Standard cloning strains DH5α, Top10, Novablue or α-Select Gold contain lacZΔM15 deletion mutation? The lacZ gene in the plasmid vector complements the lacZ deletion mutation in the host E. coli producing a functional enzyme?
If I understood it correctly the strains cannot produce functional beta gal because of the mutation and therefore complement by enzyme produce by the plasmids pUC, pGEM, pBluescript, pTZ.
or
the lacZ omega-fragment is expressed by the host strain? and lacZ alpha complements the lacZ omega-fragment?
Hi there,
alpha and omega fragment assembly results in a functional enzyme. So the host strain encodes the omega fragment and the empty plasmid encodes the alpha fragment. The blue clone results from the activity of alpha-omega assembly on X-gal, when cloning is successful alpha fragment is no longer produced resulting in white clone because X-gal is no longer degraded into blue product.
Hi again,
omega fragment (Cter) results from the full length enzyme truncated for the alpha fragment(Nter). See the dia below:
https://www.google.fr/search?q=alpha+and+omega+lacZ&tbm=isch&source=iu&ictx=1&fir=a0vyoFCWik5CMM%253A%252CM95ahMZbTnrgeM%252C_&usg=__DByg36D5EBFAJgkA90UJPjmMY8Q%3D&sa=X&ved=0ahUKEwj9pIS3lM3YAhWF6RQKHfdHCPgQ9QEIQjAF#imgrc=a0vyoFCWik5CMM:
Yes! lacZΔM15 is coding for Beta galactosidase lacking alpha peptide.
β-galactosidase cleaves the glycosidic bond in X-gal and form galactose and 5-bromo-4-chloro-3-hydroxyindole which dimerizes and oxidizes to 5,5'-dibromo-4,4'-dichloro-indigo, an intense blue product that is easy to identify and quantify.[15] It is used for example in blue white screen.[16] Its production may be induced by a non-hydrolyzable analog of allolactose, IPTG, which binds and releases the lac repressor from the lac operator, thereby allowing the initiation of transcription to proceed.
It is commonly used in molecular biology as a reporter marker to monitor gene expression. It also exhibits a phenomenon called α-complementation which forms the basis for the blue/white screening of recombinant clones. This enzyme can be split in two peptides, LacZα and LacZΩ, neither of which is active by itself but when both are present together, spontaneously reassemble into a functional enzyme. This property is exploited in many cloning vectors where the presence of the lacZα gene in a plasmid can complement in trans another mutant gene encoding the LacZΩ in specific laboratory strains of E. coli. However, when DNA fragments are inserted in the vector, the production of LacZα is disrupted, the cells therefore show no β-galactosidase activity. The presence or absence of an active β-galactosidase may be detected by X-gal, which produces a characteristic blue dye when cleaved by β-galactosidase, thereby providing an easy means of distinguishing the presence or absence of cloned product in a plasmid.
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