In vitro DNA SCRaMbLE

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

doi:10.1038/s41467-018-03743-6

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.

Highlights

The power of synthetic biology has enabled the expression of heterologous pathways in cells, as well as genome-scale synthesis projects
The loxP sites encode a symmetric spacer region (loxPsym) sites flank “transcription unit” (TU) sequences, the unit to be SCRaMbLEd in the system
To test the “chemical” feasibility of top-down in vitro SCRaMbLE, 10 loxPsym sites were evenly distributed across a 5 kb piece of DNA and assembled into a plasmid by overlap polymerase chain reaction (PCR) (Fig. 1b)

Summary

Introduction

The power of synthetic biology has enabled the expression of heterologous pathways in cells, as well as genome-scale synthesis projects. The in vivo Synthetic Chromosome Rearrangement and Modification by LoxPsym-mediated Evolution (SCRaMbLE) system, built into synthetic yeast chromosomes, has been demonstrated to generate stochastic diversity in chromosome structure, including deletions, duplications, inversions, insertions (transpositions), or translocations in synthetic chromosomes synIII and synIXR13–17 In this system, the Cre recombinase is introduced into Sc2.0 cells genetically and controlled both transcriptionally and chemically[14,15]. With the addition of Cre recombinase to the reaction, donor fragments are randomly inserted into the acceptor vector to produce a pool of diverse constructs which add one or more donor constructs to the base pathway The products of both in vitro SCRaMbLE strategies can be transferred to a host strain directly for phenotype testing and genotyping of individual SCRaMbLE derivatives. Our results indicate that in vitro SCRaMbLE is a unique and straightforward method for generating DNA libraries, and is potentially amenable to biochemical optimization in ways not achievable in vivo

Methods

Results

Conclusion