Publications by authors named "Yizhi Cai"

63 Publications

SCRaMbLE: A Study of Its Robustness and Challenges through Enhancement of Hygromycin B Resistance in a Semi-Synthetic Yeast.

Bioengineering (Basel) 2021 Mar 23;8(3). Epub 2021 Mar 23.

Manchester Institute of Biotechnology (MIB), The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.

Recent advances in synthetic genomics launched the ambitious goal of generating the first synthetic designer eukaryote, based on the model organism (Sc2.0). Excitingly, the Sc2.0 project is now nearing its completion and SCRaMbLE, an accelerated evolution tool implemented by the integration of symmetrical loxP sites (loxPSym) downstream of almost every non-essential gene, is arguably the most applicable synthetic genome-wide alteration to date. The SCRaMbLE system offers the capability to perform rapid genome diversification, providing huge potential for targeted strain improvement. Here we describe how SCRaMbLE can evolve a semi-synthetic yeast strain housing the synthetic chromosome II (synII) to generate hygromycin B resistant genotypes. Exploiting long-read nanopore sequencing, we show that all structural variations are due to recombination between loxP sites, with no off-target effects. We also highlight a phenomenon imposed on SCRaMbLE termed "essential raft", where a fragment flanked by a pair of loxPSym sites can move within the genome but cannot be removed due to essentiality restrictions. Despite this, SCRaMbLE was able to explore the genomic space and produce alternative structural compositions that resulted in an increased hygromycin B resistance in the synII strain. We show that among the rearrangements generated via SCRaMbLE, deletions of YBR219C and YBR220C contribute to hygromycin B resistance phenotypes. However, the hygromycin B resistance provided by SCRaMbLEd genomes showed significant improvement when compared to corresponding single deletions, demonstrating the importance of the complex structural variations generated by SCRaMbLE to improve hygromycin B resistance. We anticipate that SCRaMbLE and its successors will be an invaluable tool to predict and evaluate the emergence of antibiotic resistance in yeast.
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http://dx.doi.org/10.3390/bioengineering8030042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004914PMC
March 2021

Compacting a synthetic yeast chromosome arm.

Genome Biol 2021 Jan 4;22(1). Epub 2021 Jan 4.

CAS Key Laboratory of Quantitative Engineering Biology, Guangdong Provincial Key Laboratory of Synthetic Genomics and Shenzhen Key Laboratory of Synthetic Genomics, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.

Background: Redundancy is a common feature of genomes, presumably to ensure robust growth under different and changing conditions. Genome compaction, removing sequences nonessential for given conditions, provides a novel way to understand the core principles of life. The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system is a unique feature implanted in the synthetic yeast genome (Sc2.0), which is proposed as an effective tool for genome minimization. As the Sc2.0 project is nearing its completion, we have begun to explore the application of the SCRaMbLE system in genome compaction.

Results: We develop a method termed SCRaMbLE-based genome compaction (SGC) and demonstrate that a synthetic chromosome arm (synXIIL) can be efficiently reduced. The pre-introduced episomal essential gene array significantly enhances the compacting ability of SGC, not only by enabling the deletion of nonessential genes located in essential gene containing loxPsym units but also by allowing more chromosomal sequences to be removed in a single SGC process. Further compaction is achieved through iterative SGC, revealing that at least 39 out of 65 nonessential genes in synXIIL can be removed collectively without affecting cell viability at 30 °C in rich medium. Approximately 40% of the synthetic sequence, encoding 28 genes, is found to be dispensable for cell growth at 30 °C in rich medium and several genes whose functions are needed under specified conditions are identified.

Conclusions: We develop iterative SGC with the aid of eArray as a generic yet effective tool to compact the synthetic yeast genome.
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http://dx.doi.org/10.1186/s13059-020-02232-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7780613PMC
January 2021

: A fast and efficient CRISPR/Cas9 method for fission yeast.

Wellcome Open Res 2020 24;5:274. Epub 2020 Nov 24.

Wellcome Centre for Cell Biology and Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, Mayfield Road, Edinburgh, EH9 3BF, UK.

