The plasmid vector I am referring to is pCR 2.1 - TOPO. I added the vector to th
ID: 35527 • Letter: T
Question
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?
Explanation / Answer
The traditional blue/white screening is set up so that blue colonies are considered positive for the insert, and white colonies are negative. 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.