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Article ; Online: Engineering stringent genetic biocontainment of yeast with a protein stability switch.

Hoffmann, Stefan A / Cai, Yizhi

2024 Volume 15, Issue 1, Page(s) 1060

Abstract: Synthetic biology holds immense promise to tackle key problems in resource use, environmental remediation, and human health care. However, comprehensive safety measures are lacking to employ engineered microorganisms in open-environment applications. ... ...

Abstract	Synthetic biology holds immense promise to tackle key problems in resource use, environmental remediation, and human health care. However, comprehensive safety measures are lacking to employ engineered microorganisms in open-environment applications. Genetically encoded biocontainment systems may solve this issue. Here, we describe such a system based on conditional stability of essential proteins. We used a destabilizing domain degron stabilized by estradiol addition (ERdd). We ERdd-tagged 775 essential genes and screened for strains with estradiol dependent growth. Three genes, SPC110, DIS3 and RRP46, were found to be particularly suitable targets. Respective strains showed no growth defect in the presence of estradiol and strong growth inhibition in its absence. SPC110-ERdd offered the most stringent containment, with an escape frequency of <5×10
MeSH term(s)	Humans ; Saccharomyces cerevisiae/genetics ; Genetic Engineering ; Organisms, Genetically Modified/genetics ; Containment of Biohazards ; Synthetic Biology ; Estradiol/metabolism
Chemical Substances	Estradiol (4TI98Z838E)
Language	English
Publishing date	2024-02-05
Publishing country	England
Document type	Journal Article
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-024-44988-8
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Methodological advances enabled by the construction of a synthetic yeast genome.

Schindler, Daniel / Walker, Roy S K / Cai, Yizhi

Cell reports methods

2024 Volume 4, Issue 4, Page(s) 100761

Abstract: The international Synthetic Yeast Project (Sc2.0) aims to construct the first synthetic designer eukaryote genome. Over the past few years, the Sc2.0 consortium has achieved several significant milestones by synthesizing and characterizing all 16 nuclear ...

Abstract	The international Synthetic Yeast Project (Sc2.0) aims to construct the first synthetic designer eukaryote genome. Over the past few years, the Sc2.0 consortium has achieved several significant milestones by synthesizing and characterizing all 16 nuclear chromosomes of the yeast Saccharomyces cerevisiae, as well as a 17
MeSH term(s)	Genetic Engineering/methods ; Genome, Fungal/genetics ; Genomics/methods ; Saccharomyces cerevisiae/genetics ; Synthetic Biology/methods
Language	English
Publishing date	2024-04-22
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2667-2375
ISSN (online)	2667-2375
DOI	10.1016/j.crmeth.2024.100761
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Article ; Online: Searching for the optimal microbial factory: high-throughput biosensors and analytical techniques for screening small molecules.

O'Connor, Eloise / Micklefield, Jason / Cai, Yizhi

Current opinion in biotechnology

2024 Volume 87, Page(s) 103125

Abstract: High-throughput screening technologies have been lacking in comparison to the plethora of high-throughput genetic diversification techniques developed in biotechnology. This review explores the challenges and advancements in high-throughput screening for ...

Abstract	High-throughput screening technologies have been lacking in comparison to the plethora of high-throughput genetic diversification techniques developed in biotechnology. This review explores the challenges and advancements in high-throughput screening for high-value natural products, focusing on the critical need to expand ligand targets for biosensors and increase the throughput of analytical techniques in screening microbial cell libraries for optimal strain performance. The engineering techniques to broaden the scope of ligands for biosensors, such as transcription factors, G protein-coupled receptors and riboswitches are discussed. On the other hand, integration of microfluidics with traditional analytical methods is explored, covering fluorescence-activated cell sorting, Raman-activated cell sorting and mass spectrometry, emphasising recent developments in maximising throughput.
Language	English
Publishing date	2024-03-27
Publishing country	England
Document type	Journal Article ; Review
ZDB-ID	1052045-4
ISSN	1879-0429 ; 0958-1669
ISSN (online)	1879-0429
ISSN	0958-1669
DOI	10.1016/j.copbio.2024.103125
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Article ; Online: New opportunities for genetic code expansion in synthetic yeast.

