BLUE-WHITE SCREENING

INTRODUCTION The blue-white screen is a screening technique that allows for the detection of successful ligations in vectorbased gene cloning. Usuallyin molecular cloning, DNA of interest is ligated into a vector. The vector is then transformed into competent cells. However, not all the plasmids transformed into cells may contain the desired gene insert, checking each individual colony for the presence of the insert is time-consuming. One of the early methods developed for the detection of insert is blue-white screening which allows for identification of successful products of cloning reactions through the color of thebacterial colony. CLONING VECTORS FOR BLUE-WHITE SCREENING: The pUC series of plasmid cloning vectors by Vieira and Messing was developed from the M13 system and were the first plasmids constructed to take advantage of this screening method.The plasmids must contain the lacZ gene that produces the -peptide region of the -galactosidase, and examples of such plasmids are pUC19 andpBluescript. COMPETENT CELLS FOR THIS METHOD: Competent cells used for this should contain the mutant lacZ gene with deleted sequence (i.e. lacZM15), and some of the commonly-used cells with such genotype are JM109, DH5, and XL1-Blue.The -galactosidase produced by these are inactive.

MOLECULAR MECHANISM:
-galactosidase is a protein encoded by the lacZ gene of the lac operon, and it exists as a homotetramer in its active state. However, a mutant -galactosidase derived from the M15 strain of E. coli has its N-terminal residues 1141 deleted and this mutant, the -peptide, is unable to form a tetramer and is inactive. This mutant form of protein however may return fully to its active tetrameric state in the presence of an Nterminal fragment of the protein, the -peptide. MAIN CONCEPT: In this method of screening, the host E. coli strain carries the lacZ deletion mutant (lacZM15} which contains the -peptide, while the plasmids used carry the lacZ sequence which encodes the first 59 residues of galactosidase, the -peptide. When the two peptides are expressed together, as when a plasmid containing the lacZ sequence is transformed into a lacZM15 cells, they form a functional -galactosidaseenzyme. However, the plasmid also carries within the lacZ sequence an internal multiple cloning sites (MCS). This MCS within the lacZ sequence can be cut by restriction enzymes so that the foreign DNA may be inserted within the lacZ gene, thereby disrupting the gene and thus production of -peptide. Consequently, in cells containing the plasmid with an insert, no functional -galactosidase may be formed.

The presence of an active -galactosidase can be detected by X-gal, a colourless analog of lactose that may be cleaved by -galactosidase to form 5-bromo-4-chloro-indoxyl, which then spontaneously dimerizes and oxidizes to form a bright blue insoluble pigment 5,5'-dibromo-4,4'-dichloro-indigo. This results in a characteristic blue colour in cells containing a functional -galactosidase. Blue colonies therefore show that they may contain a vector with uninterrupted lacZ (therefore no insert), while white colonies, where X-gal is not hydrolyzed, indicate the presence of an insert in lacZ which disrupts the formation of an active -galactosidase as shown in the figure.

NOTE: It should also be understood that the lac operon is affected by the presence of glucose. Glucose reduces cAMP level, and cAMP is necessary for the formation of the CRP-cAMP complex required for the initiation of transcription of the lac operon. Presence of glucose therefore inactivates the lac operon, and the lac genes will only be turned on when glucose level drops low enough for the CRP-cAMP complex to form. 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 -D-1-thiogalactopyranoside (IPTG), which functions as the inducer of the lac operon, may be used in the media to enhance the production of lacZ.

Drawback
Some white colonies may not contain the desired recombinant plasmid for a number of reasons. The ligated DNA may not the correct one, and it is possible for some linearized vector to be transformed, its ends "repaired" and ligated together such that no LacZ is produced and no blue colonies may be formed. A colony with no vector at all will also appear white. It is also possible that blue colonies may contain the insert. This occurs when the insert is "in frame" with the LacZ gene and a STOP codon is absent in the insert. This can lead to the expression of a fusion protein that is still functional as LacZ. The correct recombinant construct can sometimes give lighter blue colonies which may complicate its identification.