Bacterial glutamine synthetase and preparation method thereof
A technology of glutamine and synthetase, which is applied in the field of molecular biology, can solve the problems of low yield and cumbersome cultivation, and achieve the effect of simple method and easy operation
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Embodiment 1
[0022] Acquisition of Bacterial Glutamine Synthetase Gene
[0023] 1) Obtain the strain containing the target gene
[0024] The present invention utilizes a strain of the genus Cryobacterium, which is screened from the soil of Changbai Mountain. The specific screening process is like the Chinese invention patent with the patent number 201710034491.5. The strain is named: Cryobacteriumbaishanse 02, and it is preserved in the China Type Culture Collection Center , deposit number CCTCC NO: M2016604. The date of deposit is October 31, 2016, and the deposit address is Wuhan University, Wuhan, China.
[0025] 2) Genome extraction
[0026] The genome of the Cryobacterium baishanse 02 strain was extracted using a DNA extraction kit (TAKARA Dalian) as a template, and the extracted genome samples were stored at -20°C until use.
[0027] 3) PCR amplification of the target gene
[0028] Design primers: the forward primer is shown in SEQ ID NO.3, and the reverse primer is shown in SEQ ...
Embodiment 2
[0036] Construction of Expression Vector of Bacterial Glutamine Synthetase
[0037] The PCR amplified product obtained in Example 1 was carried out to gel recovery, and the PCR amplified product was carried out double digestion reaction with restriction endonuclease BamHI and HindIII; Carrier pET21a (+ ) for double enzyme digestion reaction, and then catalyzed by high-efficiency DNA ligase High Ligation (TOYOBO) to connect the pET21a(+) and PCR amplification product after the double enzyme digestion reaction; construct the expression vector pET21a- GS, the expression vector pET21a-GS was constructed as figure 2 shown.
Embodiment 3
[0039] Expression and purification of bacterial glutamine synthetase protein
[0040] The expression vector pET21a-GS obtained in Example 2 is transformed into the competent thallus of Escherichia coli BL21, and the preparation of the competent state of Escherichia coli BL21 adopts CaCl 2 method, CaCl 2 The competent method for preparing Escherichia coli BL21 is a routine experimental method in the laboratory, so it will not be repeated here. The resulting expression vector pET21a-GS was transformed into Escherichia coli BL21 to obtain recombinant strain Ecoli BL21-pET21a-GS. Expand Ecoli BL21-pET21a-GS to OD 600 After = 0.6, add 1mM IPTG, induce culture and expression of bacterial glutamine synthetase protein at 28°C, collect the bacteria after the induction culture is over, break the bacteria and purify with Ni-NTA affinity chromatography column to obtain the purified bacteria Glutamine synthetase proteins, such as image 3 As shown, the protein molecular weight of the o...
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