What is Alpha-complementation?

The Escherichia coli enzyme beta-galactosidase is a homotetramer of the protein produced from the lacZ gene of the lac operon. Certain mutations in the 5' region of the lacZ prevent association of the subunits. Monomers lack enzyme activity. So, the failure to assemble leads to a lac- phenotype, i.e. lactose is not metabolized in such mutated bacteria.

In some such mutants, subunits assemble, and hence, enzyme activity can be restored if a small amino-terminal fragment of the lacZ protein product is present in the cell. This fragment is 26 amino acids long and is called as the "alpha-polypeptide". This reversal of the effect of mutation is known as 'alpha-complementation'.

Messing and his coworkers took advantage of alpha-complementation and constructed the M13 phage and pUC plasmid series of cloning vectors. The polycloning region of these vectors is embedded in the open reading frame of an alpha-polypeptide gene fragment (lacZalpha). The presence of the polycloning site does not affect the ability of this lacZalpha fragment to alpha-complement. This region encodes the first N-terminal 146 amino acids of the lacZ protein. The chromosome of the bacterial host of this plasmid vector carries a mutation that deletes amino acids 11-41 from the protein product (lacZdel15 allele), and hence can be subjected to alpha-complementation. The lacZalpha gene present on the plasmid will complement the chromosomal mutation, and produce a lacZ+ cell, fully capable of metabolizing lactose. (The N-terminal fragment of lacZ protein produced from lacZalpha in the vector, forms a multimer with the mutant lac protein produced by the host, forming a functional beta-galactosidase enzyme.)

Role of IPTG:

IPTG, or Isopropyl-beta-D-galactopyrano side is a synthetic analog of allolactose, a lactose metabolite that triggers transcription of lac operon. Thus it induces the activity of beta-galactosidase and is often used to induce the expression of cloned genes that are under the control of lac operon.

Role of X-gal: Blue-White Screening

The activity of beta-galactosidase can be monitored by including in the growth medium the chromogenic substrate X-gal, or 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside. Beta-galactosidase hydrolyzes X-Gal into a colorless galactose and 4-chloro-3-brom-indigo which forms a deep blue precipitate. Induction of the lacZ gene with the non-substrate (gratuitous) inducer IPTG leads to the hydrolysis of X-Gal. On solid medium, colonies that are expressing active beta-galactosidase will be blue in color, while those that are devoid of the enzyme will be colorless/white. This is known as 'blue-white screening', often utilized in distinguishing between lacZ mutants and wild-type bacteria.

Advantage of Blue-White Screening

When lacZdel15 mutant bacteria containing pUC plasmid vectors (i.e. vectors carrying the N-terminal fragment of the lacZ gene) without an insert, are plated out on agar containing IPTG and X-gal, blue colonies will be produced. However, when foreign DNA is inserted withing the polycloning site of the vector, it will disrupt the open reading frame of the lacZalpha fragment and prevent alpha-complementation. Cells containing such vectors will thus be lacZ- and hence, white. Thus, transformed cells, bearing the foreign DNA insert can be identified from the color of the colony.

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