Department of Life Science, College of Science, Rikkyo University, Tokyo 171-8501, Japan.
ACS Synth Biol. 2022 Sep 16;11(9):3088-3099. doi: 10.1021/acssynbio.2c00353. Epub 2022 Aug 23.
The physical stability of bacterial chromosomes is important for their in vitro manipulation, while genetic stability is important in vivo. However, extracted naked chromosomes in the open circular form are fragile due to nicks and gaps. Using a nick/gap repair and negative supercoiling reaction (named SCR), we first achieved the negative supercoiling of the whole genomes extracted from and cells. Supercoiled chromosomes of 0.2-4.6 megabase (Mb) were separated by size using a conventional agarose gel electrophoresis and served as DNA size markers. We also achieved the enzymatic replication of 1-2 Mb chromosomes using the reconstituted replication-cycle reaction (RCR). Electroporation-ready 1 Mb chromosomes were prepared by a modified SCR performed at a low salt concentration (L-SCR) and directly introduced into commercial electrocompetent cells. Since successful electroporation relies on the genetic stability of a chromosome in cells, genetically stable 1 Mb chromosomes were developed according to a portable chromosome format (PCF). Using physically and genetically stabilized chromosomes, the democratization of genome synthetic biology will be greatly accelerated.
细菌染色体的物理稳定性对于其体外操作很重要，而遗传稳定性对于体内操作很重要。然而，由于切口和缺口的存在，以开环形式提取的裸露染色体很脆弱。我们利用切口/缺口修复和负超螺旋反应（称为 SCR），首次实现了从 和 细胞中提取的整个基因组的负超螺旋化。使用常规琼脂糖凝胶电泳分离大小为 0.2-4.6 兆碱基（Mb）的超螺旋染色体，并将其用作 DNA 大小标记。我们还通过重新构建的复制周期反应（RCR）实现了 1-2 Mb 染色体的酶复制。通过在低盐浓度（L-SCR）下进行改良的 SCR 制备可电穿孔的 1 Mb 染色体，并直接引入商业电感受态 细胞。由于电穿孔的成功依赖于细胞中染色体的遗传稳定性，因此根据可携带染色体格式（PCF）开发了遗传稳定的 1 Mb 染色体。使用物理和遗传稳定的染色体，将极大地加速基因组合成生物学的民主化。
