Abil Zhanar, Ellefson Jared W, Gollihar Jimmy D, Watkins Ella, Ellington Andrew D
Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, Texas, USA.
Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.
Nat Protoc. 2017 Dec;12(12):2493-2512. doi: 10.1038/nprot.2017.119. Epub 2017 Nov 9.
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.
区室化配对复制（CPR）是一种基于稳健且模块化的表型-基因型关联的乳液定向进化方法。与其他体内定向进化方法不同，由于感兴趣基因的表达时间相对较短，CPR在很大程度上减轻了宿主适应性影响。CPR基于基因回路，其中从文库中选择“伙伴”功能会导致产生一种热稳定聚合酶。文库制备后，细菌产生的伙伴蛋白可能会增强转录、翻译、基因调控以及细胞代谢的其他方面，从而加强热稳定聚合酶的产生。然后在引物和脱氧核苷三磷酸存在的情况下，将单个细胞捕获在油包水乳液滴中，随后通过乳液PCR回收伙伴基因。在这一步中，含有表达促进聚合酶产生的伙伴蛋白的细胞的液滴将产生更高拷贝数的改进后的伙伴基因。所得的伙伴基因随后可重新克隆用于下一轮选择。在此，我们通过提供（i）识别正交启动子的T7 RNA聚合酶的选择和（ii）增强琥珀密码子抑制的tRNA的选择的示例，给出该程序的分步指南。一轮CPR大约需要3 - 5天，而整个定向进化根据选择效率可在3 - 10轮内完成。
