Department of Post-harvest biotechnology, Vietnam National Institute of Agricultural Engineering and Post-harvest technology.
Title of the talk: Cell-free Synthesis and Functional Assembly of the Bacteriophage T7 Replisome
One challenge in the construction of a genetically controlled synthetic cell is the replication of genomic DNA. Inspired by the DNA replication mechanism of the bacteriophage T7, we decided to reconstitute in vitro the multi-protein T7 replisome complex starting from the genes encoding for all essential enzymes and cofactors, and to express them in a cell-free transcription-translation system. We first validated the synthesis of the full-length helicase/primase (gp4), T7 DNA polymerase (gp5), single-strand DNA binding protein (SSB) and E. coli thioredoxin (trx). Using a combination of DNA replication substrates – a short dsDNA with 3’ (primed) and 5’ ssDNA overhangs, and the ssDNA M13 template – we confirmed the activity of the various proteins as documented in the literature with purified proteins. This includes: unwinding of DNA duplex by gp4, polymerase and exonuclease activities of gp5, enhancement of gp5 polymerization by trx and SSB-dependent activity of gp4. The replication of long coding DNA coupled with a minimal gene expression system will be a crucial step toward the realization of a self-replicating system. In addition, the functional reconstitution of a cell-free synthesized T7 replisome provides a novel and versatile platform for single-molecule investigations, where the dynamic molecular assembly can be examined as a function of varying concentrations of specific partners.
Huong Thi Bui has completed her Ph.D in 2005 from Osaka University, Japan and postdoctoral studies from Kansai University, Japan and Delft University of Technology, the Netherlands. She is vice head of Department of Post-harvest biotechnology, Vietnam National Institute of Agricultural Engineering and Postharvest technology. She has published more than 15 papers in international and domestic journals.