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.

Sample 1: 我们构建了携带GIsul2序列（源自福氏志贺氏菌51575菌株，IN-11、IN-05）以及pKD46骨架的供体质粒（donor plasmid）。受体质粒（recipient plasmid）携带来自大肠杆菌DH5α的attB位点（attB site）与pKF18k骨架。将两种质粒共转化至大肠杆菌DH5α中，开展整合实验。提取质粒并经XbaI酶酶切，将线性化产物在PromethION平台上开展纳米孔测序（nanopore sequencing）。供体质粒与受体质粒可在att位点间发生位点特异性重组（site-specific recombination），产生五类共整合体（cointegrates）：三类供体-受体共整合体（供体质粒携带三个att位点）、GIsul2-受体共整合体（基因组岛（genomic island）GIsul2插入受体质粒）以及ISCR2-受体共整合体（iscr2-sul2单元插入受体质粒）。XbaI酶可酶切受体质粒与这五类共整合体，但无法酶切供体质粒。过滤后的有效测序数据量为4.39 Gb。Sample 2: 为进一步验证att位点的功能，我们构建了微型供体质粒（mini donor plasmid）。该微型质粒仅包含整合酶基因（integrase gene）、三个att位点以及pKD46骨架。将该微型供体质粒与前述受体质粒共转化至大肠杆菌DH5α中并开展整合实验，提取质粒后经XbaI酶酶切，将线性化产物用于纳米孔测序，最终获得4.49 Gb的过滤后有效测序数据。