Figure 4.

Toxin gene-based genetic engineering strategies. (A) Schematic representation of the toxin gene cassette. P: Commonly-used constitutive promoter. Pr: Promoter of operator-repressor system, which is repressed by the repressor and activated by an inducer e.g. P_spac, P_xyl. The repressor gene in the cassette can also be deleted in some methods. (B) Two different strategies based on toxin gene cassettes. A: upstream sequence; B: downstream sequence; C: sequence for integration of the toxin gene cassette, in combination with A in (2); DR: direct repeat sequence. After integration of the toxin gene cassette into a target chromosome locus via double-crossover recombination [A and B in (1) or A and C in (2)] and positive selection for antibiotic resistance, the cassette is removed by a single crossover event between two DR sequences in (1) or B sequences in (2).