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Imagine that you are a graduate student in a molecular biology laboratory. You have discovered a new strain of E. coli that you name conjugation plus bacteria. During the process of conjugation, the donor (male) conjugation plus bacteria are able to transfer Gene Y and Protein A into a recipient (female) bacterial cell, which contains Gene X. Gene Y and Protein A are present only in male conjugation plus. Gene X is present only in female bacterial strains.

The male strain contains a plasmid which has the information to cause the construction of a pilus to exchange genetic and peptide information to the female recipient. This specific plasmid also has a lac gene that allows the bacterium to metabolize lactose and use it as an energy source. The male strains are sensitive to ampicillin. That means that they will die in the presence of ampicillin. The female strain lacks the plasmid, and it cannot utilize lactose; however, it is resistant to ampicillin because of a gene carried on its chromosome.

Your major advisor wants you to plan and design an experiment to determine whether the male conjugation plus is able to transfer DNA and protein to a female recipient to create a unique and more resistant bacterium.

Design your experiment, and support your decision in a logical manner that can be scientifically supported.

A

Mix donor and recipient cells together, and allow them to incubate at 37°C for 2 hours in Luria broth to allow for conjugation to take place. Transfer a drop of the conjugation culture onto MacConkey agar (containing ampicillin) plates.

To determine whether the plasmid has moved from the male to the female cell, assess whether the lac gene and Protein Y have been transferred successfully by Southern and Western blotting, respectively.

To test whether there are any lac-positive cells in a culture, observe the presence of red colonies on McConkey agar (plus ampicillin) plates.

Since this agar contains lactose, as well as pH indicator dyes that turn red in the presence of acids that are produced when lactose is metabolized, the presence of red colonies supports the transfer of the lac gene from the male to the female strain.

B

Mix donor and recipient cells together, and allow them to incubate at 37°C for 2 hours in Luria broth to allow for conjugation to take place. Transfer a drop of the conjugation culture onto MacConkey agar (containing ampicillin) plates.

To determine whether the plasmid has moved from the female to the male cell, assess whether the lac gene and Protein Y have been transferred successfully by Southern and Western blotting, respectively.

To test whether there are any lac-positive cells in a culture, observe the presence of red colonies on McConkey agar (plus ampicillin) plates.

Since this agar contains lactose, as well as pH indicator dyes that turn red in the presence of acids that are produced when lactose is metabolized, the presence of red colonies supports the transfer of the lac gene from the female to the male strain.

C

Mix donor and recipient cells together, and allow them to incubate at 37°C for 2 hours in Luria broth to allow for conjugation to take place. Transfer a drop of the conjugation culture onto MacConkey agar (containing ampicillin) plates.

To determine whether the plasmid has moved from the male to the female cell, assess whether the lac gene and Protein Y have been transferred successfully by Northern and Western blotting, respectively.

To test whether there are any lac-positive cells in a culture, observe the presence of red colonies on McConkey agar (plus ampicillin) plates.

Since this agar contains lactose, as well as pH indicator dyes that turn red in the presence of acids that are produced when lactose is metabolized, the presence of red colonies supports the transfer of the lac gene from the male to the female strain.

D

Mix donor and recipient cells together, and allow them to incubate at 37°C for 2 hours in Luria broth to allow for conjugation to take place. Transfer a drop of the conjugation culture onto MacConkey agar (containing ampicillin) plates.

To determine whether the plasmid has moved from the male to the female cell, assess whether the lac gene and Protein Y have been transferred successfully by Southern and Western blotting, respectively.

To test whether there are any lac-positive cells in a culture, observe the presence of red colonies on McConkey agar (plus ampicillin) plates.

Since this agar contains lactose, as well as pH indicator dyes that turn red in the presence of bases that are produced when lactose is metabolized, the presence of red colonies supports the transfer of the lac gene from the male to the female strain.

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