The yeast cytoplasmically localized pGKL1/TP-DNAP1 plasmid/DNA polymerase pair forms an orthogonal DNA replication system whose mutation rate can be drastically increased without influencing genomic replication, thereby supporting in vivo continuous evolution. Here, we report that the pGKL2/TP-DNAP2 plasmid/DNA polymerase pair forms a second orthogonal replication system. We show that custom genes can be encoded and expressed from pGKL2, that error-prone TP-DNAP2s can be engineered, and that pGKL2 replication by TP-DNAP2 is both orthogonal to genomic replication in Saccharomyces cerevisiae and mutually orthogonal with pGKL1 replication by TP-DNAP1. This demonstration of two mutually orthogonal DNA replication systems with tunable error rates and properties should enable new applications in cell-based continuous evolution, genetic recording, and synthetic biology at large.

中文翻译：

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体内相互正交 DNA 复制系统

酵母细胞质中定位的pGKL1/TP-DNAP1质粒/DNA聚合酶对形成正交DNA复制系统，其突变率可以大幅提高而不影响基因组复制，从而支持体内连续进化。在这里，我们报告 pGKL2/TP-DNAP2 质粒/DNA 聚合酶对形成第二个正交复制系统。我们表明，可以从 pGKL2 编码和表达定制基因，可以设计容易出错的 TP-DNAP2，并且 TP-DNAP2 的 pGKL2 复制既与酿酒酵母中的基因组复制正交，又与 TP-DNAP2 的 pGKL1 复制相互正交。 DNAP1。这种具有可调错误率和特性的两个相互正交的 DNA 复制系统的演示应该能够在基于细胞的连续进化、基因记录和合成生物学中实现新的应用。