Steroid 11 beta-hydroxylase in curvularia lunata and coding gene and application thereof
A technology for encoding and transgenic cell lines, applied in the field of steroid 11β-hydroxylase and its encoding gene and application
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Embodiment 1
[0085] Example 1, Cloning and expression of Curvularia lunae 11-position β-hydroxylase
[0086] Gene cloning and expression are divided into the following 3 steps:
[0087] 1. Extraction of total RNA from Curvularia lunae
[0088] First, Curvularia lunae AS3.4381 (ATCC12017) was cultured on a plate for several days, and a certain number of spores were harvested and inserted into 50mL potato-dextrose (PDA) medium, and cultivated overnight until a large number of bacteria were synthesized; then , centrifuge to collect the mycelia of Curvularia lunata, wash with potassium phosphate buffer (PBS), and finally resuspend with 50 mL of buffer and add the substrate hydrocortisone 21-acetate ( RSA) was induced for 2 h, and samples were taken for RNA extraction.
[0089] RNA extraction method:
[0090] (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 liquid nitrogen quick-frozen mycelia (about 1...
Embodiment 2
[0116] Embodiment 2, the construction of Saccharomyces cerevisiae engineering bacteria HC101
[0117] 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
[0118] Example 3, Saccharomyces cerevisiae engineering bacteria HC101 catalyzes the synthesis of hydrocortisone and 14α-hydroxycortisol
[0119] 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 three bottles of 500mL Erlenmeyer flasks containing 100mL of YPD liquid medium, 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 ...
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