 Purpose of this step Landing pad insertion is the first step of our two-step recombination system. We insert a “landing pad” fragment which includes a promoter (placIQ1) and a tetracycline resistance gene (tetA) flanked by I-SceI recognition sites and 20-bp landing pad regions (LP1 and LP2) into Escherichia coli chromosome via att recombination. Then the helper plasmids encoding I-SceI endonuclease and λ-Red and donor plasmid encoding various antibiotic genes flanked by I-SceI recognition sites and same landing pad regions (LP1 and LP2) are transformed, which will be introduced in detail in next part. I-SceI expression is induced via the addition of L-arabinose. I-SceI recognition sites in the donor plasmid and chromosome are cleaved. Integration of the fragment in donor plasmid is facilitated by IPTG-induced λ-Red expression. This two-step recombination method allows for the insertion of very large fragments into a specific location in Escherichia coli chromosome and in any orientation.

 Construction of landing pad Landing pad consists of the following parts: a promoter (placIQ1), two landing pad regions, two I-SceI recognition sites and a tetracycline resistance gene (tetA).