Yeast Golden Gate (yGG) for the Efficient Assembly of <i>S. cerevisiae</i> Transcription Units

Agmon, Neta; Mitchell, Leslie A.; Cai, Yizhi; Ikushima, Shigehito; Chuang, James; Zheng, Allen; Choi, Woo Jin; Martin, James A.; Caravelli, Katrina; Stracquadanio, Giovanni; Boeke, Jef D.

doi:10.1021/sb500372z

Cited by 74 publications

(83 citation statements)

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“…Having the ability to assemble SGTUs combinatorially provides many more variants for testing than could be made individually. Thus, we assembled our SG constructs as a combinatorial yeast Golden Gate (yGG) assembly (25) and screened for the best candidates in yeast. We amplified all of the parts (promoters, essential gene CDSs, and terminators) from the yeast genome with the appropriate yGG overhangs, as described (25).…”

Section: Resultsmentioning

confidence: 99%

“…Thus, we assembled our SG constructs as a combinatorial yeast Golden Gate (yGG) assembly (25) and screened for the best candidates in yeast. We amplified all of the parts (promoters, essential gene CDSs, and terminators) from the yeast genome with the appropriate yGG overhangs, as described (25). For each essential gene, we performed a "one-pot" yGG assembly, adding six distinct galactose-regulated promoters [GAL1, GAL7, GAL10, SPAL2, SPAL5, and SPAL6 (26)], GAL1 terminator, and acceptor vector (pAV10.HO5.loxP).…”

Section: Resultsmentioning

confidence: 99%

“…All 50 genes were to be potentially cloned downstream of six different promoters: GAL1, GAL10, GAL7, SPAL2, SPAL5, and SPAL6 (26). They were assembled with the GAL1 terminator and into acceptor vectors with integration cassettes (25). All combinations were examined for their ability to serve as potential SGs.…”

Section: Methodsmentioning

confidence: 99%

“…All 50 genes, six promoters, and terminator were amplified with the appropriate overhangs for yGG from the yeast genome (25), cloned into a pCR-Blunt II-TOPO vector (Invitrogen), and sequence-verified. Most genes were amplified as one-part genes; however, some were either too large to amplify as one part or had an internal BsaI site, which is incompatible with yGG assembly (25), and thus were amplified as multiple gene parts compatible with yGG assembly. All but one gene, PSA1, failed at this step, so we moved forward with the successful 49 genes.…”

Section: Methodsmentioning

confidence: 99%

“…In total, we synthesized 67 gene parts, six promoter parts, and one terminator part for the yGG assembly. As an acceptor vector, we chose to use pAV10.HO5.loxP, an integrating vector, with the LEU2 marker for integration into the HO locus (25).…”

Section: Methodsmentioning

confidence: 99%

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Low escape-rate genome safeguards with minimal molecular perturbation ofSaccharomyces cerevisiae

Agmon

Tang

Yang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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As the use of synthetic biology both in industry and in academia grows, there is an increasing need to ensure biocontainment. There is growing interest in engineering bacterial-and yeastbased safeguard (SG) strains. First-generation SGs were based on metabolic auxotrophy; however, the risk of cross-feeding and the cost of growth-controlling nutrients led researchers to look for other avenues. Recent strategies include bacteria engineered to be dependent on nonnatural amino acids and yeast SG strains that have both transcriptional-and recombinational-based biocontainment. We describe improving yeast Saccharomyces cerevisiaebased transcriptional SG strains, which have near-WT fitness, the lowest possible escape rate, and nanomolar ligands controlling growth. We screened a library of essential genes, as well as the best-performing promoter and terminators, yielding the best SG strains in yeast. The best constructs were fine-tuned, resulting in two tightly controlled inducible systems. In addition, for potential use in the prevention of industrial espionage, we screened an array of possible "decoy molecules" that can be used to mask any proprietary supplement to the SG strain, with minimal effect on strain fitness.genome safety | escape mutants | Rpd3L | histone deacetylase | yeast W ith the increasing use of genetically modified organisms, there is a growing need for biocontainment to secure biosystems from outside malice as well as to prevent propagation in an open system in the event of advertent or inadvertent release to the environment. Although recombinant DNA technology was established more than four decades ago (1), the fact that genetically modified organisms have not caused any substantial deleterious incident is due, in part, to precautionary measures taken by professional genetic engineers and the high cost of genetic engineering, largely restricting its use to academic and industrial laboratories. However, in the current era of "do-ityourself" synthetic biology, with rapid technological advances in DNA synthesis and assembly (2), genome editing (3, 4) and computational tools for design (5-9), and with the decreasing cost of DNA synthesis, the need for genomic safeguards (SGs) is clear.Early biocontainment efforts focused on the use of metabolic auxotrophy dependence (10, 11), toxin/antitoxin-dependent suicide (12-15), or both (16,17). Although top performers using these strategies do comply with the NIH standard for SGs (10

