Question

1) You are given 2 strains of E. coli, X & Y. Strain X carries a kanamycin (kan) resistance gene on a plasmid and strain Y carries a tetracyline (tetR) resistance gene on a plasmid. The 2 strains are mixed together, plated on media containing kanamycin and tetracyline and incubated. The next day you notice that there is bacterial growth on your plate indicating conjugation has ocurred. You know that one of the original strains must have carried the F factor. Given a 3rd strain, Z, that you are told is F and ampicillin resistant (amp), please describe how you would experimentally determine which strain (X or Y) was the donor, indicating media and results. NOTE: your experiment should be based on mating, not any sequencing or visualization method to identify the F pilus. (It may be helpful to draw out parts of your experiment), (3 pts)

thats why i was so confused on this one, because our teacher did not give us any surrounding context regarding experimental biotechnology

Answer 1

We know that during a conjugation between F+ strain and F- ,only the plasmid from F+ is transferred to the F- strain. Keeping this process in mind, we now proceed.

To identify experimentally, we now need to provide suitable medium for the conjugation of unknown F+ strain with the 3rd strain Z. Now we need to take a medium which has kanamycin, tetracycline and ampicillin and transfer the unknown F+ strain and Z strain. Now these two strains should conjugate (since Z is a F- strain) to produce bacteria which are resistant to all the three antibiotics. At the same time, the unknown F+ and F- Z strain strain shall die . Thus the remaining new F+ strain bacteria will only survive.

There can be two situations :

a. If X strain was F+ strain, then the first conjugation event with Y strain would produce a F+ bacterium with plasmid containing kanamycin resistance gene (kanR). When this F+ bacterium undergoes conjugation with the 3rd Z strain (which is F-), the new F+ strain produced will contain a plasmid of kanamycin resistance gene (kanR) only. Thus this bacterium will have resistance to ampicillin and kanamycin but will not have the tetracycline resistance gene (tetR) found in Y strain.

b. If Y strain was F+ strain, then the first conjugation event with X strain would produce a F+ bacterium with plasmid containing tetracycline resistance gene (tetR). When this F+ bacterium undergoes conjugation with the 3rd Z strain (which is F-), the new F+ strain produced will contain a plasmid of tetracycline resistance gene (tetR) only. Thus this bacterium will have resistance to tetracycline and ampicillin but will not have the kanamycin resistance gene (kanR) found in X strain.

Thus the resistance gene found in the new F+ bacteria plasmid will be the resistance gene found in the starting strain (X or Y) which was F+.

To find this out, we plate the final F+ bacterium, in two mediums - one containing Tetracycline and another containing Kanamycin. The medium in which the F+ bacterium survives will indicate that the resistance gene for that antibiotic is present in its plasmid and thus help us in concluding which strain (X or Y) was F+.