Question

could you please answer these questions?

1.

Which of the following is correct about why bacteria colony are blue verses white following ligations of PCR product into pGEM-T Easy plasmid and transformation?

	A.	White colonies have no PCR insert in the plasmid and a functional B-galactosidase enzyme, while blue colonies have a non-functional B-galactosidase enzyme with a PCR insert.
	B.	White colonies have a PCR insert in the plasmid and a non-functional B-galactosidase enzyme, while blue colonies have a functional B-galactosidase enzyme with no PCR insert.
	C.	White colonies have no PCR insert in the plasmid and a non-functional B-galactosidase enzyme, while blue colonies have a functional B-galactosidase enzyme with a PCR insert.
	D.	White colonies have a PCR insert in the plasmid and a functional B-galactosidase enzyme, while blue colonies have a non-functional B-galactosidase enzyme with no PCR insert.

Answer 1

1) The answer will be white colonies have a PCR insert in the plasmid & a non-functional B-galactosidase enzyme, while blue colonies have a functional B-galactosidase enzyme with no PCR insert (Option B).

Explanation: Blue-white screening is used to detect the recombination of plasmid. When no PCR insert is in the plasmid, lacZ gene in the plasmid vector can complement the lacZ mutation of the bacteria & as a result produce functional beta-galactosidase enzyme. Functional beta-galactosidase can cleave X-gal & produce blue colonies. When PCR insert is in the plasmid, lacZ gene in the plasmid vector is disrupted & no complementation can occur with the bacteria. As a result, no functional beta-galactosidase enzyme is produced. In absence of functional beta-galactosidase, bacteria will unable to cleave X-gal & will remain white. Thus, white colonies will have a PCR insert in the plasmid which blocks the functional beta-galactosidase production & blue colonies will lack PCR insert in the plasmid.