Method for producing artificial bear gall powder through engineering bacteria fermentation
A technology of engineering bacteria and bear bile powder, applied in the biological field, can solve the problems of a large number of organic solvents, cumbersome steps, and unfavorable environmental protection
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Embodiment 1
[0070] Whole-gene synthesis of codon-optimized 7α-HSDH and 7β-HSDH genes
[0071] 7α-HSDH and 7β-HSDH are 7α-hydroxysteroid dehydrogenase and 7β-hydroxysteroid dehydrogenase, respectively;
[0072]The two 7α-HSDHs were derived from: Clostridium sardiniense strain DSM599, GenBank accession number: JN191345.1) and Escherichia coli strain TW14359, GenBank accession number: CU928163.2),
[0073] The two 7β-HSDH genes were derived from: Clostridium sardiniense strain DSM599, GenBank accession number: JN191345.1) and Ruminococcus gnavus strain N53, GenBank accession number: KF052988.1.
[0074] Using http: / / www.jcat.de / online software, Escherichia coli as host bacteria, codon optimization of the whole gene sequence according to the codon preference, and whole gene synthesis, respectively recorded as α1, α2, β1 , β2.
[0075] The codon-optimized 7α-HSDH derived from Clostridium sardiniense (denoted as α 1 ) gene sequence as shown in SEQ ID No 1; codon-optimized 7α-HSDH derived fr...
Embodiment 2
[0077] Construction of engineered bacteria
[0078] 1) Expanded culture using pMD as a carrier with a synthetic target gene α 1 、α 2 , β 1 , β 2 coli, and DH5α-pETM6 strain: take 10μl sample, add 10ml of LB (Amp + ) medium, cultivated on a shaker at 37° C. for 12 to 16 hours, and the shaker speed was 225 rpm.
[0079] 2) Use the plasmid extraction kit purchased from Shanghai Chuangying Biotechnology Co., Ltd. to extract the plasmids of the above bacteria, and operate according to the operation instructions of the kit.
[0080] 3) For the extracted pMD-α 1 , pMD-α 2 , pMD-β 1 , pMD-β 2 The plasmid and the expression vector pETM6 were subjected to double enzyme digestion, and the enzyme digestion system was as follows:
[0081]
[0082] Digest at 37°C for 2 to 4 hours, and purify the target fragment with a gel recovery kit.
[0083] 4) The recovered 850bp target gene fragment α 1 、α 2 , β 1 , β 2 , respectively, and the 5.6kb pETM6 carrier fragment, the connecti...
Embodiment 3
[0108] Cultivation and fermentation of engineered bacteria
[0109] 1) Apply the engineering bacteria stored in the -80°C refrigerator to the LB solid plate medium containing 100 mg / L ampicillin by streaking method, and activate and culture at 37°C for 12 hours;
[0110] The composition of the LB solid medium is as follows: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L, agarose 15.0g / L.
[0111] 2) Pick a single colony from the LB solid plate medium, inoculate it in the LB liquid medium supplemented with 100mg / L ampicillin, and culture it with shaking at 225rpm and 37°C for 12 hours;
[0112] The composition of described LB liquid medium is as follows: tryptone 10g / L, yeast extract 5g / L, sodium chloride 10g / L;
[0113]3) The above culture was inoculated into liquid 1XM9 medium at a ratio of 1:50, cultured with shaking at 225rpm / min and 37°C for 5 hours, and 1mM isopropyl-β-D-thiogalactopyranoside ( IPTG), adjusted to 30 ° C for 3 hours;
[0114] 4) prepare the N...
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