Efficient traceless gene editing system for salmonella and use thereof

A method of constructing the targeting plasmid B, comprises the following steps:

• 1) for knock-in (or substitution):
• amplifying the targeting plasmid B backbone, the target site sgRNA expression frame, the upstream/downstream homology arms, and the knock-in (or substitution) fragment, respectively, and ligating all DNA fragments using one-step homologous recombination;
• or
• 2) for knock-out:
• amplifying the targeting plasmid B backbone, the target site sgRNA expression frame, and the upstream/downstream homology arms, respectively, and ligating all DNA fragments using one-step homologous recombination;
• or
• 3) for targeting plasmid B contain in no homology arms, amplifying the upstream/downstream homology arms respectively, and ligating them as a linear DNA template.

A use of the efficient traceless gene editing system for Salmonella in genome editing, comprises the following steps:

• 1) introducing the helper plasmid A into Salmonella and inducing a λRed recombinase expression to prepare competent cells;
• 2) introducing the targeting plasmid B and a linear DNA template to the competent cells of step 1), or introducing a targeting plasmid B comprising the template DNA;
• 3) resuscitating the cells from step 2) and coating on a plate comprising two resistances corresponding respectively to the plasmid A and the plasmid B, and screening for positive clones with double crossover;
• 4) after performing PCR or sequencing verification of the positive clones, inducing sgRNA expression of the targeting plasmid B replicon and eliminating the plasmid B;
• 5) after verifying the elimination of the plasmid B, increasing the bacterial culture temperature and eliminating the plasmid A to obtain a successfully modified Salmonella clone.