TY - JOUR
T1 - Compartmentalized partnered replication for the directed evolution of genetic parts and circuits
AU - Abil, Zhanar
AU - Ellefson, Jared W.
AU - Gollihar, Jimmy D.
AU - Watkins, Ella
AU - Ellington, Andrew D.
N1 - Funding Information:
acknowleDGMents 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.
Publisher Copyright:
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
PY - 2017/12/1
Y1 - 2017/12/1
N2 - C ompartmentalized partnered replication (CPRCPRCPR) is an emulsion-based directed evolution method based on a robust and modular phenotypegenotype linkage. In contrast to other in vivo directed evolution approaches, CPRCPRCPR largely mitigates host fitness effects due to a relatively short expression time of the gene of interest. CPRCPRCPR 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 dNTPNTPNTPs, followed by the recovery of the partner genes via emulsion PCRPCRPCR. 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 RNARNARNA polymerases that recognize orthogonal promoters and (ii) selection of tRNARNARNA for enhanced amber codon suppression. A single round of CPRCPRCPR should take 35 d, whereas a whole directed evolution can be performed in 310 rounds, depending on selection efficiency.
AB - C ompartmentalized partnered replication (CPRCPRCPR) is an emulsion-based directed evolution method based on a robust and modular phenotypegenotype linkage. In contrast to other in vivo directed evolution approaches, CPRCPRCPR largely mitigates host fitness effects due to a relatively short expression time of the gene of interest. CPRCPRCPR 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 dNTPNTPNTPs, followed by the recovery of the partner genes via emulsion PCRPCRPCR. 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 RNARNARNA polymerases that recognize orthogonal promoters and (ii) selection of tRNARNARNA for enhanced amber codon suppression. A single round of CPRCPRCPR should take 35 d, whereas a whole directed evolution can be performed in 310 rounds, depending on selection efficiency.
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U2 - 10.1038/nprot.2017.119
DO - 10.1038/nprot.2017.119
M3 - Article
C2 - 29120463
AN - SCOPUS:85036635294
SN - 1754-2189
VL - 12
SP - 2493
EP - 2512
JO - Nature Protocols
JF - Nature Protocols
IS - 12
ER -