Question

Antibiotic selection allows you to determine which bacteria contain the plasmid of interest, but do not necessarily tell you if the plasmid has taken up the recombinant DNA and contains your gene of interest. Which gene, which spans the multiple cloning site (MCS), allows you to screen for plasmids that contain your insert?

• A.

  β-lactamase (bla)

• B.

  Ori

• C.

  lacZ

• D.

  X-gal

Answer 1

Option C. lacZ

Blue white screening is a popular screening technique, which determines the presence of insert DNA in a plasmid. The technique depends upon lacZ gene, present within the plasmid, which carries a Multiple Cloning Site (MCS). lacZ gene encodes the enzyme beta galactosidase. The host E.coli strain used for this purpose is different from other strains. It has a mutation in the lacZ gene, as a result of which, only the omega peptide portion of the enzyme is made, which is non-functional. On the other hand, lacZ gene in plasmids, such as pUC19, produces the alpha fragment of the enzyme. Thus, introduction of plasmid within the bacteria, produces a fully functional beta galactosidase enzyme, which can cleave the substrate. This is known as alpha-complementation. This method is widely used to detect the presence of insert DNA within the plasmid. lacZ gene within the plasmid, carries the MCS. The MCS has several restriction endonuclease sites, which can be used to insert a foreign DNA. Insertion of foreign DNA into the MCS disrupts the lacZ gene, as a result of which, alpha-peptide of the beta galactosidase enzyme is not made. When this recombinant plasmid is introduced into the host bacterial cells, no functional beta galactosidase enzyme is made. The cells are grown in presence of X-gal, which is a substrate for beta galactosidase. X-gal is cleaved by functional beta galactosidase to produce a blue colored pigment.

Thus, if a cell carries a recombinant plasmid, which has it's lacZ gene disrupted, no functional beta galactosidase will be made, X-gal will not be cleaved, and the cells will appear white. This is known as blue-white screening. Cells carrying plasmid which do not have the gene of interest, produce blue colonies, whereas, cells carrying plasmid which have the gene of interest, produce white colonies.