Arzumanyan Garri A, Gabriel Kristin N, Ravikumar Arjun, Javanpour Alex A, Liu Chang C
Department of Biomedical Engineering , University of California , Irvine , California 92697 , United States.
Department of Molecular Biology & Biochemistry , University of California , Irvine , California 92697 , United States.
ACS Synth Biol. 2018 Jul 20;7(7):1722-1729. doi: 10.1021/acssynbio.8b00195. Epub 2018 Jul 10.
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.
酵母细胞质定位的pGKL1/TP-DNAP1质粒/DNA聚合酶对形成了一个正交DNA复制系统，其突变率可以在不影响基因组复制的情况下大幅提高，从而支持体内连续进化。在此，我们报告pGKL2/TP-DNAP2质粒/DNA聚合酶对形成了第二个正交复制系统。我们表明，可以从pGKL2编码并表达定制基因，可以设计易错的TP-DNAP2，并且TP-DNAP2介导的pGKL2复制在酿酒酵母中既与基因组复制正交，又与TP-DNAP1介导的pGKL1复制相互正交。这两个具有可调错误率和特性的相互正交DNA复制系统的证明应该能够在基于细胞的连续进化、遗传记录以及整个合成生物学中实现新的应用。