The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast  rely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a Cas9-expressing plasmid. In addition, Cas9 endonuclease has been reported to be toxic to fission yeast when constitutively overexpressed from the strong  promoter. To overcome these problems we have developed an improved system,  , that uses a synthesised Cas9 sequence codon-optimised for  expressed from the medium strength  promoter. The   system exhibits a flexible modular design where the sgRNA is fused to the 3' end of the self-cleaving hepatitis delta virus (HDV) ribozyme, allowing expression of the sgRNA cassette to be driven by RNA polymerase III from a tRNA gene sequence. Lastly, the inclusion of sites for the  I type IIS restriction enzyme flanking a GFP placeholder enables one-step Golden Gate mediated replacement of GFP with synthesized sgRNAs for expression. The   system allowed a 100% mutagenesis efficiency to be achieved when generating targeted point mutants in the   or   genes by transformation of cells from asynchronous cultures.   also permitted insertion, tagging and deletion events to be obtained with minimal effort. Simultaneous editing of two independent non-homologous loci was also readily achieved. Importantly the   system displayed reduced toxicity compared to currently available   editing systems. Thus,  provides an effective and user-friendly CRISPR/Cas9 procedure that significantly improves the genome editing toolbox for fission yeast.
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http://dx.doi.org/10.12688/wellcomeopenres.16405.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721064PMC
November 2020

SCRaMbLE-in: A Fast and Efficient Method to Diversify and Improve the Yields of Heterologous Pathways in Synthetic Yeast.

Methods Mol Biol 2020 ;2205:305-327

Manchester Institute of Biotechnology (MIB), School of Chemistry, The University of Manchester, Manchester, UK.

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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http://dx.doi.org/10.1007/978-1-0716-0908-8_17DOI Listing
March 2021

Sc3.0: revamping and minimizing the yeast genome.

Genome Biol 2020 08 13;21(1):205. Epub 2020 Aug 13.

CAS Key Laboratory of Quantitative Engineering Biology, Guangdong Provincial Key Laboratory of Synthetic Genomics and Shenzhen Key Laboratory of Synthetic Genomics. Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.

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http://dx.doi.org/10.1186/s13059-020-02130-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425127PMC
August 2020

Leveraging the Hermes Transposon to Accelerate the Development of Nonconventional Yeast-based Microbial Cell Factories.

ACS Synth Biol 2020 07 16;9(7):1736-1752. Epub 2020 Jun 16.

Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa, United States.

We broadened the usage of DNA transposon technology by demonstrating its capacity for the rapid creation of expression libraries for long biochemical pathways, which is beyond the classical application of building genome-scale knockout libraries in yeasts. This strategy efficiently leverages the readily available fine-tuning impact provided by the diverse transcriptional environment surrounding each random integration locus. We benchmark the transposon-mediated integration against the nonhomologous end joining-mediated strategy. The latter strategy was demonstrated for achieving pathway random integration in other yeasts but is associated with a high false-positive rate in the absence of a high-throughput screening method. Our key innovation of a nonreplicable circular DNA platform increased the possibility of identifying top-producing variants to 97%. Compared to the classical DNA transposition protocol, the design of a nonreplicable circular DNA skipped the step of counter-selection for plasmid removal and thus not only reduced the time required for the step of library creation from 10 to 5 d but also efficiently removed the "transposition escapers", which undesirably represented almost 80% of the entire population as false positives. Using one endogenous product (, shikimate) and one heterologous product (, ()-norcoclaurine) as examples, we presented a streamlined procedure to rapidly identify high-producing variants with titers significantly higher than the reported data in the literature. We selected , a representative nonconventional yeast, as a demo, but the strategy can be generalized to other nonconventional yeasts. This new exploration of transposon technology, therefore, adds a highly versatile tool to accelerate the development of novel species as microbial cell factories for producing value-added chemicals.
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http://dx.doi.org/10.1021/acssynbio.0c00123DOI Listing
July 2020

Probing eukaryotic genome functions with synthetic chromosomes.

Exp Cell Res 2020 05 9;390(1):111936. Epub 2020 Mar 9.

Guangdong Provincial Key Laboratory of Synthetic Genomics, Shenzhen Key Laboratory of Synthetic Genomics, Center for Synthetic Genomics, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China; College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, 518055, China. Electronic address:

The ability to redesign and reconstruct a cell at whole-genome level provides new platforms for biological study. The international synthetic yeast genome project-Sc2.0, designed by interrogating knowledge amassed by the yeast community to date, exemplifies how a classical synthetic biology "design-build-test-learn" engineering cycle can effectively test hypotheses about various genome fundamentals. The genome reshuffling SCRaMbLE system implemented in synthetic yeast strains also provides unprecedented diversified resources for genotype-phenotype study and yeast metabolic engineering. Further development of genome synthesis technology will shed new lights on complex biological processes in higher eukaryotes.
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http://dx.doi.org/10.1016/j.yexcr.2020.111936DOI Listing
May 2020

Concentration, distribution and sources of perfluoroalkyl substances and organochlorine pesticides in surface sediments of the northern Bering Sea, Chukchi Sea and adjacent Arctic Ocean.

