Firebug: A Living Cell that Propagates Plasmids Built from an Artificial Genetic System

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Our view of life is very much influenced by the life on Earth around us. All of the life on Earth (that we know of) descends from a single common ancestor, and it all has fundamentally the same biochemistry. This means that our view of biology is very much biased, reflecting only what we know of our surrounding biosphere and ourselves. The scientific goal of this project is to expand this view, something important to understand the intimate connection between the phenomenon that we know as life and the molecules that underlie it. This expanded view will be important, for example, should we encounter alien life in our search of the cosmos. It is equally important if we wish to use life-like processes in commerce, manufacturing, and medicine. The "grand challenge" to be met by this project is to create a cell that is able to use an entirely synthetic form of DNA; one built from 6 nucleotide "letters", which is two more than the number of nucleotides found in natural DNA. In pursuing this challenge, more will be learned about the chemistry of metabolism, reproduction, and genetic regulation. In addition to its scientific interest as a second example of a genetic system able to evolve, the cell will also have practical value. This cell will be used to manufacture molecules for human and animal diagnostics, for homeland security, and for manufacturing as a bio safe platform. Undergraduate students will participate in the research activities, and the findings will continue to provide useful insights and perspectives on the origins of life and the nature of extraterrestrial life. This project is co-funded by the Systems and Synthetic Biology Cluster within the Division of Molecular and Cellular Biosciences and the Chemistry of Life Processes in the Chemistry Division.This project will engineer a strain of E. coli ("Firebug") that can propagate plasmid DNA carrying nucleotides from a "second-generation" artificially expanded genetic information system (AEGIS). In its broadest form, AEGIS increases the number of independently replicable nucleotides in DNA from 4 to 12. This project will begin by adding just two AEGIS components, named (Z and P), to the four natural nucleotides (GACT), exploiting prior work that has (1) optimized second-generation AEGIS components for their chemical and enzymatic performance; (2) developed in vitro molecular biology to support AEGIS DNA, including six-letter GACTZP PCR, sequencing, and endonucleases, ligases, and kinases that use GACTZP DNA; (3) demonstrated in vitro evolution of GACTZP DNA, including generation of aptamers by evolution in the laboratory; and (4) engineered kinases that convert dZ and dP to their triphosphates inside cells. To pass the next milestone in synthetic biology that makes the expanded code functional in the cell, Firebug will be constructed. This will involve managing E. coli's error-prone polymerases and repair systems, moving enzymes encoding biosynthesis of Z and P into the chromosome, and placing the engineered cells under selection to improve their fidelity of replication of plasmid-borne Z:P pairs.