Sanders, James / Hoffmann, Stefan A / Green, Anthony P / Cai, Yizhi

Current opinion in biotechnology

2022 Volume 75, Page(s) 102691

Abstract: The synthetic yeast, Sc2.0, is nearing completion as consolidation of all 17 synthetic chromosomes into a single cell advances. This organism will be the first synthetic eukaryote and provides a highly plastic biological chassis built from the bottom-up ... ...

Abstract	The synthetic yeast, Sc2.0, is nearing completion as consolidation of all 17 synthetic chromosomes into a single cell advances. This organism will be the first synthetic eukaryote and provides a highly plastic biological chassis built from the bottom-up using principles of biological design. This synthetic approach to genome construction has allowed the genetic code to be re-wired in this background to liberate the amber stop codon as a dedicated triplet for encoding non-canonical amino acids. The availability of an expanded set of amino acid building blocks allows precise control of protein structure and function, providing new opportunities to develop protein-based therapeutics, materials and catalysts. In this article, we review the challenges facing genetic code expansion research in yeast and highlight how the development of Sc2.0 provides new and exciting opportunities to address existing limitations.
MeSH term(s)	Amino Acids/metabolism ; Codon, Terminator/genetics ; Genetic Code/genetics ; Proteins/genetics ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism
Chemical Substances	Amino Acids ; Codon, Terminator ; Proteins
Language	English
Publishing date	2022-02-10
Publishing country	England
Document type	Journal Article ; Review ; Research Support, Non-U.S. Gov't
ZDB-ID	1052045-4
ISSN	1879-0429 ; 0958-1669
ISSN (online)	1879-0429
ISSN	0958-1669
DOI	10.1016/j.copbio.2022.102691
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Article: New opportunities for genetic code expansion in synthetic yeast

Sanders, James / Hoffmann, Stefan A / Green, Anthony P / Cai, Yizhi

Current opinion in biotechnology. 2022 June, v. 75

2022

Abstract	The synthetic yeast, Sc2.0, is nearing completion as consolidation of all 17 synthetic chromosomes into a single cell advances. This organism will be the first synthetic eukaryote and provides a highly plastic biological chassis built from the bottom-up using principles of biological design. This synthetic approach to genome construction has allowed the genetic code to be re-wired in this background to liberate the amber stop codon as a dedicated triplet for encoding non-canonical amino acids. The availability of an expanded set of amino acid building blocks allows precise control of protein structure and function, providing new opportunities to develop protein-based therapeutics, materials and catalysts. In this article, we review the challenges facing genetic code expansion research in yeast and highlight how the development of Sc2.0 provides new and exciting opportunities to address existing limitations.
Keywords	amino acids ; biotechnology ; eukaryotic cells ; genetic code ; protein structure ; stop codon ; therapeutics ; yeasts
Language	English
Dates of publication	2022-06
Publishing place	Elsevier Ltd
Document type	Article
ZDB-ID	1052045-4
ISSN	1879-0429 ; 0958-1669
ISSN (online)	1879-0429
ISSN	0958-1669
DOI	10.1016/j.copbio.2022.102691
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Article: Perfluoroalkyl substances in water, sediment, and fish from a subtropical river of China: Environmental behaviors and potential risk

Wang, Siquan / Cai, Yizhi / Ma, Liya / Lin, Xiaoping / Li, Qin / Li, Yongyu / Wang, Xinhong

Chemosphere. 2022 Feb., v. 288

2022

Abstract: Perfluoroalkyl substances (PFAS) in water, sediment and fish were analyzed from a subtropical river, Jiulong River in the southeast of China, to character the sources, seasonal variations, bioconcentration and potential risk. PFAS in water, sediment, ... ...