Chemosphere 2019 Nov 1;235:959-968. Epub 2019 Jul 1.

National Institute of Oceanography, Clifton, Block-1, Karachi, 75600, Pakistan.

Perfluoroalkyl substances (PFAS) and organochlorine pesticides (OCPs) in surface sediments were investigated from the Bering Sea, the Chukchi Sea and adjacent Arctic Ocean in 2010. Total concentrations (dry weight) of ΣPFAS in surface sediments (0.85 ± 0.22 ng g) of the Bering Sea were lower than that in the Chukchi Sea and adjacent Arctic Ocean (1.27 ± 0.53 ng g). Perfluoro-butanoic acid (PFBS) and perfluoro-octanoic acid (PFOA) were the dominant PFAS in these areas. The concentrations of ΣOCPs in the sediment of the Bering Sea (13.00 ± 6.17 ng g) was slightly higher than that in the Chukchi and Arctic Ocean (12.05 ± 2.27 ng g). The most abundant OCPs were hexachlorocyclohexane isomers (HCHs) and dichlorodiphenyltrichloroethane (DDT) and its metabolites. The composition patterns of HCHs and DDTs indicated that they were mainly derived from the early residues via river runoff. Increasing trends of PFAS, HCHs and DDTs in surface sediments from the Bering Sea to the Arctic Ocean were found, indicating oceanic transport. In summary, the concentrations of OCPs were orders of magnitude greater than the observed PFAS concentrations, and the concentrations of PFAS and OCPs in surface sediments from the Bering Sea to the Chukchi Sea and adjacent Arctic Ocean are at the low to moderate levels by comparing with other coastal and marine sediments worldwide.
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http://dx.doi.org/10.1016/j.chemosphere.2019.06.219DOI Listing
November 2019

Improving Chromosome Synthesis with a Semiquantitative Phenotypic Assay and Refined Assembly Strategy.

ACS Synth Biol 2019 10 20;8(10):2203-2211. Epub 2019 Sep 20.

Key Laboratory of Industrial Biocatalysis (Ministry of Education) and Center for Synthetic and Systems Biology, School of Life Sciences , Tsinghua University , Beijing 100084 , China.

Recent advances in DNA synthesis technology have made it possible to rewrite the entire genome of an organism. The major hurdles in this process are efficiently identifying and fixing the defect-inducing sequences (or "bugs") during rewriting. Here, we describe a high-throughput, semiquantitative phenotype assay for evaluating the fitness of synthetic yeast and identifying potential bugs. Growth curves were measured under a carefully chosen set of testing conditions. Statistical analysis revealed strains with subtle defects relative to the wild type, which were targeted for debugging. The effectiveness of the assay was demonstrated by phenotypic profiling of all intermediate synthetic strains of the synthetic yeast chromosome XII. Subsequently, the assay was applied during the process of constructing another synthetic chromosome. Furthermore, we designed an efficient chromosome assembly strategy that integrates iterative megachunk construction with CRISPR/Cas9-mediated assembly of synthetic segments. Together, the semiquantitative assay and refined assembly strategy could greatly facilitate synthetic genomics projects by improving efficiency during both debugging and construction.
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http://dx.doi.org/10.1021/acssynbio.8b00505DOI Listing
October 2019

Author Correction: Building a global alliance of biofoundries.

Nat Commun 2019 Jul 11;10(1):3132. Epub 2019 Jul 11.

UK Centre for Mammalian Synthetic Biology SynthSys, School of Biological Sciences, University of Edinburgh, Edinburgh, EH93FF, UK.

The original version of this Comment contained errors in the legend of Figure 2, in which the locations of the fifteenth and sixteenth GBA members were incorrectly given as '(15) Australian Genome Foundry, Macquarie University; (16) Australian Foundry for Advanced Biomanufacturing, University of Queensland.'. The correct version replaces this with '(15) Australian Foundry for Advanced Biomanufacturing (AusFAB), University of Queensland and (16) Australian Genome Foundry, Macquarie University'. This has been corrected in both the PDF and HTML versions of the Comment.
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http://dx.doi.org/10.1038/s41467-019-10862-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624256PMC
July 2019

Evaluation of marine sediment contamination by polycyclic aromatic hydrocarbons along the Karachi coast, Pakistan, 11 years after the Tasman Spirit oil spill.

Chemosphere 2019 Oct 27;233:652-659. Epub 2019 May 27.

National Institute of Oceanography, Clifton, Block 1, Karachi-75600, Pakistan.

