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Blue white screen
The blue-white screen is a screening technique that allows for the rapid and convenient detection of recombinant bacteria in vector-based molecular cloning experiments. DNA of interest is ligated into a vector. The vector is then inserted into a competent host cell viable for transformation, which are then grown in the presence of X-gal. Cells transformed with vectors containing recombinant DNA will produce white colonies; cells transformed with non-recombinant plasmids (i.e. only the vector) grow into blue colonies. This method of screening is usually performed using a suitable bacterial strain, but other organisms such as yeast may also be used.
==Background==
Molecular cloning is one of the most commonly used procedures in molecular biology. A gene of interest may be inserted into a plasmid vector via ligation, and the plasmid is then transformed into ''Escherichia coli'' cells. However, not all the plasmids transformed into cells may contain the desired gene insert, and checking each individual colony for the presence of the insert is time-consuming, therefore a method for the detection of the insert would be useful for making this procedure less time- and labor-intensive. 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 colour of the bacterial colony.
The method is based on the principle of α-complementation of the β-galactosidase gene. This phenomenon of α-complementation was first demonstrated in work done by Agnes Ullmann in the laboratory of François Jacob and Jacques Monod, where the function of an inactive mutant β-galactosidase with deleted sequence was shown to be rescued by a fragment of β-galactosidase in which that same sequence, the α-donor peptide, is still intact. Langley ''et al.'' showed that the mutant non-functional β-galactosidase was lacking in part of its N-terminus with its residues 11—41 deleted, but it may be complemented by a peptide formed of residues 3—90 of β-galactosidase. M13 filamentous phage containing sequence coding for the first 145 amino acid was later constructed by ''Messing et al.'', and α-complementation via the use of a vector was demonstrated by the formation of blue plaques when cells containing the inactive protein were infected by the phage and then grown in plates containing X-gal.
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. In this method, DNA ligated into the plasmid disrupts the α peptide and therefore the complementation process, and no functional β-galactosidase can form. Cells transformed with plasmid containing an insert therefore form white colonies, while cells transformed with plasmid without an insert form blue colonies; result of a successful ligation can thus be easily identified by the white coloration of cells formed from the unsuccessful blue ones.
抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』
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