Loss of Tetracycline Resistant Gene from pBR322 by Direct Repeat Spontaneous Homologous Recombination
Al- Anbar Medical Journal,
2012, Volume 10, Issue 2, Pages 11-18
AbstractObjectives: 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.
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