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Volume 31 Issue 2
April 2016
    Citation: Zheng Hou, Zheng Zhou, Zonglin Wang, Gengfu Xiao. Assembly of long DNA sequences using a new synthetic Escherichia coli-yeast shuttle vector .VIROLOGICA SINICA, 2016, 31(2) : 160-167.  http://dx.doi.org/10.1007/s12250-016-3730-8
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    Assembly of long DNA sequences using a new synthetic Escherichia coli-yeast shuttle vector

    • State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China

    • Corresponding author: Gengfu Xiao, xiaogf@wh.iov.cn, ORCID: 0000-0001-9401-235X
    • These authors contributed equally to this work.
    • Received Date: 21 January 2016
      Accepted Date: 17 March 2016
      Published Date: 11 April 2016
      Available online: 01 April 2016
    • 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.
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      Assembly of long DNA sequences using a new synthetic Escherichia coli-yeast shuttle vector

        Corresponding author: Gengfu Xiao, xiaogf@wh.iov.cn
      • State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China
      • Received Date: 21 January 2016
      • Accepted Date: 17 March 2016
      • Published Date: 11 April 2016

      Abstract: 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.

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