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. 2016 Apr 11;31(2):160–167. doi: 10.1007/s12250-016-3730-8

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

Zheng Hou 1, Zheng Zhou 1, Zonglin Wang 1, Gengfu Xiao 1,✉
PMCID: PMC8193415  PMID: 27113243

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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Electronic Supplementary Material

Supplementary material is available for this article at 10.1007/s12250-016-3730-8 and is accessible for authorized users.

Keywords: synthetic biology, DNA fragment assembly, shuttle vector pGF, transformationassociated recombination (TAR)

Electronic supplementary material

12250_2016_3730_MOESM1_ESM.pdf (141.7KB, pdf)

Supplementary material, approximately 142 KB.

Footnotes

These authors contributed equally to this work.

ORCID: 0000-0001-9401-235X

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Supplementary Materials

12250_2016_3730_MOESM1_ESM.pdf (141.7KB, pdf)

Supplementary material, approximately 142 KB.

Articles from Virologica Sinica are provided here courtesy of Wuhan Institute of Virology, Chinese Academy of Sciences

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