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Nat Commun. 2018 May 22;9(1):1935. doi: 10.1038/s41467-018-03743-6.

In vitro DNA SCRaMbLE.

Wu Y1,2,3, Zhu RY1,2, Mitchell LA3, Ma L1,2, Liu R1,2, Zhao M1,2,3, Jia B1,2, Xu H1,2, Li YX1,2, Yang ZM1,2, Ma Y1,2, Li X1,2, Liu H1,2, Liu D1,2, Xiao WH1,2, Zhou X1,2, Li BZ1,2, Yuan YJ1,2, Boeke JD4.

Author information

1
Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, 300072, Tianjin, China.
2
SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, 300072, Tianjin, China.
3
Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY, 10016, USA.
4
Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY, 10016, USA. Jef.Boeke@nyumc.org.

Abstract

The power of synthetic biology has enabled the expression of heterologous pathways in cells, as well as genome-scale synthesis projects. The complexity of biological networks makes rational de novo design a grand challenge. Introducing features that confer genetic flexibility is a powerful strategy for downstream engineering. Here we develop an in vitro method of DNA library construction based on structural variation to accomplish this goal. The "in vitro SCRaMbLE system" uses Cre recombinase mixed in a test tube with purified DNA encoding multiple loxPsym sites. Using a β-carotene pathway designed for expression in yeast as an example, we demonstrate top-down and bottom-up in vitro SCRaMbLE, enabling optimization of biosynthetic pathway flux via the rearrangement of relevant transcription units. We show that our system provides a straightforward way to correlate phenotype and genotype and is potentially amenable to biochemical optimization in ways that the in vivo system cannot achieve.

PMID:
29789594
PMCID:
PMC5964173
DOI:
10.1038/s41467-018-03743-6
[Indexed for MEDLINE]
Free PMC Article

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