Method for increasing soluble expression quantity of 4, 6-alpha-glucosyltransferase
A technology of glucosyl and transferase, which is applied in the fields of protein engineering and fermentation engineering, and achieves the effect of remarkable effect, simple operation process, and improvement of soluble expression amount.
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Embodiment 1
[0053] Embodiment 1: Construction of the expression vector derived from the 4,6-α-glucosyltransferase gene (gtfB) of Lactobacillus fermentum (Lactobacillus fermentum)
[0054] Using a synthetic plasmid containing the gtfB gene (nucleotide sequence shown in SEQ ID NO.1) as a template, the target gene was obtained by PCR, and then connected to the pET15b plasmid to finally obtain the expression vector pET15b-gtfB. The specific process is as follows:
[0055] gtfB-F: 5'- CATATG CAGGCCAACGATGGTCATTG-3' (the underline is the NdeI restriction site, SEQ ID NO.3)
[0056] gtfB-R: 5'- GGATCC TTAATCATCTTCAATATTG-3' (the underline is the BamHI restriction site, SEQ ID NO.4) artificial sequence
[0057] PCR reaction system: 10xSuper PfxMasterMix 5 μL, GtfB-F (10 μM) 1 μL, gtfB-R (10 μM) 1 μL template 0.5 μL, add sterile water to make up to 50 μL. PCR amplification program: 98°C 5min pre-denaturation; (98°C 15S, 55°C 30S, 72°C 3.5min) x 30 cycles; 72°C extension 10min.
[0058] The P...
Embodiment 2
[0061] Example 2: Transformation of BL21(DE3) with the recombinant plasmid pET15-gtfB to obtain a recombinant strain
[0062] Take 2-5 μL of recombinant plasmid pET15-gtfB, add BL21(DE3) competent cells, incubate on ice for 30 min, then heat shock at 42°C for 90 s, add 800 μL LB to recover for 40 min, and coat with LB containing ampicillin (100 μg / mL) for solid culture culture base at 37°C for 12 hours, select the correct transformants into LB liquid medium, culture at 37°C and 200rpm for 10 hours, add glycerol, store the strain at -80°C, and ferment the strain BL21 / pET15-gtfB .
Embodiment 3
[0063] Example 3: Adding small molecular substances to TB medium increases the enzyme production of recombinant Escherichia coli BL21(DE3) / pET15-gtfB shake flask fermentation
[0064] (1) Seed activation stage
[0065] From the glycerol tube at -80°C, take 10 μL of the recombinant bacteria constructed in Example 2 and insert it into a 100mL Erlenmeyer flask containing 10mL of liquid LB medium, and cultivate it on a rotary constant temperature and speed-adjustable shaker, controlling the temperature at 37°C and rotating speed 200rpm, initial pH 7.0, cultured for 10h.
[0066] (2) Shake flask fermentation stage
[0067] Set up different fermentation media: add betaine, maltose, β-cyclodextrin, trehalose, and sorbitol to the TB medium.
[0068] The seed culture solution (OD 600 About 3) Inoculate 2mL / 100mL inoculum into the above-mentioned fermentation medium respectively, at a rotation speed of 200rpm and a temperature of 37°C, after culturing for 2 hours, add inducers with a...
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