IPTG
Encyclopedia
Isopropyl β-D-1-thiogalactopyranoside, abbreviated IPTG, is a molecular biology
reagent.
This compound is used as a molecular mimic of allolactose
, a lactose
metabolite
that triggers transcription
of the lac operon
. Unlike allolactose, the sulfur (S
) atom creates a chemical bond which is non-hydrolyzable by the cell, preventing the cell from "eating up" or degrading the inductant; therefore the IPTG concentration remains constant. For induction, a sterile 1 M solution of IPTG is typically added by 1:1000 dilution into a logarithmically growing bacterial culture. Higher concentrations can be used.
IPTG induces the transcription of the gene coding for beta-galactosidase
, a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. One advantage of IPTG for in vivo
studies is that since it cannot be metabolized by E. coli its concentration remains constant and the rate of expression of lac p/o-controlled genes, is not a variable in the experiment. IPTG intake is independent on the action of lactose permease
, since other transport pathways are also involved.
In cloning experiments, colonies that have been transformed with the recombinant plasmid rather than a non-recombinant need to be identified. X-gal
is a substance that can be metabolised by beta-galactosidase
to produce a blue product. Thus cells expressing beta-galactosidase
grown in the presence of X-gal
and IPTG (to induce the expression) will turn blue. Where a DNA fragment has been inserted into the LacZ (one of the genes for beta-galactosidase
) there will be no action upon X-gal
and the cells will not turn blue, thus identifying the cells that carry recombinant plasmid rather than non-recombinant plasmid.
Many regulatory elements of the lac operon are used in inducible recombinant protein systems; IPTG is an effective inducer in the concentration range of 100 μM to 1.5 mM.
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
reagent.
This compound is used as a molecular mimic of allolactose
Allolactose
Allolactose is a disaccharide similar to lactose. It consists of the monosaccharides D-galactose and D-glucose linked through a β1-6 glycosidic linkage instead of the β1-4 linkage of lactose. It may arise from by the occasional transglycosylation of lactose by β-galactosidase.It is an inducer of...
, a lactose
Lactose
Lactose is a disaccharide sugar that is found most notably in milk and is formed from galactose and glucose. Lactose makes up around 2~8% of milk , although the amount varies among species and individuals. It is extracted from sweet or sour whey. The name comes from or , the Latin word for milk,...
metabolite
Metabolite
Metabolites are the intermediates and products of metabolism. The term metabolite is usually restricted to small molecules. A primary metabolite is directly involved in normal growth, development, and reproduction. Alcohol is an example of a primary metabolite produced in large-scale by industrial...
that triggers transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
of the lac operon
Lac operon
The lac operon is an operon required for the transport and metabolism of lactose in Escherichia coli and some other enteric bacteria. It consists of three adjacent structural genes, lacZ, lacY and lacA. The lac operon is regulated by several factors including the availability of glucose and of...
. Unlike allolactose, the sulfur (S
Sulfur
Sulfur or sulphur is the chemical element with atomic number 16. In the periodic table it is represented by the symbol S. It is an abundant, multivalent non-metal. Under normal conditions, sulfur atoms form cyclic octatomic molecules with chemical formula S8. Elemental sulfur is a bright yellow...
) atom creates a chemical bond which is non-hydrolyzable by the cell, preventing the cell from "eating up" or degrading the inductant; therefore the IPTG concentration remains constant. For induction, a sterile 1 M solution of IPTG is typically added by 1:1000 dilution into a logarithmically growing bacterial culture. Higher concentrations can be used.
IPTG induces the transcription of the gene coding for beta-galactosidase
Beta-galactosidase
β-galactosidase, also called beta-gal or β-gal, is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins...
, a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. One advantage of IPTG for in vivo
In vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
studies is that since it cannot be metabolized by E. coli its concentration remains constant and the rate of expression of lac p/o-controlled genes, is not a variable in the experiment. IPTG intake is independent on the action of lactose permease
Lactose permease
Lactose permease has a structure that consists of two halves, each of which comprises six membrane-spanning alpha helices.The two halves are well separated and are joined by a single stretch of polypeptide....
, since other transport pathways are also involved.
In cloning experiments, colonies that have been transformed with the recombinant plasmid rather than a non-recombinant need to be identified. X-gal
X-gal
X-gal is an organic compound consisting of galactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and collaborators in Detroit, MI, in 1964. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl...
is a substance that can be metabolised by beta-galactosidase
Beta-galactosidase
β-galactosidase, also called beta-gal or β-gal, is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins...
to produce a blue product. Thus cells expressing beta-galactosidase
Beta-galactosidase
β-galactosidase, also called beta-gal or β-gal, is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins...
grown in the presence of X-gal
X-gal
X-gal is an organic compound consisting of galactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and collaborators in Detroit, MI, in 1964. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl...
and IPTG (to induce the expression) will turn blue. Where a DNA fragment has been inserted into the LacZ (one of the genes for beta-galactosidase
Beta-galactosidase
β-galactosidase, also called beta-gal or β-gal, is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins...
) there will be no action upon X-gal
X-gal
X-gal is an organic compound consisting of galactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and collaborators in Detroit, MI, in 1964. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl...
and the cells will not turn blue, thus identifying the cells that carry recombinant plasmid rather than non-recombinant plasmid.
Many regulatory elements of the lac operon are used in inducible recombinant protein systems; IPTG is an effective inducer in the concentration range of 100 μM to 1.5 mM.