题目

linearized pUC18 vector and DNA insert.
linearized pUC18 vector and of DNA insert are added in a LB/ampicillin agar plate.And IPTG and X-gal are also added.Why we see one white point in this agar?please account the reason.

提问时间：2021-04-07

答案

This is refer to the blue-white screen.It is a molecular technique that allows for the detection of successful ligations in vector-based gene cloning.DNA of interest is ligated into a vector (in this case pUC18).The vector is then transformed into competent cell (bacteria).The competent cells are grown in the presence of X-gal.If the ligation was successful,the bacterial colony will be white; if not,the colony will be blue.
The molecular mechanism for blue/white screening is based on a genetic engineering of the lac operon in the Escherichia coli laboratory strain serving as a host cell combined with a subunit complementation achieved with the cloning vector.The vector encodes the α subunit of LacZ protein with an internal multiple cloning site (MCS),while the chromosome of the host strain encodes the remaining Ω subunit to form a functional β-galactosidase enzyme.The MCS can be cleaved by different restriction enzymes so that the foreign DNA can be inserted within the lacZα gene,thus disrupting the production of functional β-galactosidase.The chemical required for this screen is X-gal,a colourless modified galactose sugar that is metabolized by β-galactosidase to form an insoluble product (5-bromo-4 chloroindole) which is bright blue,and thus functions as an indicator.Isopropyl β-D-1-thiogalactopyranoside (IPTG),which functions as the inducer of the Lac operon,can be used in some strains to enhance the phenotype,although it is with many common laboratory strains unnecessary.The hydrolysis of colourless X-gal by the β-galactosidase causes the characteristic blue colour in the colonies; it shows that the colonies contain vector without insert.White colonies indicate insertion of foreign DNA and loss of the cells' ability to hydrolyse the marker.
Bacterial colonies in general,however,are white,and so a bacterial colony with no vector at all will also appear white.These are usually suppressed by the presence of an antibiotic in the growth medium.A resistance gene on the vector allows successfully transformed bacteria to survive despite the presence of the antibiotic.