Serine Integrase-based Recombination Enables Direct Plasmid Assembly In Vivo

Serine integrases are emerging as one of the powerful tools for synthetic biology. They have been widely developed across genome engineering, biological part construction, genetic circuit design, and in vitro DNA assembly. However, the strategy of in vivo DNA assembly by serine integrases has not yet been reported. To address this opportunity, here we developed a serine integrase-based in vivo DNA (plasmid) assembly approach. First, we demonstrated that the engineered “Acceptor” plasmids could be assembled with diverse “Donor” plasmids by serine integrases (Bxb1 and phiC31) in Escherichia coli (E. coli). Then, by programming the “Donor” plasmids and the host E. coli cells, we established an assembly cascade procedure and finally constructed plasmids that could constitutively express three different fluorescent proteins. Moreover, we used this approach to assemble different chromoprotein genes and generated colored E. coli cells. We anticipate that this approach will enrich the serine integrase-based biotechnology toolbox, and accelerate multiple plasmid assembly for synthetic biology with broad applications.