4. Transformation with the recombinant pBR322 plasmid will not always be success

Question

4. Transformation with the recombinant pBR322 plasmid will not always be successful. Bacteria that retain the wild-type plasmid look the same as those that carry the recombinant plasmid, but the antibiotic genes in the pBR322 plasmid can be used to detect bacteria that carry the recombinant plasmid using replica plating.+ Replica plating is a technique in which a population of bacteria is first grown on a non-selective agar medium called the master plate. Each bacterial cell on the plate gives rise to a single colony of identical cells. A sterile piece of velvet is then pressed down lightly on the surface of the master plate and the velvet picks up cells wherever there is a colony. The cells clinging to the velvet are inoculated onto the replica plates in the same relative positions as those on the master plate. The replica plates contain selective agar and transformed bacteria can be identified based on their ability or inability to grow on the replica plates. + In this example, the master plate, which contains no antibiotic, was inoculated with the transformed bacteria. The replica plates contain either ampicillin or tetracycline (but not both) The results observed on the replica plates determine which bacterial colonies possess the recombinant plasmid. The diagrams below represent replica plating to detect the presence of bacteria that carry the recombinant pBR322 plasmid with the human somatostatin gene. Master Plate Agar contains neither ampicillin nor tetracycline Replica Test Plate A+ Agar contains Replica Test Plate B+ Agar contains ampicillin only-' tetracycline only* 1+ a. The bacterial colonies growing on replica plate A or replica plate B (choose one):+ contain the recombinant plasmid. (2) b. Identify the specific colonies on this replicate plate (the one you identified above) that contain the recombinant plasmid. (Choose all that are correct.) (2)- Colony 1+ Colony 2+ Colony 3+ Colony 4 Colony 5+ Explain how you determined which colonies contain bacteria with the recombinant plasmid. (HINT: Recall that d BamHl were used to remove a piece of the pBR322 plasmid to make room for the human somatostatin gene. (3) c.

Explanation / Answer

A). the correct answer is Replica plate B, because only three colonies were found insted of five. this indicates the colonies present in the plate B are resistant for both antibiotics ( ampicillin and tetracyclin).

B). correct answers are Colony 1, 2 and 5

C) pBR322 has two resistance genes those are ampicillin resistance gene and tetracyclin resistance gene, so the newly formed or recombinant bacteria has resistance to both the antibiotics.

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