Journal
NATURE PROTOCOLS
Volume 12, Issue 12, Pages 2493-2512Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2017.119
Keywords
-
Categories
Funding
- Welch Foundation [F-1654]
- DOD Air Force Research Laboratory [FA9550-14-1-0089]
- Firebird Biomolecular Sciences [1R41GM119434-01A1]
- John Templeton Foundation [54466]
Ask authors/readers for more resources
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 similar to 3-5 d, whereas a whole directed evolution can be performed in 3-10 rounds, depending on selection efficiency.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available