Double gene coexpression plasmid, construction method and application thereof
A dual-gene, co-expression technology, applied in the field of molecular biology, can solve problems such as transformation reaction interference
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Embodiment 1
[0040] Experimental steps for the construction of recombinant plasmid pMSS
[0041] The molecular biology methods involved in this example, such as enzyme digestion, ligation, preparation of competent cells, and electric shock transformation, all refer to "Molecular Cloning-A laboratory Manual" ed.by J.Sambrook, E.F.Fritsch and T.Maniatis, 1989, CSHL Press.
[0042] Step 1. Completely digest the plasmid pKKCAIS with restriction endonucleases EcoRI and HindIII, and recover the 2.5 kb acylase gene (acy) fragment produced after digesting pKKCAIS with the gel recovery kit of Huashun Company.
[0043] Step 2. Digest plasmid pET28b with restriction enzymes EcoRI and HindIII. After the enzyme reaction is finished, the enzyme reaction solution is extracted with phenol and phenol:chloroform (1:1) to remove the enzyme protein in the reaction solution. Afterwards, twice the volume of ethanol was added to precipitate the DNA, and the DNA was redissolved in redistilled water.
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Embodiment 2
[0051] Experimental steps for the construction of recombinant plasmid pMSS
[0052] The molecular biology methods involved in this example, such as enzyme digestion, ligation, preparation of competent cells, and electric shock transformation, all refer to "Molecular Cloning-A laboratory Manual" ed.by J.Sambrook, E.F.Fritsch and T.Maniatis, 1989, CSHL Press.
[0053] Step 1. Completely digest the plasmid pKKCAIS with restriction endonucleases EcoRI and HindIII, and recover the 2.5 kb acylase gene (acy) fragment produced after digesting pKKCAIS with the gel recovery kit of Huashun Company.
[0054] Step 2. Use restriction endonucleases NotI and NdeI to embodiment 1, the plasmid pMST obtained in step 4 is completely digested, and the 2.5kb acylase gene produced after pMST digestion is reclaimed with the gel recovery kit of Huashun Company (acy) fragment.
[0055] Step 3. Digest plasmid pALTER-Ex1 with restriction enzymes NotI and NdeI. After the enzyme reaction is finis...
Embodiment 3
[0063] Experimental procedure for the determination of GL-7ACA acylase activity
[0064] References for this experimental procedure: Wei Zhongdi, Yang Yunliu, Jin Zhikun, etc., Chinese Patent, Publication No. 1995CN1104255A
[0065] Take 0.5ml of fermentation broth, centrifuge at 4000rpm for 10 minutes, discard the supernatant, and dilute 6 times with 3ml of phosphate buffer. Then take 0.5ml of diluted bacterial solution, preheat at 37°C for 10 minutes, then add 0.5ml of preheated substrate (5mg / ml GL-7-ACA, freshly prepared with the same phosphate buffer), and react in a 37°C water bath for 30 minutes. Add 3ml stop solution and 0.5ml color development solution. After standing for 30 minutes, centrifuge at 4000 rpm for 15 minutes. The supernatant was taken to measure the absorbance at 415 nm. For the blank control, the stop solution was added first, followed by the substrate, and the rest were the same as above.
[0066] Enzyme activity (u / ml)=k×O.D. 415 ×Dilutio...
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