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Study on the induction of IPTG |367-93-1|

2015-06-08 来源:亚科官网
Abstract: Propylthiogalactopyranoside (IPTG) is an extremely potent inducer that is not metabolized by bacteria and is very stable. Therefore, it has been widely used in laboratories.
Key words: IPTG, inducing principle, using method, inducing experiment, research
Foreword
IPTG, Chinese name: isopropyl-β-D-thiogalactopyranoside, CAS number: 367-93-1, molecular formula: C9H18O5S, molecular weight: 238.30, application: IPTG is activity inducing substances of β-galactosidase. Based on this characteristic, when vector DNA (or other vector carrying lacZ gene) of pUC series is transformed with lacZ-deficient cells as a host or transfected with M13 phage vector DNA, if X-gal and IPTG were added to the medium, gene recombinants can be conveniently selected based on whether white colonies (or plaques) are present due to the a-complement of [beta]-galactosidase. In addition, it can also be used as an expression inducer of an expression vector having a promoter such as lac or tac.
1. Induction principle
First of all
The lactose operon of E. coli contains three structural genes, Z, Y and A, encoding galactosidase, dialysate and acetyltransferase, respectively, and a manipulation sequence O, a start sequence P and a Regulate genes. The I gene encodes a repressor protein, which binds to the O sequence, blocking the operator (meta) and leaving it off. There is also a catabolite (CAP) binding site upstream of the priming sequence P. By the P sequence, O sequence and CAP binding site together constitute the lac operon regulatory region, the three enzymes encoding genes that regulate the same regulatory region, to achieve the coordinated expression of the gene product.
Second
In the absence of lactose, the lac operator (meta) is in a repressed state. In this case, the Lac repressor expressed by the I sequence under the PI priming sequence binds to the O sequence, preventing the RNA polymerase from binding to the P sequence and inhibiting the transcription initiation. When there is lactose, lac operon (yuan) can be induced. In this operon (meta) system, the real inducer is not lactose itself. Lactose enters cells, the β-galactosidase-catalyzed conversion to isolactose. The latter, as an inducer, binds to the repressor molecule and changes the conformation of the protein, resulting in the dissociation of the repressor protein from the O-sequence and transcription is begin. Isopropylthiogalactoside (IPTG) is the same as lactose, is a highly effective inducer, is not metabolized by bacteria and very stable, it is widely used in laboratories.
2 IPTG induction
2.1 How to use IPTG
IPTG was first prepared as an aqueous solution of 23.83mg/mL (100mM) and sterilized by filtration. Then 100 μl of the above solution, 200 μl of X-Gal (20mg/ml in dimethylformamide (DMF) solution) and 100 μl of Amp (100 mg/ml) were added to 100 ml of agar medium, IPTG, X-Gal, Amp plate medium was made. After the DNA fragment has been inserted into the pUC series vector (or other vector carrying lacZ, Amp gene) and then transformed into lacZ-deficient cells, the IPTG, X-Gal and Amp plate media described above are applied, , and easily selected gene recombinants (white for DNA recombinants with DNA inserts) according to the blue-white. Induction of foreign gene expression, it commonly used in prokaryotic expression system, so that its expression increased, the product is stable, easy to identify, easy to purify.
2.2 IPTG induction experiment
Effects of IPTG induction on the expression of recombinant
The positive strain that remained was took out from a refrigerator at 86°C, streaked onto LB plates containing Amp, picked a single colony, inoculated in 5mL LB liquid medium containing Amp, cultured overnight at 37°C with shaking; positive transformants (1:100 ratio) were inoculated with 100 mL of LB medium flask, 230r·min-1, cultured at 37℃, OD value equal to 0.5, take pre-induction strain preservation. In addition, IPTG was added to a final concentration of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 mmol L-1, respectively, induced 2.5h, while testing DD value. The bacterial of 1 mL pre-induced and post-induction were centrifuged at 12000r·min-1 for 5 min. The supernatant was discarded, according to the OD value, 1 × PBS (OD600 × 50 μL) and 2 × SDS Gelatin loading buffer (including DTT), mix well, boil for 5min. Take 10 μL, the protein standard molecular weight as a reference, the separation gel concentration of 12% SDS PAGE electrophoresis analysis [1]. BandScan software was used to analyze protein expression. And transferred to nitrocellulose membrane to do the Westem_blot [2].
Conclusions
Listeria monocytogenes is one of the most important bacteria causing foodborne diseases. It is very resistant to the environment and grows well at both low (4°C) and high salt (20%) [3]. Currently, there are reports of contaminated food around the world. So this bacterium gets more and more attention by our country and the scientist all over the world. The availability of recombinant hemolysin has opened up new avenues for the detection of listeria and the diagnosis of listeriosis [4]. In this study, we demonstrated that the best concentration of recombinant Lactolide induced by lgG was 0.6mmo L-1 in liquid culture medium of LB medium, on the basis of which the increase of concentration did not increase the expression level. This not only saves IPTG costs in industrialized production but also reduces the impact of IPrrG's own toxicity on the use of genetically engineered drugs. In addition, there are many factors that affect the expression of the extracellular hemolysin, such as the culture medium, the culture temperature, the OD value of the bacterial liquid before induction, and the like. It still need further experiments.
References
[1] Lu Sheng Dong. Modern molecular biology experimental techniques [M]. 2 version. Beijing: China Union University Press, 1999.
[2] Sam Brooke J, Russell D w. Molecular cloning experiment guidelines [M]. 3 version. Beijing: Science Press, 2002.
[3] Palmer M. The family of thiol-activated, cholesteIDl-binding cy-tolysins [J]. Toxicon, 200 l, 39: 1681-1689
[4] Low J C, Donachie W. A review of Listeria monocytogenens and listeriosos [J]. Vet, 1997,153: 9-29
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