Experimental principle:
E.coli's lactose operon (unit) contains three structural genes, Z, Y and A, encoding galactosidase, permease and acetyltransferase, respectively, in addition to a manipulation sequence O, a promoter sequence P and a Regulate gene I. The I gene encodes a repressor protein that binds to the O sequence, causing the operon (meta) to be repressed and in a closed state. There is also a decomposition (metabolism) gene activating protein (CAP) binding site upstream of the initiation sequence P. The P sequence, the O sequence and the CAP binding site together constitute the regulatory region of the lac operon, and the coding genes of the three enzymes are regulated by the same regulatory region to achieve coordinated expression of the gene product.
The lac operon (meta) is in a repressed state in the absence of lactose. At this time, the Lac repressor expressed by the I sequence under the manipulation of the PI promoter sequence binds to the O sequence, hinders binding of the RNA polymerase to the P sequence, and inhibits transcription initiation. The lac operon (member) can be induced when lactose is present. In this operon (meta) system, the true inducer is not lactose itself. Lactose enters the cell and is converted to isolose by β-galactosidase. The latter acts as an inducer molecule that binds to a repressor protein, causing a conformational change in the protein, resulting in dissociation and transcription of the repressor protein from the O sequence. Isopropylthiogalactoside (IPTG) has the same effect as that of isolactose. It is a highly potent inducer and is not stable by bacterial metabolism. Therefore, it is widely used in laboratories.
Experimental reagents:
1. LB (Luria-Bertani) medium: Yeast extract 5 g, Peptone 10 g, NaCl 10 g, Agar 1-2%, Distilled water 1000 ml, pH 7.0.
2. IPTG stock solution: 2 g IPTG is dissolved in 10 mL of distilled water, filtered through a 0.22 μm filter, dispensed into 1 mL / part, and stored at -20 °C.
3. Gel electrophoresis loading buffer: 50 mmol / L Tris -CI (pH 6.8), 50 mmol / L DTT, 2 % SDS (electrophoresis grade), 0.1% bromophenol blue, 10% glycerol.
Experimental steps:
1. Digestion of the vector DNA and the gene of interest with an appropriate restriction endonuclease;
2. The target gene and vector are ligated according to the ligation step and transformed into transformed DH5α competent cells;
3. Single colonies were picked and inoculated into 5 mL LB (0.1 g·L-1 ampicillin) and cultured overnight at 37 °C.
4. Transfer to 2 ml LB (0.1 g·L-1 ampicillin) in a volume ratio of 2%, continue to culture for 2.5 hr at 37 °C, to the logarithmic growth phase of the bacteria, add 0.1 mmol/L IPTG 2 μl to induce 3- 4 hr, at the same time set up without IPTG induction as a control;
5, take 1 mL of the above bacterial solution, centrifuge at 12000 rpm for 10 min, collect and precipitate;
6. Resuspend in ice in 100 μl PBS and add PMSF to a final concentration of 10 mmol/L. The shaker was shaken and mixed. Add 2× loading buffer, boil for 10 min, and centrifuge at 12,000 rpm for 10 min.
7. Take 10 μl of the sample before and after induction for SDS-PAGE electrophoresis analysis.
(PS: If the prokaryotic promoter of the expression vector is a PL promoter, culture at 30 -32 °C for several hours, the OD600 of the culture solution is 0.4-0.6, and the temperature is rapidly raised to 42 °C for 3 - 5 hours; if expressed The prokaryotic promoter of the vector is tac, etc., and the bacteria are cultured at 37 °C for several hours to reach the logarithmic growth phase, then IPTG is added to a final concentration of 1 mmol / L, and the culture is continued for 3 -5 h)
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