Assembly of long DNA sequences using a new synthetic Escherichia coli-yeast shuttle vector  

作　　者：Zheng Hou Zheng Zhou Zonglin Wang Gengfu Xiao 

机构地区：[1]State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences

出　　处：《Virologica Sinica》2016年第2期160-167,共8页中国病毒学（英文版）

基　　金：supported by the 973 program,Grant No.2012CB721102

摘　　要：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 p GF(plasmid Genome Fast) for DNA assembly in vivo. The BAC plasmid p CC1 BAC, 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 p BS313, 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 p GF 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.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 p GF（plasmid Genome Fast） for DNA assembly in vivo. The BAC plasmid p CC1 BAC, 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 p BS313, 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 p GF 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.

关 键 词：yeast plasmid shuttle DNA recombination Genome assembly inserted amplification homologous 

分 类 号：Q93[生物学—微生物学] Q78