September 22, 2019  |  

In vitro DNA SCRaMbLE.

Authors: Wu, Yi and Zhu, Rui-Ying and Mitchell, Leslie A and Ma, Lu and Liu, Rui and Zhao, Meng and Jia, Bin and Xu, Hui and Li, Yun-Xiang and Yang, Zu-Ming and Ma, Yuan and Li, Xia and Liu, Hong and Liu, Duo and Xiao, Wen-Hai and Zhou, Xiao and Li, Bing-Zhi and Yuan, Ying-Jin and Boeke, Jef D

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 ß-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.

Journal: Nature communications
DOI: 10.1038/s41467-018-03743-6
Year: 2018

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