The synthetic chromosome rearrangement and modification by LoxP-mediated evolution (SCRaMbLE) system is a key component of the synthetic yeast genome (Sc2.0) project, an international effort to construct an entire synthetic genome in yeast. SCRaMbLE involves the introduction of thousands of symmetrical LoxP (LoxPsym) recombination sites downstream of every nonessential gene in all 16 chromosomes, enabling numerous genome rearrangements in the form of deletions, inversions, duplications, and translocations by the Cre-LoxPsym recombination system. We highlight a two-step protocol for SCRaMbLE-in (Liu, Nat Commun 9(1):1936, 2018), a recombinase-based combinatorial method to expedite genetic engineering and exogenous pathway optimization, using a synthetic β-carotene pathway as an example. First, an in vitro phase uses a recombinase toolkit to diversify gene expression by integrating various regulatory elements into the target pathway. This combinatorial pathway library can be transformed directly into yeast for traditional screening. Once an optimized pathway which is flanked by LoxPsym sites is identified, it is transformed into Sc2.0 yeast for the in vivo SCRaMbLE phase, where LoxPsym sites in the synthetic yeast genome and Cre recombinase catalyze massive genome rearrangements. We describe all the conditions necessary to perform SCRaMbLE and post-SCRaMbLE experiments including screening, spot test analysis, and PCRTag analysis to elucidate genotype-phenotype relationships.

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Swidah R, Auxillos J, Liu W, Jones S, Chan TF, Dai J, Cai Y

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