Induction of beta galactosidase gene in Escherichia coli

Induction of beta galactosidase gene in Escherichia coli

Aim

To study the induction of beta galactosidase gene in Escherichia coli

Principle

Lac operon in the gene cluster is responsible for the regulation of three enzymes needed for the metabolism of lactose. It consist of three structural genes and a control region that contains a promoter and an operator site.  RNA polymerase binds to the promoter site and this is enhanced by a cyclic AMP receptor protein which also binds to this region. Immediately adjacent to this area in the operator is the binding site for the repressor coded by the regulatory gene.  In the absence of the inducer, the repressor binds to the operator site and blocks RNA polymerase so that the structural genes can not be expressed.  However if the inducer is present, it binds to the repressor protein converting it to an inactive form that can not bind to the operator site so that the Lac operon can be transcribed.

Enzyme induction or repression is generally brought about by substrate on which the enzyme can act.  But in some instance there are molecules which resemble the substrate which can also act as inducers.  A typical kind of this induction is by IPTG (iso propyl thio galactoside) on Lac operon.  These inducers which are not substrate themselves can bring about induction because of their similarity with the substrate and are known as gratuitous inducers.  

5-Bromo-4-chloro-3-indolyl b-D-galactopyranoside, commonly known as X-Gal, is a chromogenic substrate for b-galactosidase.  X-Gal is cleaved by b-galactosidase to yield an insoluble blue precipitate.  X-Gal is particularly useful in molecular biology applications to detect the activity of b-galactosidase which is frequently used as a reporter gene.  In cloning, X-Gal is used to detect insertion of foreign DNA into the lacZ region of plasmid DNA using a complementation which is based on vectors such as the pUC and the M13mp series that carry a fragment of the b-galactosidase gene encoding b-galactosidase. Insertion of DNA into the lacZ region results in the loss of b-galactosidase activity. Lac+ bacterial colonies resulting from complementation will appear blue whereas bacterial colonies containing plasmid with DNA inserted in the lacZ region will be incapable of complementation and will appear white.  Many other applications also use X-Gal as a substrate to detect b-galactosidase activity. These include beta galactosidase-antibody linked immunoassays and immunohistochemistry, coliphage detection based on b-galactosidase induction, and the detection of micrometastasis formation during tumor progession.

X-Gal in conjunction with IPTG is used for blue/white colony screening.  X-Gal is an inert chromogenic substrate for beta-galactosidase which hydrolyzes X-Gal into colorless galactose and 4-chloro-3-brom-indigo, forming an intense blue precipitate. Induction of the lacZ gene with IPTG leads to the hydrolysis of X-Gal and to the development of blue colonies.

Beta galactosidase

 

Induction of the lacZ gene with IPTG leads to the production of Beta galactosidase and thus hydrolysis of X-Gal and to the development of blue colonies.

Materials and methods

1. Log phase culture of E. coli

2. 20 mg/ml solution of IPTG in dimethylsulfoxide (DMSO)

3. 20 mg/ml stock solution of X-Gal in dimethylsulfoxide (DMSO)

4. Nutrient agar plates

Procedure

1. Two sets of nutrient agar plates were preapared.  In the first set of plate, 20 µl IPTG and 100 µl of X-gal were spread.

2. 100 µl of log phase culture of E. coli was spread over the plate using a sterile L rod.

3. In the second set of plates 100 µl of X-gal alone was spread followed by spreading 100 µl of log phase culture of E. coli.

4. The plates were incubated overnight at 37^o C.

Observation

In the plates spread with X-gal and IPTG, blue coloured colonies appeared.  But no colour was observed in plates spread with X-gal alone.

Result

The development of blue coloured colonies is due to the degradation of X-gal.  Beta galactosidase production took place in the presence of the inducer IPTG and caused the degradation of X-gal.