Precise control of SCRaMbLE in synthetic haploid and diploid yeast

Jia, Bin; Wu, Yi; Li, Bing Zhi; Mitchell, Leslie A.; Liu, Hong; Pan, Shuo; Wang, Juan; Zhang, Hao Ran; Nan, Jun; Ли, Бо; Shen, Michael; Xie, Ze‐Xiong; Liu, Duo; Cao, Ying; Li, Xia; Qi, Hao; Boeke, Jef D.; Yuan, Ying

doi:10.1038/s41467-018-03084-4

Cited by 123 publications

(111 citation statements)

References 69 publications

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“…From another angle, we need this kind of de novo designed artificial promoters as barcodes to mark gene expression at genome level to research the genome rearrangement [18,19]. The artificial barcodes of promoters will further assist the analysis of synthetic genomic evolution.…”

Section: Open Accessmentioning

confidence: 99%

Engineering yeast artificial core promoter with designated base motifs

Liu

et al. 2020

Microb Cell Fact

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Background: Synthetic biology requires toolbox of promoters to finely tune gene expression levels for building up efficient cell factories. Yeast promoters owned variable core promoter regions between the TATA-box and transcriptional starting site (TSS) at the length mostly around 20-80 bases. This region allowed flexible design of artificial promoter but potentially demand special base motifs to maintain or enhance the promoter's strength.Results: Here, we designed and screened the base motifs and tested the activities of yeast artificial core promoters. Different 30 bases of artificial sequences led to variable expression levels of CrtY enzyme which determined the lycopene-carotene compositions, represented in the colony-color spectrum of red-orange-yellow. The upstream sequences of two strong promoter P EXP1 and P GPD and two starting strains with distinguishable lycopene production levels were utilized to characterize the promoter sequences. Different partition designs of T-rich or G/C-rich base motifs led to distinguishable colony-color distributions. Finally, we screened a champion promoter with a highest 5.5-fold enhancement of lycopene-carotene transformation. Another selected promoter generated a highest betacarotene production as 7.4 mg/g DCW. Conclusions:This work offered an approach to redesign promoter with artificial sequences. We concluded that the core promoter region could be designated as 30 bases and different base motifs would enhance or weaken the promoter's strength. Generally, more T-rich elements, higher %T and lower G/C percentage were beneficial to enhance the strength of artificial core promoter.

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Section: Open Accessmentioning

confidence: 99%

Engineering yeast artificial core promoter with designated base motifs

Liu

et al. 2020

Microb Cell Fact

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“…More recently, researchers have developed several new methods based on SCRaMbLE, including Reporter of SCRaMbLEd cells using Efficient Selection, Multiplex SCRaMbLE Iterative Cycling, diploid SCRaMbLE, SCRaMbLE‐in, in vitro SCRaMbLE, and L‐SCRaMbLE, which explore the SCRaMbLE system in various applications. However, due to the limited number of completed synthetic chromosomes, SCRaMbLE has only been applied in strains with one or two synthetic chromosomes.…”

Section: Recombinase‐based Genome Engineeringmentioning

confidence: 99%

Whole‐Genome Regulation for Yeast Metabolic Engineering

Jiang

Dai

2019

Small Methods

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As the most thoroughly investigated eukaryotic microorganism, Saccharomyces cerevisiae is widely used as a cell factory for producing valuable compounds. Currently, strain construction and optimization still cost a vast amount of capital and time to achieve industrially relevant parameters. To efficiently excavate the genetic factors required for desired traits, many high‐throughput genome engineering tools are developed. This review focuses on the genome‐wide approaches that are applied in yeast. Through multiplex genome editing and/or transcription regulation, these tools generate cell populations with great genetic diversity, enabling identification of the critical factors and synergistic interactions in a short time.

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“…Distinct from most genome synthesis projects, the goal of Sc2.0 is to write a dramatically modified version of the 12 Mb genome in order to be able to address otherwise unapproachable biological questions. Thus far multiple synthetic chromosomes have been completed (7)(8)(9)(10)(11)(12)(13)(14) along with widespread use of the semisynthetic cells to investigate genome structure-function relationship using the inducible evolution system SCRaMbLE (synthetic chromosome rearrangements and modification by loxP-mediated evolution) (15)(16)(17)(18)(19)(20).…”

Section: Introductionmentioning

confidence: 99%

De novoassembly, delivery and expression of a 101 kb human gene in mouse cells

Mitchell¹,

McCulloch²,

Pinglay³

et al. 2018

Preprint

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Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here we describe a pipeline for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kb. The DNA assembly step is supported by an integrated robotic workcell. We assembled the 101 kb human HPRT1 gene in yeast, delivered it to mouse embryonic stem cells, and showed expression of the human protein from its full-length gene. This pipeline provides a framework for producing systematic, designer variants of any mammalian gene locus for functional evaluation in cells. Significance StatementMammalian genomes consist of a tiny proportion of relatively well-characterized coding regions and vast swaths of poorly characterized "dark matter" containing critical but much less well-defined regulatory sequences. Given the dominant role of noncoding DNA in common human diseases and traits, the interconnectivity of regulatory elements, and the importance of genomic context, de novo design, assembly, and delivery can enable large-scale manipulation of these elements on a locus scale. Here we outline a pipeline for de novo assembly, delivery and expression of mammalian genes replete with native regulatory sequences. We expect this pipeline will be useful for dissecting the function of non-coding sequence variation in mammalian genomes.

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Precise control of SCRaMbLE in synthetic haploid and diploid yeast

Cited by 123 publications

References 69 publications

Engineering yeast artificial core promoter with designated base motifs

Engineering yeast artificial core promoter with designated base motifs

Whole‐Genome Regulation for Yeast Metabolic Engineering

De novoassembly, delivery and expression of a 101 kb human gene in mouse cells

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