Transformation of Antibiotic Resistant Gene from Staphylococcus Species to E.coli Dh5 Alpha Sensitive Strain

Author Name: *U. Manasa,T.V Sai Mohan,T. Narendra,K. Soumya lakshmi,B. Anusha kumara
Author Email: manasamanu1679@gmail.com

Abstract

Natural genetic transformation is the active uptake of free DNA by bacterial cells and the heritable incorporation of its genetic information. In the current work, Transformational insertion of an Antibiotic resistant gene (Ampicillin) from the native bacteria transformed into the sensitive strain of E. coli DH5 alpha. The bacteria were isolated from stagnant water and were tested for their antibiotic resistance. The resistant bacteria which showed a good growth in the antibiotic media is pure cultured and used for further analysis. The resistant bacteria were identified based on Biochemical analysis as per Bergey’s manual. The bacterium was identified to be Staphylococcus aureus, whose blaR1gene was responsible for antibiotic resistance.  The Plasmid DNA was extracted from the resistant strains and was inserted into the previously prepared competent sensitive E coli DH5 alpha strains. The transformation was performed and the cells were further incubated for division and multiplication. The final cells obtained were tested for the transformation efficiency using Blue White Screening on X Gal media. The presence of all white colonies indicated a positive and successful transformation. In order to confirm the transformation efficiency at the molecular level the gene blaR1 was targeted. In case of successful transformation this gene must be available in the transformed culture. Thus the gene sequence was retrieved from the database and the primer was designed. The plasmid DNA from the transformed cells was extracted and was amplified using the above designed primer. Successful amplification and band in the post PCR Gel electrophoresis indicates a successful transformation at the molecular level. The work can be used for the transformation of the genes responsible for some special user interested qualities into the existing strains so as to meet the industrial demands and produce recombinant strains of bacteria.

Keywords

Transformation, Competent, Resistance, Plasmid, Genomic DNA

Introduction

Bacterial transformation: In molecular biology, TRANSFORMATION is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material (exogenous DNA) from its surroundings and taken up through the cell membrane(s). Transformation occurs naturally in some species of bacteria. For transformation to happen, bacteria must be in a state of competence which might occur as a time-limited response to environmental conditions such as starvation and cell density.

Plasmid: Apart from genomic DNA in bacteria, it also contains extra-chromosomal DNA in them. This extra-chromosomal DNA is called as PLASMID. These plasmids are known to have the genes for the many antibiotics. There may be antibiotic susceptible genes and antibiotic resistant genes or even multi-drug resistant genes. Normally bacteria cannot grow in presence of antibiotics as they are susceptible to them. These are called as antibiotic susceptible bacteria. But there are some bacteria that are capable of growing in presence of some antibiotics. These are called as antibiotic resistant bacteria

Staphylococcus species: Staphylococcus species which is resistant to ampicillin is a genus of Gram-positive bacteria. Under the microscope, they appear round (cocci), and form in grape-like clusters. It is a facultative aerobe. The Staphylococcus genus includes at least 40 species. Of these, nine have two subspecies and one has three subspecies. Most are harmless and reside normally on the skin and mucous membranes of humans and other organisms. Found worldwide, they are a small component of soil microbial flora. Ampicillin is an antibiotic thatb acts as an irreversible inhibitor of the enzyme trans peptidase, which is needed by bacteria to make their cell walls It inhibits the third and final stage of bacterial cell wall synthesis in binary fission, which ultimately leads to cell lysis; therefore ampicillin is usually bactericidal. In recent years, antibiotics resistant bacteria have become a great concern to the medical community. There has been a great increase in the number of species that have acquired resistance to antibiotics, as well as an increase in the kind of antibiotics.

Antibiotics work in variety of ways, some antimicrobial agents inhibit bacterial cell wall synthesis. These agents include β-lactam compounds such as penicillin (e.g. penicillin G, ampicillin and methicillin), cephalosporins and as well as monolactams and β-lactamase inhibitors.  Beta-lactams inhibit the final stage of murein synthesis. This, by some undetermined mechanism, triggers murein hydrolases to lyse the cell. A related group of antibiotics that prevent a different step in cell wall synthesis are the glycopeptides, vancomycin etc. Other agents have an antibacterial effect by inhibiting protein synthesis. Representatives of this group include the aminoglycosides, tetracyclines, macrolides and chloramphenicol which interfere with ribosome function. In addition, there are antibiotics that inhibit DNA synthesis, including quinolones, fluoroquinolones and sulfonamides.

Conclusion

The current work aimed to study the role of genetic transformation in imparting a user desired quality into the known microbe for its use at the commercial scale. Here the sample collected was stagnant water from a nearby area at LB Nagar. The sample was used for the isolation of the organisms from the source. The organisms thus isolated were screed for the antibiotic resistance by culturing on a specialized media with antibiotic under study. The bacteria that were resistant are used for further study by replicating into a pure culture. The bacteria were used for the extraction of Plasmid as the antibiotic resistant gene would be present in the extra chromosomal material only. The extracted plasmid was used to transform the E coli DH5 alpha strain which were made competent in the earlier step. The transformed cells were then checked for the success in the technique. Several molecular techniques including PCR was used for the confirmation of transformation. The study also focused on the use of various molecular biology techniques in the identification of chirality in the DNA samples

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