On July 27, 2003, a spill of approximately 31,000 tons of Iranian light crude oil affected the coast of Karachi, Pakistan. Approximately 11 years after the spill, we analyzed polycyclic aromatic hydrocarbons (PAHs) and their alkylated homologues (alkyl-PAHs) as the indicators to evaluate the residual effect of oil spill to the sediment along the Karachi coast. The total concentrations (dry weight) of parent PAHs and alkyl-PAHs ranged from 121.9 to 735.4 and 42.3-1149.9 ng/g, respectively. The estuary and harbor were the two regions with the highest levels of PAHs in the sediment. Conversely, sedimentary PAHs in the oil spill areas and remote coastal areas showed significantly lower levels. Although the results of the source identification indicated the up to 75.2% of the contribution from petroleum and its derivatives, this could only reflect the direct impact of the Karachi city on the presence of PAHs in the coastal sedimentary environment and did not indicated that the oil spill continues to stay 11 years later. Compared with 11 years ago, the sharply reduced PAH content, great changed composition, and the degradation driven trend of diagnostic ratios all indicated a sharp decrease in the influence of PAHs caused by the oil spill. Finally, the ecological risk caused by the PAH residual in the marine sedimentary ecosystem had disappeared along the Karachi coasts, Pakistan.
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http://dx.doi.org/10.1016/j.chemosphere.2019.05.217DOI Listing
October 2019

Multiplex Genome Engineering for Optimizing Bioproduction in Saccharomyces cerevisiae.

Biochemistry 2019 03 8;58(11):1492-1500. Epub 2019 Mar 8.

Manchester Institute of Biotechnology (MIB), School of Chemistry , The University of Manchester , 131 Princess Street , Manchester M1 7DN , United Kingdom.

The field of synthetic biology is already beginning to realize its potential, with a wealth of examples showcasing the successful genetic engineering of microorganisms for the production of valuable compounds. The chassis Saccharomyces cerevisiae has been engineered to function as a microfactory for producing many of these economically and medically relevant compounds. However, strain construction and optimization to produce industrially relevant titers necessitate a wealth of underpinning biological knowledge alongside large investments of capital and time. Over the past decade, advances in DNA synthesis and editing tools have enabled multiplex genome engineering of yeast, permitting access to more complex modifications that could not have been easily generated in the past. These genome engineering efforts often result in large populations of strains with genetic diversity that can pose a significant challenge to screen individually via traditional methods such as mass spectrometry. The large number of samples generated would necessitate screening methods capable of analyzing all of the strains generated to maximize the explored genetic space. In this Perspective, we focus on recent innovations in multiplex genome engineering of S. cerevisiae, together with biosensors and high-throughput screening tools, such as droplet microfluidics, and their applications in accelerating chassis optimization.
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http://dx.doi.org/10.1021/acs.biochem.8b01086DOI Listing
March 2019

EMMA assembly explained: A step-by-step guide to assemble synthetic mammalian vectors.

Methods Enzymol 2019 13;617:463-493. Epub 2019 Feb 13.

Manchester Institute of Biotechnology (MIB), School of Chemistry, The University of Manchester, Manchester, United Kingdom. Electronic address:

Construction of expression vectors is imperative for many areas of biological research and the biotechnology industry. Modular cloning systems for expression vector construction offer a labor- and cost-effective alternative to overcome drawbacks associated with traditional cloning methods. We developed an Extensible Mammalian Modular Assembly toolkit (EMMA) as an efficient and versatile tool to facilitate the construction of functionally diverse mammalian expression vectors from a standardized library of DNA parts. This system supports both hierarchical and combinatorial assembly, and is adaptable for automation. In this chapter, we describe the protocols to construct libraries of DNA parts and assemble these parts into mammalian expression vectors using EMMA.
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http://dx.doi.org/10.1016/bs.mie.2018.12.017DOI Listing
November 2019

Whole genome engineering by synthesis.

Sci China Life Sci 2018 12 20;61(12):1515-1527. Epub 2018 Nov 20.

Shenzhen Key Laboratory of Synthetic Genomics and Center for Synthetic Genomics, Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.

