10. E. Coli Cloning Vector Pbr322

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10. E. coli cloning vector pBR322 Important questions based on it: A. (a) Name the organism in which the vector shown is inserted to get the copies of the desired gene. (b) Mention the area labelled in the vector responsible for controlling the copy number of the inserted gene. (c) Name and explain the role of a selectable marker in the vector shown. (AI 2010) B. (a) Identify the selectable markers in the diagram of E. coli vector shown below. (b) How is the coding sequence of -galactosidase considered a better marker than the ones identified by you in the diagram? Explain. (Delhi 2009) A. Explain the importance of (a) ori, (b) ampR and (c) rop in the E. coli vector shown below. (AI 2008) B. Draw pBR322 cloning vector. Label ‘ori’, ‘rop’ and any one antibiotic resistance site on it and state their functions. (AI 2015C) C. Draw a schematic diagram of the E. coli cloning vector pBR322 and mark the following in it: (a) ori (b) rop (c) ampicillin resistance gene (d) tetracycline resistance gene (e) restriction site BamHI (f) restriction site EcoR I (AI 2014C) D. Draw a schematic sketch of pBR322 plasmid and label the following in it: (a) Any two restriction sites. (b) Ori and rop genes. (c) An antibiotic resistant gene. (Delhi 2012) E. Identify A, B, C and D in the given diagram. (a) A-ori, B-ampR, C-tetR, D-HindIII (b) A-HindIII, B-tetR, C-ampR, D-ori (c) A-ampR, B-tetR, C-HindIII, D-ori (d) A-tetR, B-HindIII, C-ori, D-ampR (COMEDK) F. The given figure is the diagrammatic representation of the E. coli vector pBR 322. Which one of the given options correctly identifies its certain component(s)? (a) ori-original restriction enzyme (b) rop-reduced osmotic pressure (c) HindIII, EcoRI - selectable markers (d) ampR, tetR–antibiotic resistance genes. (AIPMT Prelims) .

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Ampicillin Versus Tetracycline in the Selection of Pbr322 Transformants
Journal of Experimental Microbiology and Immunology (JEMI) Vol. 2:189-193 Copyright April 2002, M&I UBC The Effects of Ampicillin versus Tetracycline in the Selection of pBR322 Transformants JOYCE S. LAW, SHARON LEE, AMY TSENG, KO WING TSUI, AND NANCY YU Department of Microbiology and Immunology, UBC Escherichia coli DH5α cells were transformed with pBR322 plasmids, which encode both ampicillin and tetracycline resistance genes. After 45- and 60-minute recovery periods, the transformed cells were plated on three types of selective plates, containing 1) ampicillin only, 2) tetracycline only, and 3) ampicillin plus tetracycline. It was found that tetracycline selection from these recovery periods resulted in a lower level of transformant survival than ampicillin selection. However, after the transformants were allowed to recover for several generations, their survival rates were similar on ampicillin plates and on tetracycline plates. The pBR322 plasmid is a commonly used cloning vector that contains both the ampicillin and tetracycline resistance genes as selectable markers. Escherichia coli cells transformed with pBR322 plasmids have a selective advantage in antibiotic-treated environments. Results from a previous experiment indicate that different selective pressures affect the survival of transformed cells, as considerably more transformants survived ampicillin-only selection than ampicillin-tetracycline selection (1). Many possible reasons can explain this difference. Firstly, the mechanisms of ampicillin resistance and tetracycline resistance encoded on the pBR322 plasmid are very different. Ampicillin resistance on the pBR322 plasmid encodes the enzyme β-lactamase, which is secreted to the periplasm and acts by binding and inactivating the ampicillin molecule (http://wine1.sb.fsu.edu/bch5425/lec13/lect13.htm).
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Download Author Version (PDF)
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