Abstract	Perfluoroalkyl substances (PFAS) in water, sediment and fish were analyzed from a subtropical river, Jiulong River in the southeast of China, to character the sources, seasonal variations, bioconcentration and potential risk. PFAS in water, sediment, muscle and liver tissues of fish ranged from 2.5 to 410 ng L⁻¹, 0.24–1.9 ng g⁻¹ dw, 25–100 and 35–1100 ng g⁻¹ ww, respectively. Generally, perfluorohexanoic acid (PFHxA) was the dominant compound in water, while, perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) were the dominant compounds in sediment and fish tissues. High concentrations of PFAS in water were found near the machinery manufacturing and paper packaging plants in the north branch of Jiulong River. PFAS during the dry season were significantly (P < 0.01) higher than that during the normal season and wet season. The Kd of PFAS increased with the carbon chain length, and perfluoroalkyl sulfonic acids (PFSAs) exhibited higher Kd values than perfluoroalkyl carboxylic acids (PFCAs), indicating these long chain PFAS tended to be adsorbed by sediment. Long chain PFAS exhibited high bioconcentration factors (BCFs), while short and medium carbon chain PFAS had weak bioconcentration capacity. The hazard ratios (HR) suggested that frequent consumption of river fish may pose health risks to local population.
Keywords	carbon ; dry season ; fish ; liver ; muscles ; paper ; perfluorohexanoic acid ; perfluorooctane sulfonic acid ; perfluorooctanoic acid ; risk ; rivers ; sediments ; wet season ; China
Language	English
Dates of publication	2022-02
Publishing place	Elsevier Ltd
Document type	Article
ZDB-ID	120089-6
ISSN	1879-1298 ; 0045-6535 ; 0366-7111
ISSN (online)	1879-1298
ISSN	0045-6535 ; 0366-7111
DOI	10.1016/j.chemosphere.2021.132513
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Article ; Online: EMMA-CAD: Design Automation for Synthetic Mammalian Constructs.

Luo, Yisha / James, Joshua S / Jones, Sally / Martella, Andrea / Cai, Yizhi

ACS synthetic biology

2022 Volume 11, Issue 2, Page(s) 579–586

Abstract: Computational design tools are the cornerstone of synthetic biology and have underpinned its rapid development over the past two decades. As the field has matured, the scale of biological investigation has expanded dramatically, and researchers often ... ...

Abstract	Computational design tools are the cornerstone of synthetic biology and have underpinned its rapid development over the past two decades. As the field has matured, the scale of biological investigation has expanded dramatically, and researchers often must rely on computational tools to operate in the high-throughput investigational space. This is especially apparent in the modular design of DNA expression circuits, where complexity is accumulated rapidly. Alongside our automated pipeline for the high-throughput construction of Extensible Modular Mammalian Assembly (EMMA) expression vectors, we recognized the need for an integrated software solution for EMMA vector design. Here we present EMMA-CAD (https://emma.cailab.org), a powerful web-based computer-aided design tool for the rapid design of bespoke mammalian expression vectors. EMMA-CAD features a variety of functionalities, including a user-friendly design interface, automated connector selection underpinned by rigorous computer optimization algorithms, customization of part libraries, and personalized design spaces. Capable of translating vector assembly designs into human- and machine-readable protocols for vector construction, EMMA-CAD integrates seamlessly into our automated EMMA pipeline, hence completing an end-to-end design to production workflow.
MeSH term(s)	Algorithms ; Animals ; Automation ; DNA/genetics ; Humans ; Mammals/genetics ; Software ; Synthetic Biology/methods
Chemical Substances	DNA (9007-49-2)
Language	English
Publishing date	2022-01-20
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2161-5063
ISSN (online)	2161-5063
DOI	10.1021/acssynbio.1c00433
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Article ; Online: Automation and Expansion of EMMA Assembly for Fast-Tracking Mammalian System Engineering.

James, Joshua S / Jones, Sally / Martella, Andrea / Luo, Yisha / Fisher, David I / Cai, Yizhi

ACS synthetic biology

2022 Volume 11, Issue 2, Page(s) 587–595

Abstract: With applications from functional genomics to the production of therapeutic biologics, libraries of mammalian expression vectors have become a cornerstone of modern biological investigation and engineering. Multiple modular vector platforms facilitate ... ...

