Conrad.Y
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Hey everyone, I just have a question on AQA Biology A-level Recombinant DNA.

So I understand that we use plasmids with two antibiotic resistant genes. E.g. Gene A codes for antibiotic A and gene B (marker gene) codes for antibiotic B. We then "inactivate" gene B by inserting the desired gene into the middle of it. This means we can grow the bacteria in a medium containing antibiotic A and all bacteria that have failed to take up the plasmid will die. But some bacteria can survive by taking up plasmids which didn't successfully combine with the desired gene. Now we have to use the marker gene (gene B) to test for bacteria containing the desired gene with replica plating.

What I don't understand is why not skip straight to replica plating? What's the point in having gene A in the plasmid if some of the bacteria will survive exposure to antibiotic A despite not containing the desired gene? If we just use the marker gene, all the bacteria which have either failed to take up the plasmid at all, or taken up a plasmid without the desired gene, will die. This immediately tells us which bacteria are viable and which aren't.
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ch3mz
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The point of having two antibiotic genes is so that when you replicate your bacterial colonies over from antibiotic A to antibiotic B, you would be able to tell which colonies actually contain the plasmid WITH your desired gene, as these colonies would not be able to grow on antibiotic B, as the B gene is disrupted by your inserted desired gene. Thus, you could go back to the original plate of antibiotic A and isolate a colony which contains your desired gene.

If you were to only use one marker gene (e.g. antibiotic B) and then inactivate it by inserting your desired gene, then yes, you would be able to tell if your bacterial culture may contain the desired gene if you were to immediately spread it on a plate containing antibiotic B as there would be no growth since the gene B is disrupted by your desired gene. BUT this is basically useless because then only colonies which don't contain your desired gene would grow (you want growth and expression of your new bacteria which express your desired gene)
Last edited by ch3mz; 7 months ago
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