Article
Biochemical Research Methods
Gerrich Behrendt, Jonas Frohwitter, Maria Vlachonikolou, Steffen Klamt, Katja Bettenbrock
Summary: Zymo-Parts is a modular toolbox based on Golden-Gate cloning that enables efficient construction of genetically modified strains. It offers a collection of promoters, terminators, ribosomal binding sites, and reporter genes for achieving desired levels of gene expression. The GoldenBraid-based system allows for the assembly of operons with multiple genes. The utility of Zymo-Parts for metabolic engineering applications is demonstrated by overexpressing a heterologous lactate dehydrogenase gene in Z. mobilis.
ACS SYNTHETIC BIOLOGY
(2022)
Article
Biotechnology & Applied Microbiology
Ming Luo, Liqiong Xie, Soma Chakraborty, Aihua Wang, Oadi Matny, Michelle Jugovich, James A. Kolmer, Terese Richardson, Dhara Bhatt, Mohammad Hoque, Mehran Patpour, Chris Sorensen, Diana Ortiz, Peter Dodds, Burkhard Steuernagel, Brande B. H. Wulff, Narayana M. Upadhyaya, Rohit Mago, Sambasivam Periyannan, Evans Lagudah, Roger Freedman, T. Lynne Reuber, Brian J. Steffenson, Michael Ayliffe
Summary: Through the introduction of a transgene cassette containing five resistance genes into bread wheat, researchers have successfully generated wheat lines with highly resistant to aggressive and highly virulent Pgt isolates from around the world. This approach simplifies breeding efforts and enhances resistance levels, although strategic deployment of gene stacks may be needed to maintain effectiveness against new Pgt isolates.
NATURE BIOTECHNOLOGY
(2021)
Article
Plant Sciences
Hagen Stellmach, Robert Hose, Antonia Raede, Sylvestre Marillonnet, Bettina Hause
Summary: Fluorescence-based approaches with fusion proteins enable efficient localization of proteins in plant cells and provide insights into their physiological functions. We have developed a set of vectors containing markers for basic cell organelles using Golden Gate cloning, allowing for easy insertion of the gene of interest and fusion with a fluorescent protein. These vectors can be used for localization studies and transient transformation experiments.
Review
Biochemical Research Methods
Jasmine E. Bird, Jon Marles-Wright, Andrea Giachino
Summary: Golden Gate assembly is a widely used technique for DNA part assembly. While there are different standards and toolboxes available, choosing the right tools can be a challenge for both new users and experienced scientists.
ACS SYNTHETIC BIOLOGY
(2022)
Article
Genetics & Heredity
Leticia F. Ferigolo, Mateus H. Vicente, Fabio T. S. Nogueira
Summary: In this study, a new cloning strategy named BiG was developed, which allows the merged use of Gateway system and other systems. The BiG method showed high cloning efficiency and ensured the correct reading frame.
Article
Multidisciplinary Sciences
Minakshi Mukherjee, Emily Caroll, Zhen Q. Wang
Summary: The Golden Gate cloning method allows rapid, scar-less, directional, and modular assembly of multiple genes. Using a hierarchical workflow enables easy cloning of various multi-gene constructs without the need for further sequencing. This method is highly applicable for yeast metabolic engineering and other situations requiring multi-gene plasmid cloning.
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
(2021)
Article
Biotechnology & Applied Microbiology
Dongdong Zeng, Cuiyuan Jing, Lin Tang, Peng He, Jie Zhang
Summary: In this study, a PSM system combining Gibson assembly and Gateway cloning was developed to assemble multiple transgenes into a single T-DNA. The system demonstrated flexibility, simplicity, and reliability in transgenic analysis using Arabidopsis, making it a promising tool for genetic engineering and improvement of multiple traits.
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
(2023)
Article
Biochemical Research Methods
Sonja Billerbeck, Rianne C. Prins, Malte Marquardt
Summary: Candida glabrata is an emerging drug-resistant human pathogen that causes severe infections in immunocompromised individuals and is a valuable biotechnology host. However, there is a lack of well-characterized cloning toolkit for this yeast. In this study, a CgTK was developed by characterizing a yeast molecular cloning toolkit and adding missing components, which can be used for genome-wide library screens.
