Corynebacterium glutmicum synthase gene cloning and L183Q site directed mutagenesis method
A technique of Corynebacterium glutamicum and site-directed mutagenesis, which can be used in genetic engineering, plant genetic improvement, botany equipment and methods, etc., can solve the problem that the catalytic activity has not been improved, and increase the amount of substrate biosynthesis and enhance catalytic activity. Active, enhanced metabolic flux effect
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[0041] The gene cloning of Corynebacterium glutamicum DAHP synthase and the L183Q site-directed mutagenesis method are implemented in the following manner: the material used in the method is Corynebacterium glutamicum LG-332, Escherichia coli DH5α;
[0042] The gene cloning of the Corynebacterium glutamicum DAHP synthase, the gene cloning steps are as follows:
[0043] 1) Extraction of the genomic DNA of Corynebacterium glutamicum LG-332, a single colony of Corynebacterium glutamicum LG-332 was inoculated into 10ml LB liquid medium, cultured on a shaker at 30°C for 12h, and 2% of the inoculation volume was connected the next day 100ml LB liquid medium; the steps for extracting the genome are as follows:
[0044] (1) 100ml of bacterial overnight culture solution, centrifuged at 5000rpm for 10 minutes, and remove the supernatant.
[0045] (2) Add 9.5ml TE to suspend the precipitate, and add 0.5ml 10% SDS, 50μl 20mg / ml (or 1mg dry powder) proteinase K, mix well, and incubate at 37°C for ...
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