Overview:
Isopropyl thiogalactoside (IPTG) is a highly potent inducer that is stable and not metabolized by bacteria, so it is widely used in laboratories. IPTG is often used in cloning experiments where the induction of β-galactosidase activity is required. It is often used in combination with X-Gal or Bluo-Gal for blue-white screening of recombinant bacterial colonies that can induce expression of the lac operon in Escherichia coli. IPTG binds to the lacI repressor protein and changes its conformation to prevent inhibition of lacZ, the gene encoding β-galactosidase.
Principle of induction:
The lactose operon (unit) of E.coli contains three structural genes, Z, Y and A, which encode galactosidase, permease and acetyltransferase, respectively. In addition, there is an operator sequence O, a promoter sequence P and a regulatory gene I. The I gene encodes a repressor protein that binds to the O sequence, keeping the operon (element) repressed and closed. There is also a catabolizer gene activator protein (CAP) binding site upstream of the initiator sequence P. The P sequence, O sequence and CAP binding site together constitute the lac operon regulatory region, and the coding genes of the three enzymes are regulated by the same regulatory region, so that the coordinated expression of gene products is realized. In the absence of lactose, the lac operon is under repression. At this point, the Lac repressor protein expressed by the I sequence under the manipulation of the PI initiator sequence binds to the O sequence, hindering RNA polymerase from binding to the P sequence and inhibiting transcription initiation. The lac operon can be induced in the presence of lactose. In this operon (meta) system, the real inducer is not lactose itself. Lactose enters the cell and, catalyzed by β-galactosidase, is converted to isogalactose. As an inducer, the latter binds to the repressor protein and causes a conformational change, leading to the dissociation of the repressor protein from the O sequence and transcription. Isopropyl thiogalactoside (IPTG), which has the same effect as isolactose, is a strong inducer and is very stable without bacterial metabolism, so it is widely used in laboratories.
Advantages:
1. Molecules that induce the synthesis of enzymes, but are not broken down, are called placebo inducers. Because lactose can induce the synthesis of enzymes, but then decompose them, resulting in many complicated kinetic problems, people often use placebo inducers to conduct various experiments. 2.IPTG is not metabolized by the bacteria and is a lactose analogue. Once it enters the cells, it will produce a sustained and long-term induction effect, so the induction efficiency of IPTG is high, and often only a small amount (1mmol/L) can achieve the ideal induction effect. Since IPTG is not metabolized, it specifically induces the expression of foreign protein in the cell. It was not affected by cell metabolism, and the induction effect was sustained and stable. Lactose has a dual effect, which can be used as the precursor of the inducer isogalactose, and can be used as a carbon source. During the induction process, it is very unstable. IPTG is suitable for experimental research because of its excellent stability. IPTG could be efficiently copied in the absence of lacY gene. 4. Sulfur is substituted for oxygen in the galactosidic bond and the hydrolytic activity is lost, but the affinity of thiogalactoside and its homologous oxo compounds to the enzyme site is the same. Although IPTG is not recognized by β-galactosidase, it is a very effective inducer of lac gene cluster. Usage: First, IPTG was prepared into 23.83mg/ml(100mM) aqueous solution, filtered and stored. Then, in 100ml AGAR medium, 100μl of the above solution, 200μl of X-Gal(20mg/ml of dimethylformamide (DMF) solution) and 100μl of Amp(100mg/ml) were added to make IPTG, X-Gal and Amp plate medium. When dna fragments were inserted into pUC vectors (or other vectors containing lacZ and Amp genes) and then transformed into lacz-null cells, they were coated with IPTG, X-gal and Amp plates as described above. And conveniently pick out gene recombinants (white is gene recombinants with DNA inserts).
| Melting point |
105 °C |
| Specific optical rotation |
-31 º (c=1, water) |
| Boiling point |
350.9°C (rough estimate) |
| Density of density |
1.3329 (rough estimate) |
| Index of refraction |
1.5060 (estimate) |
| Storage conditions |
2-8°C |
| Degree of solubility |
Dissolved in water or methanol. |
| Coefficient of acidity (pKa) |
13.00±0.70(Predicted) |
| form |
White powder crystallization |
| Solubility of water |
soluble |
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