ACS SYNTHETIC BIOLOGY
(2023)
Article
Biochemical Research Methods
Dong Hun Kang, Sung Cheon Ko, Yu Been Heo, Hyun Jeong Lee, Han Min Woo
Summary: Efficient and versatile DNA assembly frameworks have had a significant impact on promoting synthetic biology. However, the traditional MoClo system for multiple DNA assemblies in a biofoundry workflow has limitations in terms of simplicity and feasibility. In this study, a robot-assisted MoClo (RoboMoClo) system was proposed to accelerate synthetic biology projects with multiple gene expressions. The RoboMoClo framework combines hierarchical gene assembly and iterative gene assembly strategies, and requires fewer destination vectors. The application of RoboMoClo in an industrial bacterium, C. glutamicum, demonstrated its potential in laboratory automation for synthetic biology.
ACS SYNTHETIC BIOLOGY
(2022)
Article
Biochemical Research Methods
Christopher Szent-Gyorgyi, Lydia A. Perkins, Brigitte F. Schmidt, Zhen Liu, Marcel P. Bruchez, Robert van de Weerd
Summary: There is a need in synthetic biology for flexible fully designed vectors to address challenging cloning strategies. This article presents a new method for plasmid synthesis that paves the way for exploring protein interactions.
ACS SYNTHETIC BIOLOGY
(2022)
Article
Chemistry, Multidisciplinary
Zrinka Raguz Nakic, Christin Peters
Summary: The conventional cloning strategies for preparing expression vectors are time-consuming and laborious, which are not suitable for designing metabolic pathways or enzyme cascades. However, Modular Cloning, a novel cloning technique in synthetic biology, provides a rapid and efficient method for preparing individual expression vectors using Golden Gate assembly. This technique has successfully been used to generate strains capable of producing complex compounds involving up to 20 enzymes. Although industrial applications of Modular Cloning are still rare, the lack of high-throughput screening methods limits its potential. Future developments should address the bottleneck in screening capacity to promote the establishment of Modular Cloning in industrial applications.
Article
Biochemical Research Methods
Yingjia Tong, Jingwen Zhou, Liang Zhang, Peng Xu
Summary: Violacein, a naturally occurring anticancer therapeutic compound, was produced in Y. lipolytica using a library of strains constructed with Golden Gate assembly method. Gene expression analysis revealed that strong expression of VioB, VioC, and VioD favored violacein production, while weak expression of VioD was associated with high deoxyviolacein production. By optimizing culture conditions, violacein reached a maximum of 70.04 mg/L with 5.28 mg/L of deoxyviolacein in shake flasks.
ACS SYNTHETIC BIOLOGY
(2021)
Article
Biochemical Research Methods
Jonas De Saeger, Mattias Vermeersch, Christophe Gaillochet, Thomas B. Jacobs
Summary: Oligo stitching is an efficient method that can convert Golden Gate parts between different assembly standards and assemble incompatible Golden Gate parts without PCR amplification. It can also be used to assemble de novo sequences and perform plasmid modification and mutagenesis.
ACS SYNTHETIC BIOLOGY
(2022)
Article
Biotechnology & Applied Microbiology
Ya-Wen Li, Cai-Ling Yang, Qi Shen, Qian-Qian Peng, Qi Guo, Zhi-Kui Nie, Xiao-Man Sun, Tian-Qiong Shi, Xiao-Jun Ji, He Huang
Summary: In this study, a Golden Gate modular cloning system (YALIcloneNHEJ) was established to develop a robust DNA assembly platform in Yarrowia lipolytica. By optimizing key factors, the assembly efficiency of multiple fragments reached up to 90%. The system was successfully applied for the overproduction of (-)-alpha-bisabolol, achieving the highest titer reported in yeast to date. This study expands the toolbox of metabolic engineering and enables efficient production of various terpenoids.
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
(2022)
Article
Energy & Fuels
Ting Yang, Feng Zhai, HaiQing Xu, Wei Li
Summary: This paper proposes a PIoT-oriented WSNs communication model and designs an identity authentication protocol for multi-gateway WSNs. Through security analysis, the protocol is proven to be more secure and efficient compared to other multi-user access protocols.
