TY - CHAP

T1 - Scramble-in

T2 - A fast and efficient method to diversify and improve the yields of heterologous pathways in synthetic yeast

AU - Swidah, Reem

AU - Auxillos, Jamie

AU - Liu, Wei

AU - Jones, Sally

AU - Chan, Ting Fung

AU - Dai, Junbiao

AU - Cai, Yizhi

N1 - Funding Information: Special thanks to Dr. Eva Garcia-Ruiz for the invaluable advice and discussion for using the Echo® 550/555 Liquid Handler and Certus-Flex and Dr. Daniel Schindler for his help with the technical issues, and BBSRC for funding BB/P02114X/1 (to Y.C.) and Royal Society Newton Advanced Fellowship R123288 (to J.D. and Y.C.). Publisher Copyright: © Springer Science+Business Media, LLC, part of Springer Nature 2020.

PY - 2020

Y1 - 2020

N2 - 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.

AB - 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.

KW - Metabolic engineering

KW - SCRaMbLE

KW - SCRaMbLE-in

KW - Synthetic biology

KW - Yeast

UR - http://www.scopus.com/inward/record.url?scp=85089638997&partnerID=8YFLogxK

U2 - 10.1007/978-1-0716-0908-8_17

DO - 10.1007/978-1-0716-0908-8_17

M3 - Book chapter

C2 - 32809206

AN - SCOPUS:85089638997

T3 - Methods in Molecular Biology

SP - 305

EP - 327

BT - Methods in Molecular Biology

PB - Humana Press

ER -