Hou Zheng, Zhou Zheng, Wang Zonglin, Xiao Gengfu
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China.
Virol Sin. 2016 Apr;31(2):160-7. doi: 10.1007/s12250-016-3730-8. Epub 2016 Apr 11.
Synthetic biology is a newly developed field of research focused on designing and rebuilding novel biomolecular components, circuits, and networks. Synthetic biology can also help understand biological principles and engineer complex artificial metabolic systems. DNA manipulation on a large genome-wide scale is an inevitable challenge, but a necessary tool for synthetic biology. To improve the methods used for the synthesis of long DNA fragments, here we constructed a novel shuttle vector named pGF (plasmid Genome Fast) for DNA assembly in vivo. The BAC plasmid pCC1BAC, which can accommodate large DNA molecules, was chosen as the backbone. The sequence of the yeast artificial chromosome (YAC) regulatory element CEN6-ARS4 was synthesized and inserted into the plasmid to enable it to replicate in yeast. The selection sequence HIS3, obtained by polymerase chain reaction (PCR) from the plasmid pBS313, was inserted for screening. This new synthetic shuttle vector can mediate the transformation-associated recombination (TAR) assembly of large DNA fragments in yeast, and the assembled products can be transformed into Escherichia coli for further amplification. We also conducted in vivo DNA assembly using pGF and yeast homologous recombination and constructed a 31-kb long DNA sequence from the cyanophage PP genome. Our findings show that this novel shuttle vector would be a useful tool for efficient genome-scale DNA reconstruction.
合成生物学是一个新发展起来的研究领域，专注于设计和重建新型生物分子组件、电路和网络。合成生物学还可以帮助理解生物学原理并构建复杂的人工代谢系统。在全基因组范围内进行DNA操作是一项不可避免的挑战，但却是合成生物学的必要工具。为了改进用于合成长DNA片段的方法，我们构建了一种名为pGF（质粒基因组快速）的新型穿梭载体，用于体内DNA组装。选择能够容纳大DNA分子的BAC质粒pCC1BAC作为骨架。合成酵母人工染色体（YAC）调控元件CEN6-ARS4的序列并将其插入质粒，使其能够在酵母中复制。通过聚合酶链反应（PCR）从质粒pBS313获得的筛选序列HIS3被插入用于筛选。这种新的合成穿梭载体可以介导酵母中大片段DNA的转化相关重组（TAR）组装，并且组装产物可以转化到大肠杆菌中进行进一步扩增。我们还使用pGF和酵母同源重组进行了体内DNA组装，并从蓝藻噬菌体PP基因组构建了一个31 kb长的DNA序列。我们的研究结果表明，这种新型穿梭载体将是高效基因组规模DNA重建的有用工具。
