Blue white screen
The blue - white selection is a method of genetic engineering for the identification of bacterial clones containing a transgene.
Principle
The aim of the blue - white selection is the identification of transgenic bacteria with desired modifications. Firstly, those bacteria are identified that have taken up a plasmid and on the other is answered by the blue - white selection, the question of whether the plasmid was successfully modified. For the determination of plasmid -containing bacterial plasmids carry a gene which confers antibiotic resistance does not already own of the bacterial strain used themselves. If the bacteria are cultured in a culture medium with the appropriate antibiotic resistance to so survive there ideally only those bacteria that have taken up a plasmid.
For blue - white selection certain plasmids are used as vectors, which at the position for insertion of the transgene into the plasmid ( in the multiple cloning site ), containing the gene for B -galactosidase ( lacZ gene). The gene for galactosidase is used as the reporter gene.
By introducing a transgene into the multiple cloning site, the galactosidase is inactivated. Characterized by a transformation of the plasmids containing the transgenic organisms, in contrast to non-transgenic organisms no functional galactosidase, provided that the bacterial strain used for transformation contains no additional gene for galactosidase. This specific mutant strains can be used ( eg, E. coli strains JM109, DH5 and XL1-Blue ) containing a mutation (e.g., lacZΔM15, E. coli M15) which suppresses the expression of a functional galactosidase. LacZΔM15 the mutant galactosidase has a deletion of amino acids 11 to 41, whereby the galactosidase can not form more a homotetramer, which is the active form of the enzyme galactosidase. LacZΔM15 the mutant of galactosidase ( synonymous ω - peptide) may, however, 1 to 59 ( α - peptide) are added to a functional form of a protein of the amino acids, whose gene is located in this case on the plasmid.
The glycosidase is a galactosidase and the yellow dye can XGal split into a blue dye (5,5 ' -Dibromo -4, 4' -dichloro- indigo) and galactose. The lacZ gene of the galactosidase is from a promoter controlled, wherein the gene expression by an inducer (such as lactose, allolactose, the strongest known inducer IPTG ) is introduced, as the lac repressor LacI release by binding of the inducer the gene. When using XGal in the culture medium for about an hour produced after induction by the galactosidase contained in the bacteria of a blue color. In the transgenic organisms of the galactosidase is inactivated, thus the colonies remain unstained and they can be isolated based on their lack of color. As the import of lactose on the Lactasepermease ( lacY gene ) is inhibited by glucose, the culture medium should not contain glucose.
Not every white colony is a transgenic organism (eg due to lacZ inactivation by DNA repair is not ligated plasmids ) and not everyone transgenic organism containing the desired gene to be cloned, but other DNA fragments. If too little antibiotic or too old to use culture media for selection for the resistance gene of the plasmid, grow white colonies without plasmid. For these reasons, further analysis, eg by polymerase chain reaction (mostly colony polymerase chain reaction ) or by DNA extraction with restriction digestion, both followed by agarose gel electrophoresis. XGal is light sensitive, so the dye itself and XGal -containing agar plates are stored in the dark.
History
The blue - white selection was first used in complementation, ie by insertion of a previously removed piece of DNA, the function of galactosidase should be restored. The blue - white selection for cloning was first used in pUC plasmids (eg, pUC19, pBluescript ).