A method for enhancing the gene editing efficiency of actinomycetes and its application
A gene editing and efficiency technology, applied in the field of genetic engineering and genetic modification, can solve the problems of genetic modification failure, off-target effect and toxic activity hindering the application, and the reduction of transformants, so as to improve editing efficiency, inhibit cytotoxicity and off-target probability Effect
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[0033] The bacterial species selected in Example 1 is Streptomyces coelicolor A3 (2) (Bentley SD et al.Nature, 2002) (preservation number: CGMCC 4.7168), its whole genome sequence (sequence number: GenBank: NC_003888.3 ). Taking the knockout of actII-ORF4 gene in Streptomyces coelicolor A3(2) by this method as an example, the present invention will be described in detail, wherein Streptomyces coelicolor A3(2) is used as a model bacterium. Specific steps are as follows:
[0034] (1) According to literature reports on the regulation of related Streptomyces promoters (He Huang et al.Acta BiochimBiophys Sin, 2015), the tipAp promoter and ermEp* promoter induced by thiostrepton were screened out to obtain the Constitutive promoter of strain Streptomyces coelicolor A3(2) and strong promoter of atpD gene expression. (See Table 1 and attached figure 1 )
[0035] (2) According to the plasmid library of Streptomyces coelicolor A3(2) and the size and position of the determined elemen...
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