Recombinant strain as well as construction method and application thereof
A technology for recombinant strains and construction methods, applied in the field of microorganisms, can solve the problems of insufficient NADPH, accumulation of NADH, and inability to apply
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Embodiment 1
[0056] Example 1 Construction of expression plasmid pXMJ19-gapN
[0057] 1. With the genomic DNA of Streptococcus mutans (streptococcusmutans) ATCC25175 as template, carry out PCR amplification with the primer pair that gapNF and gapNR form, obtain PCR amplification product:
[0058] gapNF: 5-'T GGATCC ATGACAAAACAATATAAAATTA-3' (the underline is the recognition site for BamHI digestion), as shown in SEQ ID No.2;
[0059] gapNR: 5-'C GAATTC TTATTTGATATCAAATACGACGG-3' (the underline is the recognition site for EcoRI digestion), as shown in SEQ ID No.3;
[0060] 2. Recover the PCR product of step 1 and connect it to the vector pEASY TM -T5 (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), the recombinant plasmid pT5-gapN was obtained, and the gapN gene sequence was obtained by sequencing, as shown in SEQIDNo.1, and its protein sequence was shown in SEQIDNo.4;
[0061] 3. Use two restriction endonucleases (BamHI and EcoRI) to double digest the plasmid pT5-gapN and...
Embodiment 2
[0063] Example 2 Construction of Engineering Bacteria CGMCC1.299 / pXMJ19-gapN
[0064] 1. Preparation of electric shock transformation competent cells of Corynebacterium;
[0065] 2. Electric shock transformation: Add 3-8 μL of pXMJ19-gapN plasmid to competent cells, place on ice for 10 minutes; transfer to a 1 mm electric shock cup, and electric shock at 1.8 or 2.1 kV for 5-7 ms;
[0066] 3. Add 1 mL of LB medium, incubate at 31°C and 150 rpm for 1 hour; spread it on a brain-heart medium plate containing 25 μg / mL kanamycin after concentration, and incubate at a constant temperature of 31°C for 36-48 hours;
[0067] 4. Screen the positive clones, and carry out PCR identification with a primer pair consisting of gapNF / gapNR (the one with a specific band of about 1.4 kb is positive) to obtain recombinant bacteria.
Embodiment 3
[0068] Example 3 Engineering bacteria CGMCC1.299 / pXMJ19-gapN (MHZ-1011) fermented to produce L-valine
[0069] 1. Medium
[0070] Seed activation medium: yeast extract 1%, peptone 1%, sodium chloride 0.5%, glucose 0.5%, agar 2%, pH7.2;
[0071] Seed medium: corn steep liquor 2.5%, glucose 1.0%, ammonium sulfate 0.4%, magnesium sulfate 0.04%, potassium dihydrogen phosphate 0.1%, urea 0.1%, CaCO 3 0.5%, pH7.2;
[0072] Fermentation medium: corn steep liquor 0.5%, glucose 12.0%, ammonium sulfate 4.0%, magnesium sulfate 0.04%, potassium dihydrogen phosphate 0.1%, CaCO 3 4%, V H 50ug / L, V B1 ·HCl100μg / L, pH7.2;
[0073] 2. Production of L-valine by shake flask fermentation
[0074] (1) Seed culture: Pick the engineering strain MHZ-1011 slant seeds 1 loop and connect them to a 500mL Erlenmeyer flask containing 20mL seed culture medium, and shake at 33°C and 220r / min for 16-22h;
[0075] (2) Fermentation culture: inoculate 2 mL of seed solution into a 500 mL Erlenmeyer flask c...
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