Ampicillin is a bacteriostatic antibiotic. (Instead of killing bacteria, it inhibits their growth.) Ramon plated bacteria transformed with a plasmid that confers ampicillin resistance. He plated the bacteria on Thursday and left them in a 37 oC incubator overnight. On Friday, he observed moderately sized bacterial colonies. He decide to leave them in the incubator a little longer before picking the colonies that he wanted to work with further. Unfortunately, he forgot about the plate and left it in the incubator over the weekend. On Monday, his plate had large colonies that were each surrounded by very small colonies

A) Interpret and explain Ramon’s observations.

B) Predict what he will find when he picks some large and some small colonies and follows the plasmid isolation protocol for each of the colonies.

A) That resistance in bacteria is produced due to inactivation of ampicillin by the beta lactamase enzyme. This enzyme is expressed by the bla gene found in the plasmid. This enzyme is secreted into the culture medium, thereby inactivating ampicillin. Thanks to this inactivation, the bacteria colonies will be able to develop. After a day of incubation, only those bacteria that took the plasmid that gives them resistance to ampicillin will grow after transformation. After prolonged incubation, two types of colonies can be observed in the culture medium. One large colony with ampicillin resistance, and another small colony, both of which are sensitive to ampicillin.

B) Large colonies are characterized by being resistant to ampicillin. When Ramón isolates the plasmid, he will have the gene that provides resistance to antibiotics. Said plasmid can be used again on those bacteria that are sensitive to ampicillin.

On the other hand, satellite colonies are sensitive to ampicillin. These types of colonies do not have the plasmid that contains the gene that gives ampicillin resistance. It is not possible to isolate any plasmids from these satellite colonies. These satellite bacteria will not be able to grow if they are transferred to a plate containing fresh ampicillin, while large colonies, which possess the plasmid that gives them resistance to ampicillin, will be able to grow on that plate.

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