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Low escape-rate genome safeguards with minimal molecular perturbation ofSaccharomyces cerevisiae

Agmon

Tang

Yang

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Oligonucleotide abundance biases aid design of a type IIS synthetic genomics framework with plant virome capacity

Pasin

2021

Biotechnology Journal

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Synthetic genomics-driven dematerialization of genetic resources facilitates flexible hypothesis testing and rapid product development. Biological sequences have compositional biases, which, I reasoned, could be exploited for engineering of enhanced synthetic genomics systems. In proof-of-concept assays reported herein, the abundance of random oligonucleotides in viral genomic components was analyzed and used for the rational design of a synthetic genomics framework with plant virome capacity (Syn-ViP). Type IIS endonucleases with low abundance in the plant virome, as well as Golden Gate and No See'm principles were combined with DNA chemical synthesis for seamless viral clone assembly by one-step digestion-ligation. The framework described does not require subcloning steps, is insensitive to insert terminal sequences, and was used with linear and circular DNA molecules. Based on a digital template, DNA fragments were chemically synthesized and assembled by one-step cloning to yield a scar-free infectious clone of a plant virus suitable for Agrobacterium-mediated delivery. SynViP allowed rescue of a genuine virus without biological material, and has the potential to greatly accelerate biological characterization and engineering of plant viruses as well as derived biotechnological tools. Finally, computational identification of compositional biases in biological sequences might become a common standard to aid scalable biosystems design and engineering. K E Y W O R D SDNA chemical synthesis, Golden Gate cloning, plant virome, type IIS restriction enzyme, viral infectious clone assembly INTRODUCTIONViruses are relatively simple biological entities, with genomic components whose size is compatible both in terms of technical feasibility and costs with current advances in de novo DNA synthesis. [1,2] Synthetic genomics is becoming commonplace for the study and engineering of animal viruses, especially those with medical interest and human pandemic potential. [3][4][5][6][7] In plant virology, synthetic genomics can help to demonstrate correctness of genomic sequences, rescue of viruses that might not be physically available (e.g., ancient or environmental samples), as well as to accelerate virus reverse genetics, host-virus interaction studies, biological characterization of emerging viruses, or engineering of biotechnological devices. [8][9][10][11] Adoption of this powerful approach for plant virology has nonetheless lagged and is reported only in a handful of studies. [9] The first reports of synthetic genomic approaches in plant virology included the use of oligonucleotides to

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A well‐characterized polycistronic‐like gene expression system in yeast

Mukherjee

Wang

2022

Biotech & Bioengineering

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Efficient expression of multiple genes is critical to yeast metabolic engineering because of the increasing complexity of engineered pathways. A yeast polycistronic expression system is of particular interest because one promoter can drive the expression of multiple genes. Polycistronic expression thus requires less genetic material and minimizes undesirable recombination due to repeated use of the same promoter. It also decreases the number of DNA parts necessary for cloning a multi-gene construct. 2A viral peptides enable the co-translation of multiple proteins from one mRNA by ribosomal skipping. However, the wide adaptation of this strategy for polycistronic-like gene expression in yeast awaits in-depth characterizations.Additionally, a one-step assembly of such a polycistronic-like system is highly desirable. To this end, we have developed a MoClo compatible 2A peptide-based polycistronic-like system capable of expressing multiple genes from a single promoter in yeast. Characterizing the bi-, tri, and quad-cistronic expression of fluorescent proteins showed high cleavage efficiencies of three 2A peptides. The expression level of each protein decreases as it moves away from the .

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Yeast Golden Gate (yGG) for the Efficient Assembly of S. cerevisiae Transcription Units

Cited by 74 publications

References 16 publications

Low escape-rate genome safeguards with minimal molecular perturbation ofSaccharomyces cerevisiae

Low escape-rate genome safeguards with minimal molecular perturbation ofSaccharomyces cerevisiae

Oligonucleotide abundance biases aid design of a type IIS synthetic genomics framework with plant virome capacity

A well‐characterized polycistronic‐like gene expression system in yeast

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