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Concept
Blue–white screen
Résumé
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. This method of screening is usually performed using a suitable bacterial strain, but other organisms such as yeast may also be used. DNA of transformation
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.
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.
Source officielle
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