Blue white screen

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File:Blue White Screen2.JPG
A LB agar plate showing the result of a blue white screen.

The blue-white screen 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. 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. This technique allows for the quick and easy detection of successful ligation, without the need to individually test each colony.

Background

Cloning, alongside PCR, is one of the most common techniques in molecular biology. Blue white screening makes this procedure less time and labour intensive by allowing for the screening of successful cloning reactions through the color of the bacteria colony.

The molecular mechanism for blue white screening is based on the Lac operon. The vector (e.g. pBluescript) contains the Lac Z gene with an internal Multiple cloning site(MCS). The MCS can be cleaved by different restriction enzymes so that the foreign DNA can be inserted within Lac Z gene, thus disrupting the activity of the β-galactosidase when the protein is expressed. The chemical required for this screen is X-gal, a colorless modified galactose sugar that is metabolized by β-galactosidase the products are a bright blue, and thus functions as an indicator, and Isopropyl β-D-1-thiogalactopyranoside(IPTG), which functions as the inducer of the Lac operon in the absence of lactose. The hydrolysis of colourless X-gal by the β-galactosidase causes the characteristic blue color in the colonies; it shows that the colonies contain unligated vector. 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.

The correct type of vector and competent cells are important considerations when planning a blue white screen.

It is also important to understand the lac operon is regulated by cAMP levels and the binding of cAMP to CAP. This CAP-cAMP complex promotes the binding of RNA polymerase to the lac promoter, which leads to transcription of the lac genes. cAMP levels are regulated by the cell's incorporation of glucose. Since most bacteria preferentially utilize glucose even in the presence of lactose, the lac genes will only be turned on when glucose levels drop low enough to allow the CAP-cAMP complex to form.

Drawback

Some white colonies may not contain the recombinant vector. The LacZ, in the vector transformed into the cells of colonies, may be non-functional and cannot produce beta-galactosidase. As a result, these cells cannot convert X-gal to the blue substance.

See also



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