Abstract	With applications from functional genomics to the production of therapeutic biologics, libraries of mammalian expression vectors have become a cornerstone of modern biological investigation and engineering. Multiple modular vector platforms facilitate the rapid design and assembly of vectors. However, such systems approach a technical bottleneck when a library of bespoke vectors is required. Utilizing the flexibility and robustness of the Extensible Mammalian Modular Assembly (EMMA) toolkit, we present an automated workflow for the library-scale design, assembly, and verification of mammalian expression vectors. Vector design is simplified using our EMMA computer-aided design tool (EMMA-CAD), while the precision and speed of acoustic droplet ejection technology are applied in vector assembly. Our pipeline facilitates significant reductions in both reagent usage and researcher hands-on time compared with manual assembly, as shown by system Q-metrics. To demonstrate automated EMMA performance, we compiled a library of 48 distinct plasmid vectors encoding either CRISPR interference or activation modalities. Characterization of the workflow parameters shows that high assembly efficiency is maintained across vectors of various sizes and design complexities. Our system also performs strongly compared with manual assembly efficiency benchmarks. Alongside our automated pipeline, we present a straightforward strategy for integrating gRNA and Cas modules into the EMMA platform, enabling the design and manufacture of valuable genome editing resources.
MeSH term(s)	Animals ; Automation ; CRISPR-Cas Systems ; Gene Editing ; Gene Library ; Genetic Vectors/genetics ; Mammals/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics
Chemical Substances	RNA, Guide, CRISPR-Cas Systems
Language	English
Publishing date	2022-01-21
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2161-5063
ISSN (online)	2161-5063
DOI	10.1021/acssynbio.1c00330
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Article ; Online: Methods to Synthesize Large DNA Fragments for a Synthetic Yeast Genome.

Cai, Yizhi / Dai, Junbiao

Cold Spring Harbor protocols

2017 Volume 2017, Issue 3

Abstract: De novo DNA synthesis is one of the key enabling technologies for synthetic biology. Methods for large-scale DNA synthesis, in particular, have transformed many facets of life science research, supporting new discoveries in biology through the design of ... ...

Abstract	De novo DNA synthesis is one of the key enabling technologies for synthetic biology. Methods for large-scale DNA synthesis, in particular, have transformed many facets of life science research, supporting new discoveries in biology through the design of novel synthetic biological systems. This protocol describes in detail the methods currently being used to synthesize and assemble large pieces of DNA for the synthetic yeast genome project. The protocol includes instructions for building block synthesis as well as chunk assembly, each of which can be used as a stand-alone procedure to generate a synthetic DNA of interest.
MeSH term(s)	DNA, Fungal/biosynthesis ; DNA, Fungal/chemistry ; DNA, Fungal/genetics ; Genome, Fungal ; Molecular Biology/methods ; Molecular Weight ; Saccharomyces cerevisiae/genetics ; Synthetic Biology/methods
Chemical Substances	DNA, Fungal
Language	English
Publishing date	2017-03-01
Publishing country	United States
Document type	Journal Article
ISSN	1559-6095
ISSN (online)	1559-6095
DOI	10.1101/pdb.prot080978
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Article ; Online: Safety by design: Biosafety and biosecurity in the age of synthetic genomics.

Hoffmann, Stefan A / Diggans, James / Densmore, Douglas / Dai, Junbiao / Knight, Tom / Leproust, Emily / Boeke, Jef D / Wheeler, Nicole / Cai, Yizhi

iScience

2023 Volume 26, Issue 3, Page(s) 106165

Abstract: Technologies to profoundly engineer biology are becoming increasingly affordable, powerful, and accessible to a widening group of actors. While offering tremendous potential to fuel biological research and the bioeconomy, this development also increases ... ...

Abstract	Technologies to profoundly engineer biology are becoming increasingly affordable, powerful, and accessible to a widening group of actors. While offering tremendous potential to fuel biological research and the bioeconomy, this development also increases the risk of inadvertent or deliberate creation and dissemination of pathogens. Effective regulatory and technological frameworks need to be developed and deployed to manage these emerging biosafety and biosecurity risks. Here, we review digital and biological approaches of a range of technology readiness levels suited to address these challenges. Digital sequence screening technologies already are used to control access to synthetic DNA of concern. We examine the current state of the art of sequence screening, challenges and future directions, and environmental surveillance for the presence of engineered organisms. As biosafety layer on the organism level, we discuss genetic biocontainment systems that can be used to created host organisms with an intrinsic barrier against unchecked environmental proliferation.
Language	English
Publishing date	2023-02-10
Publishing country	United States
Document type	Journal Article ; Review
ISSN	2589-0042
ISSN (online)	2589-0042
DOI	10.1016/j.isci.2023.106165
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