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Science. 2017 Mar 10;355(6329). pii: eaaf3981. doi: 10.1126/science.aaf3981.

Engineering the ribosomal DNA in a megabase synthetic chromosome.

Author information

1
Key Laboratory for Industrial Biocatalysis (Ministry of Education) and Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China.
2
Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
3
Peking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program, College of Life Science, Peking University, Beijing 100871, China.
4
BGI-Shenzhen, Shenzhen 518083, China.
5
School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, UK.
6
BGI-Qingdao, Qingdao 266555, China.
7
Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, New York University Langone Medical Center, New York, NY 10011, USA.
8
High-Throughput Biological Center and Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.
9
Key Laboratory of Systems Bioengineering (Ministry of Education), SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
10
James D. Watson Institute of Genome Sciences, Hangzhou 310058, China.
11
Wuxi Qinglan Biotechnology Inc., Yixing, Jiangsu 214200, China.
12
Key Laboratory for Industrial Biocatalysis (Ministry of Education) and Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China. jbdai@tsinghua.edu.cn.

Abstract

We designed and synthesized a 976,067-base pair linear chromosome, synXII, based on native chromosome XII in Saccharomyces cerevisiae SynXII was assembled using a two-step method, specified by successive megachunk integration and meiotic recombination-mediated assembly, producing a functional chromosome in S. cerevisiae. Minor growth defect "bugs" detected in synXII, caused by deletion of tRNA genes, were rescued by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit used to regenerate rDNA at three distinct chromosomal locations. The signature sequences within rDNA, which can be used to determine species identity, were swapped to generate a Saccharomyces synXII strain that would be identified as Saccharomyces bayanus by standard DNA barcoding procedures.

PMID:
28280149
DOI:
10.1126/science.aaf3981
[Indexed for MEDLINE]

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