Orthogonal replication systems for rapid continuous evolution

We have engineered an orthogonal DNA replication (OrthoRep) system in yeast consisting of an error-prone DNA polymerase that replicates a special DNA plasmid without increasing the mutation rate of the genome. Genes of interest encoded on the special DNA plasmid can therefore rapidly mutate and evolve entirely in vivo. We have applied OrthoRep to evolve enzymes, biosensors, and antibodies, and to study the “solution space” for drug resistance.

Recording non-genetic information into DNA with rapid mutation systems

We have invented a genetic system called CHYRON (Cell HistorY Recording by Ordered iNsertion) that progressively accumulates short insertions of random nucleotides (nts) at a synthetic locus in the DNA of mammalian cells. Since daughter cells inherit the CHYRON locus of their parents and then add additional nts to distinguish themselves, deep lineage relationships can be deduced by NGS of CHYRON loci. We have shown that CHYRON can be used to accurately record lineage relationships among groups of cells subject to complex splitting processes as well as the exposure of cells to arbitrary signals. We are using CHYRON to study normal and cancer development in animals.

Directed evolution using synthetically expanded genetic codes

Nature’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? We explore this possibility by using cells with expanded genetic codes to engineer and evolve novel functional proteins, including highly-sulfated antibodies and, most recently, peptides that trigger plant immunity. This effort will not only allow us to generate useful biomolecules, it also 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

A major goal in synthetic biology is to generalize ribosomal translation of proteins to 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 serves as the basis of OrthoRep, which is the primary reason it is interesting to us. However, the p1/p2 system also has has a number of unusual biochemical and molecular properties that make it unique among DNA replication systems. Studying the basic biology of this plasmid system may give us fundamental insights on the mechanisms of DNA replication, repair, and segregation. Some of p1/p2’s unusual properties may also be useful for applications in biotechnology and gene therapy.