacterial cells and the heritable incorporation of its genetic information is one of the most adventurous mechanism that can be manipulated with the technology to produce genetically modified and manipulated organisms that can fulfil the needs of the user.. Only more recently has it been considered that transformation may be a powerful mechanism of horizontal gene transfer in natural bacterial populations. With the advent of the technologies like RFLP, Amplification, Electrophoresis the act of addition and deletion of the genes into the native genome of the organisms has been taking up a major hand. The current work is an attempt to add the important antibiotic resistant R plasmid having penicillin resistance gene from the natural host to the E. coli cells which lack the property of Penicillin resistance. Once the transformation is performed the cells are cultured and tested for their antibiotic resistant property. If the cells are successfully transformed they too exhibit the resistant action against the antibiotic penicillin.

Keywords

transformation, plasmid, penicillin gene, vector

Introduction

“Transformation” is simply the process where bacteria manage to “uptake” or bring in a piece of external DNA (somehow or another) [1]. Usually, this process is used in the laboratory to introduce a small piece of PLASMID DNA into a bacterial cell .Nearly everything we know about transformation and competence comes from experiments carried out on four species: S. pneumoniae, Bacillus subtilis, Haemophilus influenzae, and Neisseria gonorrhoeae. Thus we can view the process of transformation as passing through four stages: (i) Competent cells sporting a limited number of DNA-binding receptors attach duplex DNA to the cell surface. (ii) The DNA is processed by endonucleolytic cleavage and, usually, by exonucleolytic removal of one of the strands. (iii) The other strand is drawn through a pore into the cytoplasm, possibly in concert with the exonucleolytic degradation [2]. The entering single strand of DNA undergoes homologous recombination with the chromosome, creating a new recombinant if the recombining partners carry different alleles. A plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA [3]. They are double-stranded and, in many cases, circular. Plasmids, usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms.
There are two types of plasmid integration into a host bacterium: Non-integrating plasmids replicate as with the top instance, whereas episomes, the lower example, integrate into the host chromosome. Plasmids used in genetic engineering are called vectors [4]. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site(MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location [5]. The plasmids are later inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria that take up copies of the plasmid survive, since the plasmid makes them resistant.

Conclusion

The objective of the work is to apply the mechanism of natural gene transfer, transformation to impart the antibiotic resistant nature to the sensitive cells. This technique being the natural mechanism would enable the sensitive strains to survive and fight against the antibiotic producing fungi. This technique may be employed in producing the resistant strains for these organisms that can act as an important bioreactors or sources for the production of industrially important enzymes or proteins.

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