Whole genome engineering is now feasible with the aid of genome editing and synthesis tools. Synthesizing a genome from scratch allows modifications of the genomic structure and function to an extent that was hitherto not possible, which will finally lead to new insights into the basic principles of life and enable valuable applications. With several recent genome synthesis projects as examples, the technical details to synthesize a genome and applications of synthetic genome are addressed in this perspective. A series of ongoing or future synthetic genomics projects, including the different genomes to be synthesized in GP-write, synthetic minimal genome, massively recoded genome, chimeric genome and synthetic genome with expanded genetic alphabet, are also discussed here with a special focus on theoretical and technical impediments in the design and synthesis process. Synthetic genomics will become a commonplace to engineer pathways and genomes according to arbitrary sets of design principles with the development of high-efficient, low-cost genome synthesis and assembly technologies.
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http://dx.doi.org/10.1007/s11427-018-9403-yDOI Listing
December 2018

Hydrogen Peroxide-Based Fluorometric Assay for Real-Time Monitoring of SAM-Dependent Methyltransferases.

Front Bioeng Biotechnol 2018 18;6:146. Epub 2018 Oct 18.

Institute of Molecular Plant Sciences, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom.

Methylated chemicals are widely used as key intermediates for the syntheses of pharmaceuticals, fragrances, flavors, biofuels and plastics. In nature, the process of methylation is commonly undertaken by a super-family of S-adenosyl methionine-dependent enzymes known as methyltransferases. Herein, we describe a novel high throughput enzyme-coupled assay for determining methyltransferase activites. Adenosylhomocysteine nucleosidase, xanthine oxidase, and horseradish peroxidase enzymes were shown to function in tandem to generate a fluorescence signal in the presence of S-adenosyl-L-homocysteine and Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine). Since S-adenosyl-L-homocysteine is a key by-product of reactions catalyzed by S-adenosyl methionine-dependent methyltransferases, the coupling enzymes were used to assess the activities of RI methyltransferase and a salicylic acid methyltransferase from in the presence of S-adenosyl methionine. For the RI methyltransferase, the assay was sensitive enough to allow the monitoring of DNA methylation in the nanomolar range. In the case of the salicylic acid methyltransferase, detectable activity was observed for several substrates including salicylic acid, benzoic acid, 3-hydroxybenzoic acid, and vanillic acid. Additionally, the synthesis of the relatively expensive and unstable cosubstrate, S-adenosyl methionine, catalyzed by methionine adenosyltransferase could be incorporated within the assay. Overall, the assay offers an excellent level of sensitivity that permits continuous and reliable monitoring of methyltransferase activities. We anticipate this assay will serve as a useful bioanalytical tool for the rapid screening of S-adenosyl methionine-dependent methyltransferase activities.
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http://dx.doi.org/10.3389/fbioe.2018.00146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200863PMC
October 2018

YeastFab: High-Throughput Genetic Parts Construction, Measurement, and Pathway Engineering in Yeast.

Methods Enzymol 2018 6;608:277-306. Epub 2018 Jul 6.

Manchester Institute of Biotechnology (MIB), School of Chemistry, The University of Manchester, Manchester, United Kingdom. Electronic address:

For many years, researchers have devised elegant techniques to assemble genetic parts into larger constructs. Recently, increasing needs for complex DNA constructs has driven countless attempts to optimize DNA assembly methods for improved efficiency, fidelity, and modularity. These efforts have resulted in simple, robust, standardized, and fast protocols that enable the implementation of high-throughput DNA assembly projects for the fabrication of large synthetic genetic constructs. Recently our groups have developed the YeastFab assembly, a highly efficient method for the design and construction of DNA-building blocks based on the native elements from Saccharomyces cerevisiae. Furthermore, these standardized DNA parts can be readily characterized and assembled into transcriptional units and pathways. In this chapter, we describe the protocols to assemble pathways from characterized standardized yeast parts using YeastFab.
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http://dx.doi.org/10.1016/bs.mie.2018.05.003DOI Listing
June 2019

Artificial Protein Scaffold System (AProSS): An efficient method to optimize exogenous metabolic pathways in Saccharomyces cerevisiae.

Metab Eng 2018 09 23;49:13-20. Epub 2018 Jul 23.

Key Laboratory of Industrial Biocatalysis (Ministry of Education) and Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China; Shenzhen Key Laboratory of Synthetic Genomics and Center for Synthetic Genomics, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China. Electronic address:

