Question

Describe in detail how the blue-white screen works. In your answer make sure to include information on the genes that are involved, the locations of these genes and the protein products of these genes. Why is the E. coli DH5-α strain ideal for performing blue-white screens following transformations with pUC18? What are the 3 necessary factors that must be included in the media that the bacteria are grown on in order to perform a blue-white screen? What is the purpose of each of these factors?  

Answer 1

Blue-white screen: A screening technique that helps in detecting the recombinant bacteria in vector based molecular cloning experiment. DNA is ligated into vector which is inserted into host cell. It is grown in the presence of x-gal. Cells transformed with vectors containing recombinant DNA will produce white colonies; and the rest show blue colored colonies.  α-complementation of the β-galactosidase gene is one of the gene used. β-galactosidase is a protein encoded by the lacZ gene of the lac operon, and it exists as a homotetramer in its active state. The correct type of vector and competent cells are important considerations when planning a blue white screen. The plasmid must contain the lacZα, and examples of such plasmids are pUC19 and pBluescript. The E. coli cell should contain the mutant lacZ gene with deleted sequence (i.e. lacZΔM15), and some of the commonly used cells with such genotype are JM109, DH5α, and XL1-Blue.

It should also be understood that the lac operon is affected by the presence of glucose. The protein EIIA^Glc, which is involved in glucose import, shuts down lactose permease when glucose is being transported into the cell. The media used in agar plate therefore should not include glucose.

X-gal is light-sensitive and therefore its solution and plates containing X-gal should be stored in the dark. Isopropyl β-D-1-thiogalactopyranoside (IPTG), which functions as the inducer of the lac operon, may be used in the media to enhance the expression of LacZ.

X-gal is an expensive material, thus other methods have been developed in order to screen bacteria. GFP has been developed as an alternative to help screen bacteria. The concept is similar to α-complementation in which a DNA insert can disrupt the coding sequence within a vector and thus disrupt the GFP production resulting in non-fluorescing bacteria. Bacteria that have recombinant vectors (vector + insert), will be white and not express the GFP protein, while non-recombinant (vector), will and fluoresce under UV light.