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Compartmentalized partnered replication for the directed evolution of genetic parts and circuits

Abstract

Compartmentalized partnered replication (CPR) is an emulsion-based directed evolution method based on a robust and modular phenotype–genotype linkage. In contrast to other in vivo directed evolution approaches, CPR largely mitigates host fitness effects due to a relatively short expression time of the gene of interest. CPR is based on gene circuits in which the selection of a 'partner' function from a library leads to the production of a thermostable polymerase. After library preparation, bacteria produce partner proteins that can potentially lead to enhancement of transcription, translation, gene regulation, and other aspects of cellular metabolism that reinforce thermostable polymerase production. Individual cells are then trapped in water-in-oil emulsion droplets in the presence of primers and dNTPs, followed by the recovery of the partner genes via emulsion PCR. In this step, droplets with cells expressing partner proteins that promote polymerase production will produce higher copy numbers of the improved partner gene. The resulting partner genes can subsequently be recloned for the next round of selection. Here, we present a step-by-step guideline for the procedure by providing examples of (i) selection of T7 RNA polymerases that recognize orthogonal promoters and (ii) selection of tRNA for enhanced amber codon suppression. A single round of CPR should take 3–5 d, whereas a whole directed evolution can be performed in 3–10 rounds, depending on selection efficiency.

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Figure 1: General CPR concept.
Figure 2: Overview and time line of experiments.
Figure 3: Schematic of the two Recovery strategies.
Figure 4: Anticipated results.

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Acknowledgements

This work was supported by the Welch Foundation (F-1654 to A.D.E.), the DOD Air Force Research Laboratory (FA9550-14-1-0089), Firebird Biomolecular Sciences (1R41GM119434-01A1), and the John Templeton Foundation (54466). The opinions expressed in this publication are those of the authors and do not necessarily reflect the views of the John Templeton Foundation.

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Contributions

Z.A. and A.D.E. wrote the manuscript. Z.A., J.W.E., J.D.G., E.W. and A.D.E. contributed technical detail to the protocol, and read, edited, and approved the final manuscript.

Corresponding author

Correspondence to Andrew D Ellington.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Basic plasmid schemes.

pACYC-Taq was modified to make pACYC-Taq.1Amb, pACYC-GFP, pACYC-GFP1Amb, and pACYC-GFPM2 by cloning the appropriate coding DNA sequence (CDS) in the place of the Taq DNAP CDS by isothermal (Gibson) assembly. Promoter mutations and amber mutations were made in pACYC plasmids by isothermal assembly with mutagenic primers. pRST.11B-AS3.4 encodes suppressor tRNA that was previously rationally engineered from WT yeast suppressor tRNA for improved amber suppression. This plasmid was used as a parental plasmid for construction of tRNA synthetase and tRNA libraries for CPR selections. Reprinted by permission from Macmillan Publishers Ltd: Nat Biotechnol, copyright 2014. (Ellefson et al. Directed evolution of genetic parts and circuits by compartmentalized partnered replication. Nat Biotechnol 32, 97-101, doi:10.1038/nbt.2714 (2014).).

Supplementary Figure 2 Assembly of the full-length T7 RNAP gene by overlap extension PCR.

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Supplementary Figures 1 and 2, Supplementary Note and Supplementary Table 1. (PDF 508 kb)

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Abil, Z., Ellefson, J., Gollihar, J. et al. Compartmentalized partnered replication for the directed evolution of genetic parts and circuits. Nat Protoc 12, 2493–2512 (2017). https://doi.org/10.1038/nprot.2017.119

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