Recombinant corynebacterium crenatum for over expression of N-acetylglutamate kinase and application thereof
A technology of Corynebacterium blunt-toothed and glutamic acid kinase, applied in the field of genetic engineering, can solve problems such as research reports on the genetic expression system of Coryne bacillus blunt-toothed, and achieve the effect of promoting utilization and increasing yield
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Embodiment 1
[0050] Embodiment 1: Construction of the genetic expression system of Corynebacterium blunt tooth with chloramphenicol acetyltransferase gene cat as reporter gene:
[0051] According to the cat sequence of chloramphenicol acetyltransferase gene published by GenBank, primers were designed, and the cat sequence was amplified from plasmid pAC by PCR technique. Primers were designed as follows:
[0052] pCm1100F Eco RI: 5'-CGC GAATTC TTCGAATTTCTGCCATTCATC-3'
[0053] pCm1100R Hind III: 5'-CGC AAGCTT GCGGTGCTTTTGCCGTTACG-3'
[0054] Using plasmid pAC as template, pCm1100F EcoRI and pCm1100R Hind III as primers, the reaction system was 1 μL of DNA template, 5 μL of 10×ExPCR buffer, 1 μL of upstream and downstream primers, 4 μL of dNTPs, and 0.5 μL of ExTaq DNA polymerase. The volume is 50 μL. The PCR amplification parameters were denaturation at 94°C for 1.5 min, renaturation at 55°C for 1 min, extension at 72°C for 1 min, and 30 cycles; PCR obtained a cat fragment containing ...
Embodiment 2
[0063] Example 2: Preliminary evaluation of the genetic expression system of Corynebacterium blunt-toothed with the chloramphenicol acetyltransferase gene cat as the reporter gene:
[0064] Pick the colony of Corynebacterium blunt tooth growing on the plate containing 20 μg / mL kanamycin to inoculate LB medium, and culture it on a shaker at 30°C until OD 600 After reaching 0.6, add IPTG to a final concentration of 20 μmol / L, and induce for 4 hours. The induced bacteria were inoculated on different concentrations of chloramphenicol-resistant plates, cultured and grown, and the strength of the promoter was preliminarily determined.
[0065] It was found that the recombinant pJC1-tac-cat before and after induction could grow on the chloramphenicol-resistant plate, while the control (without recombinant plasmid pJC1-tac-cat) could not grow on the chloramphenicol-resistant plate. It shows that the tac promoter has the function of promoter in Corynebacterium bacillus. And the tac p...
Embodiment 3
[0070] Example 3: Cloning and expression of the key enzyme N-acetylglutamate kinase (NAGK) in the synthesis of arginine by C.crenatum in Corynebacterium crenatum:
[0071] According to the Corynebacterium glutamicum coded acetylglutamate kinase gene argB nucleotide sequence published in GenBank, the primers are designed as follows:
[0072] Upstream primer P1: 5'-CGC GTC GAC GGTCATCAGTGACGGTTGC-3'(SalI) downstream primer P2: 5'-CGC GAATTC ATGAATGACTTGATCAAAG-3' (EcoRI)
[0073] Using C. crenatum genomic DNA as a template, the reaction system was 1 μL of DNA template, 5 μL of 10×ExPCR buffer, 1 μL of upstream and downstream primers, 4 μL of dNTPs, and 0.5 μL of ExTaq DNA polymerase. The total reaction volume was 50 μL. PCR amplification parameters were denaturation at 94°C for 1.5 min, renaturation at 55°C for 1 min, extension at 72°C for 1.5 min, and 35 cycles; the obtained fragment gel was recovered and ligated with the cloning vector pMD 18-T vector, transformed into E.co...
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