β-galactosidase produced by wild-type E. coli can cleave the colorless compound X-gal (5-bromo-4-chloro-3-indole-β-D-galactoside) into galactose and dark blue Substance 5-bromo-4-indigo. Colored substances can change the color of the entire cultured colony, and the color change is the most intuitive and effective method for identification and screening.
The genetically engineered bacteria suitable for blue-white screening were designed as β-galactosidase-deficient strains. The mutation of the gene encoding β-galactosidase in the chromosomal genome of this host bacteria causes the encoded β-galactosidase to lose a short peptide of 146 amino acids in the normal N segment and thus has no biological activity, that is, it cannot act on X-gal produces a blue substance. The vector used for blue-white screening has a gene called lacz', which includes: a β-galactosidase promoter; a segment encoding an α-peptide chain; and a multiple cloning site (MCS). MCS is located in the segment encoding the α-peptide chain, which is a selective insertion site for exogenous DNA, but itself does not affect the functional activity of the vector-encoded α-peptide chain. Although the genomes of the above-mentioned defective strains cannot encode active β-galactosidase alone, when the cells contain a plasmid with lacz', the α-peptide chain encoded by the plasmid lacz' gene is complementary to the N-terminal defective β-galactosidase mutant expressed in the strain genome, which has the same effect as the intact β-galactosidase and the ability of X-gal to generate blue substances. This phenomenon is called alpha-complementarity.
In operation, IPTG (isopropylthio-β-D-galactoside) was added to activate the promoter of β-galactosidase in lacz', and the colonies appeared blue in solid plate medium containing X-gal. The above are the phenotypes produced by the strain carrying the empty vector.When exogenous DNA (that is, the target fragment) is connected to the vector containing lacz', it will be inserted into MCS, and the reading frame of the α-peptide chain will be destroyed. This recombinant plasmid no longer expresses the α-peptide chain, and when it is introduced into the host-deficient strain, there is no α-complementary effect and no active β-galactosidase is produced. That is, X-gal in the non-decomposable medium produces blue color, and the culture phenotype shows white colonies.
In experiments, blue-and-white screening is usually used together with resistance screening. The plate medium containing X-gal also contains one or more antibiotics corresponding to the resistance carried by the carrier. In this way, one screening can determine: the untransformed bacteria have no resistance and do not grow; the bacteria transformed with the empty vector, that is, the bacteria without the recombinant plasmid, grow into blue colonies; the bacteria transformed with the recombinant plasmid, the target recombinant bacteria, grow into white colonies.