3. (20 points) You are given the assignment of developing a new plasmid for gene

Question

3. (20 points) You are given the assignment of developing a new plasmid for genetic engineering which contains resistance for kankamycin and ampicillin. Your advisor suggests that you start with pBR322 and elhat tetracycline resistance and replace it with kankamycin resistance. A bacterial gene which co which catalyzes hydrolysis of kankamycin was recently discovered in another bacterial plasmid (pBlo2b4 Tetracycline resistance in pBR322 can be cut out using a combination of EcoRV and Pvu I for pBIO264 is provided below. Using this map in conjunction with the restriction map for pBR322 prov outline the steps involved for developing a new ampicilin plus kankamycin resistant cloning vector. Be specific/list all standard cloning steps (Please write this in outline form or bulleted list, not in paragiap form), ns antibiotic des for a protein I. A restriction map raph BR3 Sall 2500 20 PvulI HindIII 180 Pvu I pBIO264 2100 -Sall 390 Gene for BamH1 1600 Pvull resistance to 800 kankamycin

Explanation / Answer

Take pBIO264 in a test tube. Treat this with PvuII. Take pBR322 and treat with PvuII and EcoRV. This will remove tetracycline resistance gene. Mix the contents of the two test tubes. Transform E.coli with this DNA. Plate these on nutrient agar media (NAM). Replica plate on NAM containing ampicillin and kanamycin . Collect the appropriate colonies from the master plate. Prepare pure cultures of these colonies via streaking.

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