Abstract
Most existing directed evolution methods, both in vivo1,2,3 and in vitro4,5,6, suffer from inadvertent selective pressures (i.e., altering organism fitness), resulting in the evolution of products with unintended or suboptimal function. To overcome these barriers, here we present compartmentalized partnered replication (CPR). In this approach, synthetic circuits are linked to the production of Taq DNA polymerase so that evolved circuits that most efficiently drive Taq DNA polymerase production are enriched by exponential amplification during a subsequent emulsion PCR step. We apply CPR to evolve a T7 RNA polymerase variant that recognizes an orthogonal promoter and to reengineer the tryptophanyl tRNA-synthetase:suppressor tRNA pair from Saccharomyces cerevisiae7 to efficiently and site-specifically incorporate an unnatural amino acid into proteins. In both cases, the CPR-evolved parts were more orthogonal and/or more active than variants evolved using other methods. CPR should be useful for evolving any genetic part or circuit that can be linked to Taq DNA polymerase expression.
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Acknowledgements
This work was supported by the National Security Science and Engineering Faculty (FA9550–10-1-0169) the Welch Foundation (F–1654 to A.D.E. and F-1155 to J.S.B.), the National Science Foundation (CHE1012622 to J.S.B.), and the Defense Advanced Research Projects Agency (HR–0011-12-C-0066). J.S.B. thanks Thermo Fisher Scientific with helping on the modifications to the Orbitrap Elite mass spectrometer to allow UVPD.
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J.W.E. conceived of the selection scheme. A.J.M. carried out experiments with T7 RNA polymerase. J.W.E. carried out experiments on the ScWRS and tRNA with input from R.A.H. J.R.C. and J.S.B. performed mass spectrometry and analysis. J.W.E., A.J.M. and A.D.E. wrote the manuscript with contributions from R.A.H., J.R.C. and J.S.B.
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Ellefson, J., Meyer, A., Hughes, R. et al. Directed evolution of genetic parts and circuits by compartmentalized partnered replication. Nat Biotechnol 32, 97–101 (2014). https://doi.org/10.1038/nbt.2714
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DOI: https://doi.org/10.1038/nbt.2714
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