Cointegrates formed by site-specific recombination between engineered donor and recipient plasmids

Sample 1: We constructed donor plasmid that carries GIsul2 (from Shigella flexneri 51575, IN-11, IN-05) sequence and pKD46 backbone. The recipient plasmid harbors attB site (from E. coli DH5a) and pKF18k backbone. The two plasmids were co-transformed into E.coli DH5a and the integration experiment was performed. Plasmids were extracted and digested by XbaI enzyme. The linearized products were used for nanopore sequencing on PromethION platform. The donor and recipient plasmids can conduct site-specific recombinationbetween att sites, and produce five types of cointegrates: three donor-recipient cointegrates (donor bearing three att sites), GIsul2-recipient (genomic island GIsul2 insert into recipient), and ISCR2-recipient (iscr2-sul2 unit insert into recipient). TheXbaI enzyme can digest recipient and the five types of cointegrates, but not donor plasmid. The filtered reads data are 4.39Gb.Sample 2: In order to further demonstrate the role of the att sites we constructed mini donor plasmid. The mini plasmid only contains integrase gene, the three att sites and pKD46 backbone. The mini donor and the same recipient plasmid were co-transformed into E. coli DH5a andthe integration experiment was performed. Plasmids were extracted and digested by XbaI. The linearized product was used for nanopore sequencing and obtained 4.49 Gb filter reads data.