This NSF grant was part of a larger ERA SynBio collaboration aiming to build the underlying systems needed for replicating unnatural nucleic acid polymers in cells. The consortium included the Liu Laboratory at the University of California, Irvine, as well as collaborators from the UK, France, and Belgium. Our NSF-supported contributions to the project was to better understand an orthogonal DNA replication system developed in our lab and to reconstitute the replication system in vitro in order to enable direct characterization and engineering of its ability to replicate nucleic acids containing unnatural building blocks. Since our orthogonal DNA replication system is capable of durably propagating in living cells without interfering with the cell’s genomic replication, it is a broadly useful platform for expanding the properties of DNA replication in vivo. The intellectual merit of the project focuses on the premise that if we can train our replication system to utilize unnatural building blocks, we can then replicate these building blocks in vivo, showing that new nucleic acids besides DNA and RNA can take the role of information storage, propagation, and encoding of functional molecules in life. We have successfully developed the genetics of orthogonal DNA replication in this project to understand the basis of orthogonality in vivo and the minimal components needed for the replication system to work in vitro. We have also successfully recapitulated the early replication steps of the system in vitro through biochemical reconstitution and have obtained evidence for a novel replication initiation mechanism involving self-proteolysis of the DNA polymerase that converts it from an initiation state to an elongation state. The in vitro reconstitution sets the stage for testing and engineering unnatural building block polymerization capabilities with other members of the consortium. Our studies led to the publication of 5 papers in leading journals and more to come. Beyond science and engineering, the proposal supported 1 postdoctoral scholar and partially supported 5 PhD students, all of whom received valuable training in science, engineering, and associated skills such as writing and presentation at conferences and meetings. The postdoctoral scholar on this project is now a PI running his own lab that continues on work started in this project, and the PhD students have progressed well toward their degree. During the course of the grant, the PI was actively involved not only in scientific activities and conferences, but also in larger discussions on synthetic biology through an art-science collaboration where the PI’s lab hosted an artist resulting in works featured in a synthetic biology museum exhibit and associated public forum panels. The PI also has been involved in teaching synthetic biology to the local community through a lifelong learning course for adults. Finally, the continued collaboration of members of this consortium, seeded through this project, serves the goal of advancing the field of synthetic biology at the international level. 

Last Modified: 08/15/2020
Modified by: Chang C Liu