Scaffold proteins influence cellular signaling by orchestrating multiple enzymes, receptors or ion channels, and could be tailored to enhance the efficiency of biochemical reactions by positioning related enzymes physically together. However, the number of applicable domains remains small, and the construction of scaffold proteins with optimal domain ratio could be tedious and time-consuming. In this study, we outlined a modular design to quickly assemble scaffold proteins using protein interaction domains, which have been constructed into a standardized vector. We generated multiple protein interaction domains and ligands for making artificial scaffold proteins. At the same time, we developed a robust Golden-Gate-based molecular toolkit for the construction of artificial scaffold proteins, allowing a variance of domain types, number, and positions. The synthesized domain-ligand interaction was verified by yeast two-hybrid and split-GFP assays. Using synthetic scaffolds, we demonstrated an increase in the yield of two target products by 29% and 63% respectively. Moreover, we demonstrated that the synthetic scaffold could be applied to rewire the metabolic flux. Our system could be a useful tool for metabolic engineering and beyond.
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http://dx.doi.org/10.1016/j.ymben.2018.07.006DOI Listing
September 2018

Rapid pathway prototyping and engineering using in vitro and in vivo synthetic genome SCRaMbLE-in methods.

Nat Commun 2018 05 22;9(1):1936. Epub 2018 May 22.

Center for Synthetic Genomics, Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, 518055, Shenzhen, China.

Exogenous pathway optimization and chassis engineering are two crucial methods for heterologous pathway expression. The two methods are normally carried out step-wise and in a trial-and-error manner. Here we report a recombinase-based combinatorial method (termed "SCRaMbLE-in") to tackle both challenges simultaneously. SCRaMbLE-in includes an in vitro recombinase toolkit to rapidly prototype and diversify gene expression at the pathway level and an in vivo genome reshuffling system to integrate assembled pathways into the synthetic yeast genome while combinatorially causing massive genome rearrangements in the host chassis. A set of loxP mutant pairs was identified to maximize the efficiency of the in vitro diversification. Exemplar pathways of β-carotene and violacein were successfully assembled, diversified, and integrated using this SCRaMbLE-in method. High-throughput sequencing was performed on selected engineered strains to reveal the resulting genotype-to-phenotype relationships. The SCRaMbLE-in method proves to be a rapid, efficient, and universal method to fast track the cycle of engineering biology.
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http://dx.doi.org/10.1038/s41467-018-04254-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964202PMC
May 2018

Identifying and characterizing SCRaMbLEd synthetic yeast using ReSCuES.

Nat Commun 2018 05 22;9(1):1930. Epub 2018 May 22.

Center for Synthetic Biology Engineering Research, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, China.

SCRaMbLE is a novel system implemented in the synthetic yeast genome, enabling massive chromosome rearrangements to produce strains with a large genotypic diversity upon induction. Here we describe a reporter of SCRaMbLEd cells using efficient selection, termed ReSCuES, based on a loxP-mediated switch of two auxotrophic markers. We show that all randomly isolated clones contained rearrangements within the synthetic chromosome, demonstrating high efficiency of selection. Using ReSCuES, we illustrate the ability of SCRaMbLE to generate strains with increased tolerance to several stress factors, such as ethanol, heat and acetic acid. Furthermore, by analyzing the tolerant strains, we are able to identify ACE2, a transcription factor required for septum destruction after cytokinesis, as a negative regulator of ethanol tolerance. Collectively, this work not only establishes a generic platform to rapidly identify strains of interest by SCRaMbLE, but also provides methods to dissect the underlying mechanisms of resistance.
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http://dx.doi.org/10.1038/s41467-017-00806-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964233PMC
May 2018

Temporal trends and transport of perfluoroalkyl substances (PFASs) in a subtropical estuary: Jiulong River Estuary, Fujian, China.

Sci Total Environ 2018 Oct 19;639:263-270. Epub 2018 May 19.

State Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, POPs Research Center, Tsinghua University, Beijing 100084, China.

The seasonal variations and spatial distributions of fifteen perfluoroalkyl substances (PFASs) were investigated in the water of the subtropical Jiulong River Estuary (JRE) in Fujian, China. The concentrations and composition profiles of PFASs showed significant seasonal variations. ∑PFASs concentrations ranged from 4.8 to 37.6 ng L, 12.2 to 110 ng L and 3.3 to 43.0 ng L in the dry, medium and wet seasons, respectively. Perfluorooctane sulfonate (PFOS) was found to be the most abundant PFAS in the dry season, with a composition of 33% ± 5%, Perfluorohexanoic acid PFHxA (47% ± 13%) and perfluoropentanoic acid (PFPeA) (52% ± 15%) were the dominant compounds in the medium and wet seasons, respectively. Seasonal and spatial distributions of ∑PFASs were different in the upstream and downstream sections. High concentration of PFHxA occurred in the medium season, and showed a linear decreasing trend from upstream to downstream. The majority of other PFASs did not show clear seasonal variation. Composition profiles indicated that the JRE was mainly contaminated by short-chain perfluoroalkyl carboxylic acids (PFCAs), shipbuilding industry, multiple wastewater and river runoff were identified as major potential sources.
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http://dx.doi.org/10.1016/j.scitotenv.2018.05.042DOI Listing
October 2018

Synthetic genomics: a new venture to dissect genome fundamentals and engineer new functions.

