Construction and immobilized use method of glyA genetically engineered microorganism
A genetically engineered bacterium and engineering bacterium technology, applied in the field of construction and immobilization of glyA genetically engineered bacteria, can solve the problems of continuous or repeated use of enzymes, unfavorable environmental impact, cost increase, etc., and achieve the construction and use method Simple, cost-saving, and high conversion efficiency
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0050] Including the following steps:
[0051] 1) Design primers; design primers according to the sequence GenBank of the structural gene of E.coli k-12 glyA provided in genbank: AAC75604.1 (using DNAclub software), the primer sequence is primer1: GAT CCATGG TAAAGCGTGAAATGAACAT (the underline is the NcoI restriction site), Primer2: CCC AAGCTT TTATGCGTAAACCGGGTAA (the underline is the Hind III restriction site);
[0052] 2) Cultivate Escherichia coli E.coli k-12 to the logarithmic phase, and extract the genome of Escherichia coli k-12 with a kit (purchased from Generay);
[0053] 3) Perform PCR amplification with the extracted E.coli k-12 genome as a template according to the designed primers;
[0054] 4) The PCR product was recovered by (Nco I, Hind III double enzyme digestion), connected to the expression vector pET28a (+) (purchased from Novagen), and transformed into BL21 (DE3 (purchased from Novagen);
[0055] 5) Randomly select 10 different transformants and name them...
Embodiment 2
[0066] Including the following steps:
[0067] 1) Recultivate the transformant T7, collect the cells by centrifugation, and fix them with a composite carrier of carrageenan and gelatin. Specific preparation process: take a certain amount of carrageenan and gelatin in proportion (carrageenan: gelatin mass ratio is 3: 2) and join in 0.1M phosphate buffer solution, its final concentration is 3-5%, after sterilization Cool to 50°C, then add the centrifuged bacteria and mix well (the final concentration of the bacteria is 150g / L), slowly add to 2% KCl solution, prepare into 2-3mm pellets, fix at 4°C for 8h . Obtain immobilized cell spheres, which can also be said to be immobilized cells;
[0068] 2) The immobilized cell spheres were treated with 3% glutaraldehyde at 4°C for 10 min;
[0069] 3) Under the same conditions, the enzyme activity of the immobilized engineering bacteria and the enzyme activity of the free cells were measured respectively, and the results showed that the...
Embodiment 3
[0072] Including the following steps:
[0073] 1) Recultivate the transformant T7, collect the cells by centrifugation, and fix them with a composite carrier of carrageenan and gelatin. Concrete preparation process: take a certain amount of carrageenan and gelatin in proportion (carrageenan: gelatin mass ratio is 2: 2) and join in the phosphate buffer solution of 0.1M, its final concentration is 4%, cool to 48°C, then add the centrifuged bacteria and mix well (the final concentration of the bacteria is 180g / L), slowly flow into 2% KCl solution, prepare into 2-3mm pellets, and fix at 4°C for 5h. Obtain immobilized cell spheres, which can also be said to be immobilized cells;
[0074] 2) The immobilized cell spheres were treated with 2% glutaraldehyde at 4°C for 20 min;
[0075] 3) Under the same conditions, the enzyme activity of the immobilized engineering bacteria and the enzyme activity of the free cells were measured respectively, and the results showed that the enzyme ac...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More