Supplementary Material for: High Efficiency of a Sequential Recombinase-Mediated Cassette Exchange Reaction in <i>Escherichia coli</i>

A comparison between the efficiency of recombinase-mediated cassette exchange (RMCE) reactions catalyzed in <i>Escherichia coli</i> by the site-specific recombinases Flp of yeast and Int of coliphage HK022 has revealed that an Flp-catalyzed RMCE reaction is more efficient than an Int-HK022 catalyzed reaction. In contrast, an RMCE reaction with 1 pair of <i>frt</i> sites and 1 pair of <i>att</i> sites catalyzed in the presence of both recombinases is very inefficient. However, the same reaction catalyzed by each recombinase individually supplied in a sequential order is very efficient, regardless of the order. Atomic force microscopy images of Flp with its DNA substrates show that only 1 pair of recombination sites forms a synaptic complex with the recombinase. The results suggest that the RMCE reaction is sequential.针对在大肠杆菌（Escherichia coli）中由酵母来源的Flp重组酶（Flp）与大肠杆菌噬菌体HK022来源的Int重组酶（Int）所催化的重组酶介导的盒式交换（recombinase-mediated cassette exchange, RMCE）反应效率开展的对比研究显示，经Flp催化的RMCE反应效率高于HK022 Int催化的RMCE反应。与之相反，当同时添加两种重组酶进行催化时，携带1对frt位点（frt）与1对att位点（att）的RMCE反应效率极低。然而，若按顺序单独添加两种重组酶分别催化该同一反应，则无论添加顺序如何，反应效率均极高。针对Flp与其DNA底物的原子力显微镜（atomic force microscopy, AFM）成像结果表明，仅有1对重组位点可与该重组酶形成突触复合物（synaptic complex）。上述结果提示，该RMCE反应为依次进行的分步反应。