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.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)