Ultrasonic-assisted method of transforming Escherichia coli via exogenous genes
A technology of Escherichia coli and exogenous gene, applied in the field of high-efficiency ultrasonic transformation, can solve the problems of low transformation rate, easy death, low cell viability, etc.
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Embodiment 1
[0036] (1) Culture and collection of E. coli competent cells
[0037] Pick E. coli (DE3) (purchased from Novagen) and inoculate it into LB medium, 180r / min, 35°C shaking culture for 2.5h to exponential growth phase, 12000r / min, 10min, 4°C centrifugation to collect wet bacteria body to obtain E. coli competent cells.
[0038] (2) Gene transformation study using Escherichia coli as host
[0039] The cholesterol oxidase gene choB was cloned into the pET28a vector plasmid.
[0040] Mix the wet cells obtained in step (1) with dimethyl sulfoxide to make the cells with a concentration of 1 × 10 8 cells / mL, the volume final concentration of dimethyl sulfoxide is 2% of the cell suspension, and the pET28a plasmid containing cholesterol oxidase gene choB (the plasmid concentration should not be less than 70ng / μL) is mixed with the cell suspension at a volume ratio of 1 :40 mixing, that is, take 0.5 mL of plasmid and mix with 20 mL of cell suspension, adjust the pH to 6.5; adjust the t...
Embodiment 2
[0051] (1) Culture and collection of E. coli competent cells
[0052] Pick E.coli (DE3) (purchased from Novagen) and inoculate it into LB medium, 200r / min, 36°C shaking culture for 20h to late exponential growth, 12000r / min, 10min, 4°C centrifugation to collect wet cells , to obtain E. coli competent cells.
[0053] (2) Gene transformation study using Escherichia coli as host
[0054] The cholesterol oxidase gene choB was cloned into the pET28a vector plasmid.
[0055] The wet cells obtained in step (1) are mixed with dimethyl sulfoxide to make the cell content of 1×10 8 cells / mL, the final concentration of dimethyl sulfoxide is 3% of the cell suspension, and the pET28a plasmid containing cholesterol oxidase gene choB (the plasmid concentration should not be less than 70ng / μL) is mixed with the cell suspension in a volume ratio of 1 :50 mixing, that is, take 0.5 mL of plasmid and mix with 25 mL of bacterial suspension, adjust the pH to 7.0; adjust the temperature to 36°C, t...
Embodiment 3
[0066] (1) Culture and collection of E. coli competent cells
[0067] Pick Escherichia coli E.coli (DE3) (purchased from Novagen) and inoculate it into LB medium, 220r / min, 37°C shaking culture for 8h, 12000r / min, 15min, 4°C centrifugation, collect wet bacteria, obtain large intestine Bacillus competent cells.
[0068] (2) Gene transformation study using Escherichia coli as host
[0069] The cholesterol oxidase gene choB was cloned into the pET28a vector plasmid.
[0070] The wet cells obtained in step (1) are mixed with dimethyl sulfoxide to make the cell content of 1×10 8cells / mL, the volume final concentration of dimethyl sulfoxide is 4% of the cell suspension, and the pET28a plasmid containing cholesterol oxidase gene choB (the plasmid concentration should not be less than 70ng / μL) is mixed with the cell suspension at a volume ratio of 1 :55 mixing, that is, take 0.5 mL of plasmid and mix with 27.5 mL of bacterial suspension, adjust the pH to 7.5; adjust the temperature...
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