Curr Opin Chem Biol 2018 10 9;46:56-62. Epub 2018 May 9.

Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, M1 7DN Manchester, UK; Centre for Synthetic Genomics, Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China. Electronic address:

Since the first synthetic gene was synthesized in 1970s, the efficiency and the capacity of made-to-order DNA sequence synthesis has increased by several orders of magnitude. Advances in DNA synthesis and assembly over the past years has resulted in a steep drop in price for custom made DNA. Similar effects were observed in DNA sequencing technologies which underpin DNA-reading projects. Today, synthetic DNA sequences with more than 10000bps and turn-around times of a few weeks are commercially available. This enables researchers to perform large-scale projects to write synthetic chromosomes and characterize their functionalities in vivo. Synthetic genomics opens up new paradigms to study the genome fundamentals and engineer novel biological functions.
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http://dx.doi.org/10.1016/j.cbpa.2018.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6351456PMC
October 2018

Corrigendum: Methods to Synthesize Large DNA Fragments for a Synthetic Yeast Genome.

Cold Spring Harb Protoc 2017 12 1;2017(12). Epub 2017 Dec 1.

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http://dx.doi.org/10.1101/pdb.corr103473DOI Listing
December 2017

Design and chemical synthesis of eukaryotic chromosomes.

Chem Soc Rev 2017 Nov;46(23):7191-7207

Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, P. R. China.

Following the discovery of the DNA double helix structure and the advancement of genome sequencing, we have entered a promising stage with regard to genome writing. Recently, a milestone breakthrough was achieved in the chemical synthesis of designer yeast chromosomes. Here, we review the systematic approaches to the de novo synthesis of designer eukaryotic chromosomes, with an emphasis on technologies and methodologies that enable design, building, testing and debugging. The achievement of chemically synthesized genomes with customized genetic features offers an opportunity to rebuild genome organization, remold biological functions and promote life evolution, which will be of great benefit for application in medicine and industrial manufacturing.
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http://dx.doi.org/10.1039/c7cs00208dDOI Listing
November 2017

Leaf LIMS: A Flexible Laboratory Information Management System with a Synthetic Biology Focus.

ACS Synth Biol 2017 12 13;6(12):2273-2280. Epub 2017 Sep 13.

GeneMill, University of Liverpool , Liverpool L69 7ZB, U.K.

This paper presents Leaf LIMS, a flexible laboratory information management system (LIMS) designed to address the complexity of synthetic biology workflows. At the project's inception there was a lack of a LIMS designed specifically to address synthetic biology processes, with most systems focused on either next generation sequencing or biobanks and clinical sample handling. Leaf LIMS implements integrated project, item, and laboratory stock tracking, offering complete sample and construct genealogy, materials and lot tracking, and modular assay data capture. Hence, it enables highly configurable task-based workflows and supports data capture from project inception to completion. As such, in addition to it supporting synthetic biology it is ideal for many laboratory environments with multiple projects and users. The system is deployed as a web application through Docker and is provided under a permissive MIT license. It is freely available for download at https://leaflims.github.io .
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http://dx.doi.org/10.1021/acssynbio.7b00212DOI Listing
December 2017

Using Purified Tyrosine Site-Specific Recombinases In Vitro to Rapidly Construct and Diversify Metabolic Pathways.

Methods Mol Biol 2017 ;1642:285-302

School of Biological Sciences, The University of Edinburgh, The King's Buildings, Edinburgh, EH9 3BF, UK.

The site-specific recombinase Cre was previously reported to have in vitro activity. Here, we describe the method of purifying two new tyrosine site-specific recombinases VCre and Dre along with Cre by nickel affinity chromatography. We proved the in vitro function of the VCre and Dre on their respective conditional recombination sites. We also developed a methodology to one-step construct and optimize the productivity of a biosynthetic pathway through the combinatorial integration of promoters into a plasmid-encoded pathway by simply incubating a DNA mixture with recombinase system at 37 °C in vitro.
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http://dx.doi.org/10.1007/978-1-4939-7169-5_18DOI Listing
April 2018

Orthogonal Ribosome Biofirewall.

ACS Synth Biol 2017 11 28;6(11):2108-2117. Epub 2017 Aug 28.

Key Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology, Tianjin University , Tianjin 300072, PR China.

