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Nucleic Acids Res. 2017 Oct 13;45(18):10895-10905. doi: 10.1093/nar/gkx753.

De novo design and synthesis of a 30-cistron translation-factor module.

Author information

1
Department of Cell and Molecular Biology, Uppsala University, Uppsala 751 36, Sweden.
2
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
3
Department of Physical and Analytical Chemistry, Uppsala University, Uppsala 751 23, Sweden.
4
Institute of Biology and Information Science, School of Computer Science and Software Engineering, School of Life Sciences, East China Normal University, Shanghai 200062, PR China.

Abstract

Two of the many goals of synthetic biology are synthesizing large biochemical systems and simplifying their assembly. While several genes have been assembled together by modular idempotent cloning, it is unclear if such simplified strategies scale to very large constructs for expression and purification of whole pathways. Here we synthesize from oligodeoxyribonucleotides a completely de-novo-designed, 58-kb multigene DNA. This BioBrick plasmid insert encodes 30 of the 31 translation factors of the PURE translation system, each His-tagged and in separate transcription cistrons. Dividing the insert between three high-copy expression plasmids enables the bulk purification of the aminoacyl-tRNA synthetases and translation factors necessary for affordable, scalable reconstitution of an in vitro transcription and translation system, PURE 3.0.

PMID:
28977654
PMCID:
PMC5737471
DOI:
10.1093/nar/gkx753
[Indexed for MEDLINE]
Free PMC Article

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