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Compartmentalized partnered replication for the directed evolution of genetic parts and circuits
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Compartmentalized partnered replication for the directed evolution of genetic parts and circuits
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Journal Article
Compartmentalized partnered replication for the directed evolution of genetic parts and circuits
2017
Overview
This protocol describes the procedures for compartmentalized partnered replication (CPR), an emulsion-based directed evolution method for the generation of proteins, genetic elements, and genetic circuits with improved or altered function.
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.
Publisher
Nature Publishing Group UK,Nature Publishing Group
Subject
/ 82/47
/ Amber
/ Analysis
/ Animals
/ Bacterial Proteins - genetics
/ Bacterial Proteins - metabolism
/ Biomedical Research - methods
/ Biomedical Research - trends
/ Circuits
/ Computational Biology/Bioinformatics
/ Directed Molecular Evolution
/ Droplets
/ Gene Expression Regulation, Bacterial
/ Gene Expression Regulation, Developmental
/ Genes
/ Green Fluorescent Proteins - genetics
/ Green Fluorescent Proteins - metabolism
/ Humans
/ Mutation
/ Primers
/ Proteins
/ protocol
/ Recombinant Fusion Proteins - metabolism
/ Recombinant Proteins - metabolism
/ RNA
/ tRNA