Biocontainment systems are crucial for preventing genetically modified organisms from escaping into natural ecosystems. Here, we describe the orthogonal ribosome biofirewall, which consists of an activation circuit and a degradation circuit. The activation circuit is a genetic AND gate based on activation of the encrypted pathway by the orthogonal ribosome in response to specific environmental signals. The degradation circuit is a genetic NOT gate with an output of I-SceI homing endonuclease, which conditionally degrades the orthogonal ribosome genes. We demonstrate that the activation circuit can be flexibly incorporated into genetic circuits and metabolic pathways for encryption. The plasmid-based encryption of the deoxychromoviridans pathway and the genome-based encryption of lacZ are tightly regulated and can decrease the expression to 7.3% and 7.8%, respectively. We validated the ability of the degradation circuit to decrease the expression levels of the target plasmids and the orthogonal rRNA (O-rRNA) plasmids to 0.8% in lab medium and 0.76% in nonsterile soil medium, respectively. Our orthogonal ribosome biofirewall is a versatile platform that can be useful in biosafety research and in the biotechnology industry.
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http://dx.doi.org/10.1021/acssynbio.7b00148DOI Listing
November 2017

EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors.

ACS Synth Biol 2017 07 24;6(7):1380-1392. Epub 2017 Apr 24.

School of Biological Sciences, The University of Edinburgh , The King's Buildings, Edinburgh EH9 3BF, U.K.

Mammalian plasmid expression vectors are critical reagents underpinning many facets of research across biology, biomedical research, and the biotechnology industry. Traditional cloning methods often require laborious manual design and assembly of plasmids using tailored sequential cloning steps. This process can be protracted, complicated, expensive, and error-prone. New tools and strategies that facilitate the efficient design and production of bespoke vectors would help relieve a current bottleneck for researchers. To address this, we have developed an extensible mammalian modular assembly kit (EMMA). This enables rapid and efficient modular assembly of mammalian expression vectors in a one-tube, one-step golden-gate cloning reaction, using a standardized library of compatible genetic parts. The high modularity, flexibility, and extensibility of EMMA provide a simple method for the production of functionally diverse mammalian expression vectors. We demonstrate the value of this toolkit by constructing and validating a range of representative vectors, such as transient and stable expression vectors (transposon based vectors), targeting vectors, inducible systems, polycistronic expression cassettes, fusion proteins, and fluorescent reporters. The method also supports simple assembly combinatorial libraries and hierarchical assembly for production of larger multigenetic cargos. In summary, EMMA is compatible with automated production, and novel genetic parts can be easily incorporated, providing new opportunities for mammalian synthetic biology.
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http://dx.doi.org/10.1021/acssynbio.7b00016DOI Listing
July 2017

[Changes in ultrastructure and bone morphogenetic protein expression in reconstructed mandibular condylar cartilage under continuous mandibular advancement in adult rats].

Hua Xi Kou Qiang Yi Xue Za Zhi 2016 Dec;34(6):632-638

Dept. of Stomatology, Jinan Central Hospital Affiliated to Shandong University, Jinan 250013, China.

Objective: This study investigated the reconstructed mandibular condylar cartilage and the ultrastructural variations in mandibular condylar cartilage in adult rats as a result of mandibular advancement.

Methods: Thirty 9-week-old male Sprague-Dawley rats were randomly divided into experimental and control groups. Rats in the experimental group were subjected to mandibular advancement. Rats were sacrificed on days 3, 7, 14, 21, 30. Sections were cut from condyles, and bone morphogenetic protein-2 (BMP-2) expression in condylar cartilage was examined through immunohistochemical analysis. Condylar cartilage samples were harvested, and ultrastructural changes in these samples were observed under Micro-CT and transmission electron microscope.

Results: Compared with the control group, the experimental group obviously displayed cartilage hyperplasia in the middle and rear of the condyle. Moreover, the number of BMP-2-positive cells in condylar cartilage and the gray value gradually increased in the experimental group on day 7 of the intervention. Ultrastructural changes, such as karyopyknosis, reduced microfilaments around the nucleus, reduction in size or even disappearance of lipid droplets, swelling of endoplasmic reticulum compartments, broadened and increased extracellular matrix, were observed in the condylar hypertrophic chondrocytes. Micro-CT revealed that the trabecula and the newly formed bone gradually thickened.

Conclusions: Hypertrophic remodeling of the condylar cartilage and high BMP-2 expression are observed in adult rats as a result of continuous mandibular advancement.
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http://dx.doi.org/10.7518/hxkq.2016.06.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7030868PMC
December 2016