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. 2022 Jul 7;9(7):300. doi: 10.3390/bioengineering9070300

Figure 7.

Figure 7

Construction of engineering strain EcN with araBAD deletion mutation and ST with araBAD deletion mutation. (A) The amplification of linear FRT-kanamycin-FRT fragment, band 1: linear FRT-kanamycin-FRT fragment containing 56-nt homology arms targeting the araBAD gene of EcN, band 2: linear FRT-kanamycin-FRT fragment containing 56-nt homology arms targeting the araBAD gene of ST; (B) The electrotransformation of pKD46, band 1: EcN clone, band 2: ST clone; (C,D) Colony PCR to identify the deletion of araBAD gene and the insertion of kanamycin gene of EcN or ST, and positive bands are marked in a red box; (E): The loss of pKD46 plasmid; (F): The electrotransformation of pCP20, band 1: EcNΔaraBAD::kanamycin strain, band 2: ST ΔaraBAD::kanamycin strain; (G): Excision of kanamycin gene; (H): The loss of pCP20 plasmid; (I,J): Colony PCR to identify the deletion of araBAD gene engineered strain of EcN or ST, bands 1-8: engineered strain of EcN or ST, band 9: EcN wild type or ST wild type.