The plasmid vector I am referring to is pCR 2.1 - TOPO. I added the vector to the E coli and plated them up onto LB+amp+X-gal plate, then incubated. After incubation the plates had two types of bacterial culture on them, one white and one blue.

I think that the blue cultures have accepted the plasmid as they are able to grow on ampicillin, so have the ampicillin resistance gene, and are able to convert the X-gal substrate to blue via the lac Z gene. What i'm confused about is the white colonies. If the colonies are able to grow on ampicillin, surely they have accepted the gene because they have the resistance, and surely they should to be able to convert the X-gal?

So why are these not expressing the lac Z gene? Is it that the lac Z gene and ampicillin resistance aren't on the same plasmid, or is it that the lac z gene didn't make it into the plasmid?

  • $\begingroup$ What was the strain of E. coli that you used? Was this a TA cloning kit? Did you also add insert DNA of any kind? $\endgroup$ – Alan Boyd Nov 3 '13 at 22:23
  • $\begingroup$ I believe it was the k12 strain, yes it was a TA cloning kit, and I added DNA from Aequoria victoria to the vector that caused the bacteria to fluores $\endgroup$ – Will Perry Nov 3 '13 at 22:34

The traditional blue/white screening is set up so that blue colonies are considered negative for the insert, and white colonies are positive for recombinant DNA. The gene responsible is the lacZ gene, or beta-galactosidase. This enzyme converts a synthetic substrate, X-gal, into an insoluble blue compound.

The pCR2.1 TOPO plasmid is a blue/white selection plasmid in which white colonies are considered positive for the insert. The way this works is by inserting the DNA sequence to clone inside the coding region for lacZ. By having any inserted DNA interrupting the sequences of lacZ, so that the enzyme has lost its function (because it is no longer being translated correctly in the bacteria). On the other hand, no insert present means that lacZ is ligated back together, and the entire enzyme is correctly made, and therefore can metabolize the X-gal substrate to form an insoluble blue dye.

  • $\begingroup$ This makes a lot of sense, however, if the E coli is growing on the plate, surely by default this means it has taken up the plasmid because it has the ampicillin resistance? How are the blue colonies surviving on the ampicillin if they haven't taken up the plasmid? $\endgroup$ – Will Perry Nov 3 '13 at 22:57
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    $\begingroup$ The ampicillin resistance gene is encoded on the plasmid in a different region. Every plasmid, with or without insert, contains ampicillin resistance. Every colony growing on the plate has taken up a plasmid, that's how they survive, but not all necessarily have a plasmid with the insert. $\endgroup$ – user560 Nov 4 '13 at 0:03
  • $\begingroup$ Oh I see, so it's a test to see if the plasmid has ligated together with the insert, not to see if the plasmid has been successfully inserted into the E coli? $\endgroup$ – Will Perry Nov 4 '13 at 10:37
  • $\begingroup$ That's correct! After all, the antibiotic is spread over the entire plate, so any bacteria growing must already have the resistance gene. $\endgroup$ – user560 Nov 4 '13 at 17:29

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