I have questions regarding "selection using insertional inactivation".

In a typical DNA recombination experiment using plasmid pBR322 where a new gene in inserted in the place of tet gene, it is said that entities with the recombinant plasmid can be selected by growing all the bacteria in two different medium: one with tetracycline and one with ampicillin.

I know that pBR322 contains two genes that make it resistant to both tetracycline and ampicillin, and also that the newly created plasmid will only be resistant to ampicillin, since its tet gene is no longer functional.

So in the plate with tetracycline, only entities with the original plasmid will grow. In the plate with ampicillin, however, entities with the original plasmid and the modified plasmid will grow.

How can this be a selection when two different types of entities are growing in one plate?

Am I misunderstanding the meaning of "selection" in this case?

My explanation above seems very immature, so it would be great if someone can provide me with deeper understanding.

  • $\begingroup$ Yes. I concur with canadianer. As currently described, replica plating is the way to go. IF: you had a drug that killed bacteria resistant to tetracycline, THEN: you could use that as a selection. $\endgroup$
    – mdperry
    Jul 19 '19 at 0:08

The ampicillin plate is selecting for bacteria transformed with the plasmid, either the original or recombinant, whereas the tetracycline plate is selecting only for bacteria transformed with the original plasmid. Since you are only interested in the recombinant plasmid, you need to do some form of replica plating and compare the two plates to infer which colonies contain the recombinant plasmid (those which grow on ampicillin but not on tetracycline).


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