Independent replication systems for fast evolution, synthetic biology, and cell biology

1We have engineered an orthogonal DNA replication (OrthoRep) system so that we can make major changes to the properties of DNA replication in vivo. One of our OrthoRep projects aims to achieve the rapid in vivo targeted mutation and recombination of any desired gene(s) through low-fidelity orthogonal DNA polymerases. Here, we are interested in (1) the evolution of functional RNAs, proteins, metabolic pathways, and strains; and (2) the large-scale replication of targeted evolution experiments to understand the “solution space” and dynamics of protein evolution, drug resistance, and regulatory networks. Another project aims to slow down evolution of genes on the OrthoRep system, allowing for protection of synthetic genes against evolutionary degradation. A third OrthoRep project is the creation of a non-DNA episome, which should have applications in biocontainment as well as the discovery of new functional polymers.  Finally, we are aiming to use the OrthoRep concept to achieve continuous cell barcoding for understanding cellular and developmental processes.

Directed evolution using synthetically expanded genetic codes

Expanded CodeNature’s genetic code specifies 20 amino acids for all protein synthesis. However, recent efforts have achieved organisms with synthetically expanded genetic codes that specify one or more unnatural amino acids in addition to the 20 canonical amino acids. If we let evolution use these expanded genetic systems, will new (perhaps radically new) biological function emerge? To explore this possibility, we are using cells with expanded genetic codes to evolve novel functional proteins, including highly-sulfated anti-HIV and anti-cancer agents. This effort will not only allow us to generate therapeutic leads, but also to overcome critical limitations of the immune system. More broadly, it explores the idea that synthetic genetic codes with new chemistries can be superior to nature’s code.

Entirely unnatural peptide and polymer synthesis by expanded genetic codes

The holy grail in the expanded genetic code field is to achieve the ribosomal synthesis of entirely unnatural complex polymers. We are exploring the possibility of running more than one genetic code in the cell to achieve this, focusing on engineering the mitochondrion. In the process, we also seek greater understanding of mitochondria.

Studying and exploiting the biochemistry of an unusual DNA replication system

We are interested in understanding the genetics, biochemistry, and mechanistic aspects of an autonomous DNA replication system, called the pGKL1/2 (p1/p2) plasmids. This replication system has a number of interesting properties that we believe can be broadly applied in biotechnology and gene therapy. We are also studying the basic biology of this plasmid system, as it may give us fundamental insights on the mechanisms of DNA replication, repair, and segregation.