Steroid 11β-Hydroxylase and Its Encoding Gene and Application in Absidia indica
A technique for encoding and transgenic cell lines, applied in the field of steroid 11β-hydroxylase and its encoding gene and application, can solve the problem of low chiral specificity
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Embodiment 1
[0090] Example 1. Cloning and expression of Absidia coeruleus cytochrome reductase and 11-position β-hydroxylase
[0091] Gene cloning and expression are divided into the following 3 steps:
[0092] 1. Extraction of Absidia blue total RNA
[0093] First of all, culture Agsidia blue on the plate for several days, collect a certain number of spores and insert them into 50mL potato-dextrose (PDA) medium, and cultivate them overnight until a large number of bacteria are synthesized; then, collect the blue plow by centrifugation Wash the mycelium of the head mold with potassium phosphate buffered saline (PBS), and finally resuspend it with 50 mL of buffer and add the substrate deoxyhydrocortisone 21-acetate (RSA) with a final concentration of 170 mg / L to induce it for 1 h , sampled for RNA extraction.
[0094] RNA extraction method:
[0095] (1) Add 0.5mm grinding beads (to fill the bottom of the conical tube) and 1mL Trizol into a 2.0mL screw-cap tube (RNase free), and then add...
Embodiment 2
[0121] Embodiment 2, the construction of Saccharomyces cerevisiae engineering bacteria HC001
[0122] The starting bacterium Saccharomyces cerevisiae BY4742 was cultured overnight in selection medium. The composition of the liquid screening medium is as follows: SD-Trp (Beijing Fanjinuo (Functional Genomics) Technology Co., Ltd.), 2% glucose, 0.005% His., 0.01% Leu., 0.01% Ura. (each percent sign represents g / 100mL). Take 1ml (OD about 0.6-1.0) into 1.5ml EP tubes, centrifuge at 10000g at 4°C for 1min, discard the supernatant, wash the precipitate with sterile water (4°C), centrifuge under the same conditions, and discard the supernatant. Add 1ml of treatment solution (10mM LiAc; 10mM DTT; 0.6M sorbitol; 10mM Tris-HCl (pH7.5), add DTT only when the treatment solution is used) to the bacterial cells, and place it at 25°C for 20min. Centrifuge, discard the supernatant, add 1ml of 1M sorbitol (0.22μm aqueous membrane membrane sterilization) to resuspend the bacteria, centrifug...
Embodiment 3
[0123] Embodiment 3, Saccharomyces cerevisiae engineering bacterium HC001 catalyzes the synthesis of hydrocortisone
[0124] Shake flask fermentation catalysis: in solid selection medium (recipe: solid yeast selection medium SD-Ura-Trp, 2% glucose, 0.005% His, 0.01% Leu, 1.5% agar; each percentage sign means g / 100mL) Activate the HC101 yeast strain in the medium, and prepare it in the corresponding liquid selection medium (recipe: liquid yeast selection medium SD-Ura-Trp, 2% glucose, 0.005% His, 0.01% Leu; each percentage sign represents g / 100mL) Seed solution (30°C, 250rpm, 16h), inoculate 1mL of the inoculum into 3 bottles of 500ml Erlenmeyer flasks containing 100ml of YPD liquid medium, shake culture at 30°C, 250rpm for 2 days, collect yeast cells at 5000rpm, and buffer with PBS The solution was washed and finally resuspended in a 250mL Erlenmeyer flask containing 30mL PBS, and the substrate RSA with a final concentration of 170mg / L was added to carry out the catalytic reac...
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