Loading...
Please wait, while we are loading the content...
Similar Documents
Loss of Tetracycline Resistant Gene from pBR322 by Direct Repeat Spontaneous Homologous Recombination
| Content Provider | Semantic Scholar |
|---|---|
| Author | خليف, Evan L. Khaleef ايفان لطيف |
| Copyright Year | 2012 |
| Abstract | Introduction he plasmid pBR322 was one of the first multipurpose cloning vectors to be available for efficient cloning and propagation of recombinant molecules in E. coli. This DNA molecule has been extensively used because of its simplicity and viability of its nucleotide sequence since the early days (1) . Today, pBR322 is still used as molecular cloning vehicle; although more advanced vectors have been developed from it. The plasmid pBR322 is about 4361bp carries two sets of antibiotic which resistant genes ampicillin and tetracycline. Tetracycline antibiotics inhibit bacterial protein synthesis by preventing the binding of aminoacyl-tRNA molecules to the 30S ribosomal subunit (2) . The most clinically prevalent mechanism of tetracycline resistance is an active efflux system. Resistant cells are able to grow in the presence of tetracycline because a low intracellular concentration of drug is maintained. The agent responsible for resistance is a very hydrophobic membrane-associated protein which expels tetracycline against a concentration gradient, by coupling the efflux of a proton. The overall process is lectronutral, since tetracycline is cheated to a divalent cation when expelled (3) . Homologous recombination is a subject of major interest in biology. In vivo, this mechanism is involved in DNA repair mechanism and in the generation of genetic diversity (4,5) . T Objectives: It's well known that tetracycline resistant gene in pBR322 is about 1190bp clockwise in direction and tetracycline antibiotic inhibits bacterial protein synthesis. Resistant cells are able to grow in the presence of tetracycline because of efflux system. In this study, we try to delete tetracycline resistant gene from pBR322 by spontaneous homologous recombination between direct homology flanked regions . Subject and Methods: There is direct repetitions Direct repeat between flank regions of tetrgene of pBR322 yield loss of tetrgene (Tetracycline Resistant Gene) by spontaneous homologous recombination. Restriction enzyme and T4 DNA ligation from NEB Phusion High Fidelity PCR Kit from NEB was used for PCR amplification. The measurement of rate of loss of tetrgene from pBR322 by homologous recombination was made using fluctuation test. Results: After construction of new derivative from pBR322 which includes200bp flank region upper to tetrgene homology with downstream region in same direction, the measurement of rate of loss of tetrgene by fluctuation test was 64%. Conclusion: The high rate of loss of tetrgene from pBR322 by direct repeat spontaneous homologous recombination indicated that homologous recombination between 200bp flank region for gene like tetrwhich it is about 1190bp consider as active method to delete the wanted gene. |
| Starting Page | 11 |
| Ending Page | 18 |
| Page Count | 8 |
| File Format | PDF HTM / HTML |
| DOI | 10.33091/amj.0201022012 |
| Volume Number | 10 |
| Alternate Webpage(s) | http://www.iasj.net?aId=70901&func=fulltext |
| Alternate Webpage(s) | http://amj.uoanbar.edu.iq//catalog/file/Volume%2010/Issue%202/AMJ_0201022012.pdf |
| Alternate Webpage(s) | https://doi.org/10.33091/amj.0201022012 |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
