Alginate lyase Alga and coding gene and application thereof
A technology of alginate lyase and sodium alginate, which is applied in the fields of lyase, application, and genetic engineering, and can solve problems such as time-consuming, difficult to control degradation conditions, and complicated operation
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Embodiment 1
[0030] Cultivation of Vibrioalginolyticus40B and Extraction of Strain Genomic DNA
[0031] The strain used was Vibrio W13, and the monoclonal colony of Vibriosp.W13 strain was picked and inoculated into 50ml liquid medium, and then placed in a shaker with a temperature of 30°C and a rotation speed of 220rpmin for 24 hours, and then cultured The cells were collected by centrifugation and the supernatant was discarded.
[0032] The liquid medium formula used is (g / L): beef extract 5g, glucose 15g, yeast extract powder 1.0g, NaCl5.0g, MgSO4 7H2O0.5g, CaCl20.2g, KH2PO41.0g, FeSO47H2O0.02g, The pH value is 7.0.
[0033] Take 4mL of the cultured bacterial solution, put it in a 2mL tube, centrifuge at 5000g for 10min. The supernatant was discarded, and the genome was extracted using a bacterial genome extraction kit (Thermal Scientific, USA). The operation was as follows: resuspend the bacteria with 180 μL of digestion solution (Digestion Solution), then add 20 μL of proteinase K, mi...
Embodiment 2
[0034] Example 2 Cloning and identification of the gene encoding alginate lyase Alga
[0035] The following amplification primers were designed according to the possible alginate lyase gene in the Vibrioalginolyticus40B genome: upstream primer (5'-CGCATGAAGCATATTTTCTTCAA-3') and downstream primer (5'-CCGGCCTTGGTACTTACCAAAAG-3'). Using the extracted strain genome as a template, PCR amplification was performed to obtain the full-length sequence of alginate lyase Alga gene. The PCR conditions were: pre-denaturation at 94°C for 3 minutes, followed by 30 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 2 minutes, and finally extension at 72°C for 10 minutes. Agarose gel electrophoresis showed a specific band between 1.0kb and 2.0kb, which was excised from the agarose gel and purified using a DNA gel recovery kit.
[0036] The purified DNA fragment was connected to the cloning vector pMD19-T, transformed into Escherichia coli DH5α competent cells, and cultured in LB solid medium ...
Embodiment 3
[0045] Construction of recombinant expression vector of alginate lyase Alga
[0046] According to the complete sequence of the alginate lyase gene (signal peptide removed), the upstream primer (5'-CGCGGATCCGTGGGCTGTAATAGTACGGC-3') and the downstream primer (5'-CCGCTCGAGGCCTTGGTACTTACCAAAAG-3') were designed, and PCR amplification was carried out to obtain the complete gene of the alginate lyase Alga long sequence. The PCR conditions were: pre-denaturation at 94°C for 3 minutes, followed by 30 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 2 minutes, and finally extension at 72°C for 10 minutes. The PCR product was digested with BamHI and XhoI and recovered; the Escherichia coli expression vector pET21a(+) was also digested with BamHI and XhoI and recovered, connected with the above-mentioned target gene and transformed into E. coli DH5α, and screened for the presence of ampicillin Penicillin-resistant transformants. The positive transformant plasmid was extracted with a ...
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