Article
Plant Sciences
Xuean Cui, Yanwei Wang, Jinxia Wu, Xiao Han, Xiaofeng Gu, Tiegang Lu, Zhiguo Zhang
Article
Plant Sciences
Yanwei Wang, Jing Sun, Chen Deng, Shouzhen Teng, Guoxin Chen, Zhenhua Chen, Xuean Cui, Thomas P. Brutnell, Xiao Han, Zhiguo Zhang, Tiegang Lu
Summary: This study identified the role of the SEM1 gene in regulating sucrose transport in rice by analyzing two mesophyll starch excess mutants. Through characterization and experimental validation, the relationship between sucrose and vascular cell development was revealed.
Article
Biochemistry & Molecular Biology
Yanni Li, Binglan Tan, Daoping Wang, Yongying Mu, Guiying Li, Zhiguo Zhang, Yinghong Pan, Li Zhu
Summary: In this study, nano-scale liquid chromatography mass spectrometry (nano-LC-MS/MS) technology was used to analyze the changes in protein expression profile of sorghum leaves under drought stress. The response mechanism of drought-sensitive sorghum leaves was found to be related to the upregulation of proteins involved in tyrosine metabolism pathway, while drought-resistant sorghum leaves responded to drought by promoting the TCA cycle, enhancing sphingolipid biosynthesis, interfering with triterpenoid metabolite synthesis, and influencing aminoacyl-tRNA biosynthesis. Additionally, quantitative real-time PCR (qRT-PCR) validated some important candidate proteins related to drought stress.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2022)
Review
Plant Sciences
Kaining Jin, Guoxin Chen, Yirong Yang, Zhiguo Zhang, Tiegang Lu
Summary: This review summarizes the strategies for improving C-3 photosynthesis, including enhancing Rubisco properties and reducing photorespiratory limitations. It also discusses the potential of synthetic biology approaches in constructing photorespiratory bypasses. These strategies have the potential to enhance C-3 photosynthesis and boost crop production.
PLANT CELL AND ENVIRONMENT
(2023)
Article
Microbiology
Xing Chen, Chunmei Liu, Baoli Zhu, Wenxue Wei, Rong Sheng
Summary: This study investigated the functional differentiations of nitrate reducers containing narG or napA in the genus Pseudomonas in relation to environmental changes. The growth characteristics, nitrate consumption, and dissimilatory nitrate-reducing activity were assessed under varied nitrate and oxygen concentrations. The results showed that the growth and nitrate consumption generally increased with increasing oxygen and nitrate concentrations, but the dissimilatory nitrate-reducing activities were restricted by oxygen supply. The narG-containing nitrate reducers mainly consumed nitrate through dissimilatory nitrate reduction in both anaerobic and aerobic environments, while the A-type nitrate reducers primarily consumed nitrate through assimilation by cell growth under aerobic conditions.
MICROBIOLOGY SPECTRUM
(2022)
Article
Biochemistry & Molecular Biology
Qiming Liu, Shouzhen Teng, Chen Deng, Suting Wu, Haoshu Li, Yanwei Wang, Jinxia Wu, Xuean Cui, Zhiguo Zhang, William Paul Quick, Thomas P. Brutnell, Xuehui Sun, Tiegang Lu
Summary: C-3 and C-4 grasses are crucial for human diet, but our understanding of the molecular mechanisms driving photosynthesis in these grasses is limited. A genetic circuit involving SHR, IDD, and PIN family members controls vein formation and ground cell proliferation in leaves of both C-3 and C-4 grasses. This circuit regulates auxin transport through negative regulation of PIN expression and plays a role in minor vein patterning in grasses.
Article
Plant Sciences
Shouzhen Teng, Qiming Liu, Guoxin Chen, Yuan Chang, Xuean Cui, Jinxia Wu, Pengfei Ai, Xuehui Sun, Zhiguo Zhang, Tiegang Lu
Summary: This study demonstrates the importance of the bHLH transcription factor OsbHLH92 in regulating plant architecture morphogenesis in rice, and its knockout lines show erect leaves, which can increase planting density and crop productivity. Additionally, new components of the brassinosteroid (BR) signaling pathway are identified, expanding our knowledge of typical and atypical bHLH family members in rice.