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We have adapted the Golden Gate DNA assembly method to the assembly of transcription units (TUs) for the yeast Saccharomyces cerevisiae, in a method we call yeast Golden Gate (yGG). yGG allows for the easy assembly of TUs consisting of promoters (PRO), coding sequences (CDS), and terminators (TER). Carefully designed overhangs exposed by digestion with a type IIS restriction enzyme enable virtually seamless assembly of TUs that, in principle, contain all of the information necessary to express a gene of interest in yeast. We also describe a versatile set of yGG acceptor vectors to be used for TU assembly. These vectors can be used for low or high copy expression of assembled TUs or integration into carefully selected innocuous genomic loci. yGG provides synthetic biologists and yeast geneticists with an efficient new means by which to engineer S. cerevisiae.
ACS Synth Biol 2015 Jul 17
PMID:Yeast Golden Gate (yGG) for the Efficient Assembly of S. cerevisiae Transcription Units. 2575 91

Improved plants are necessary to meet human needs. Agrobacterium-mediated transformation is the most common method used to rewire plant capabilities. For plant gene delivery, DNA constructs are assembled into binary T-DNA vectors that rely on broad host range origins for bacterial replication. Here we present pLX vectors, a set of mini binary T-DNA plasmids suitable for Type IIS restriction endonuclease- and overlap-based assembly methods. pLX vectors include replicons from compatible broad host range plasmids. Simultaneous usage of pBBR1- and RK2-based pLX vectors in a two-plasmid/one-Agrobacterium strain strategy allowed multigene delivery to plants. Adoption of pLX vectors will facilitate routine plant transformations and targeted mutagenesis, as well as complex part and circuit characterization.
ACS Synth Biol 2017 10 20
PMID:Multiple T-DNA Delivery to Plants Using Novel Mini Binary Vectors with Compatible Replication Origins. 2865 30

A modular and hierarchical DNA assembly platform for synthetic biology based on Golden Gate (Type IIS restriction enzyme) cloning is described. This enabling technology, termed MIDAS (for Modular Idempotent DNA Assembly System), can be used to precisely assemble multiple DNA fragments in a single reaction using a standardized assembly design. It can be used to build genes from libraries of sequence-verified, reusable parts and to assemble multiple genes in a single vector, with full user control over gene order and orientation, as well as control of the direction of growth (polarity) of the multigene assembly, a feature that allows genes to be nested between other genes or genetic elements. We describe the detailed design and use of MIDAS, exemplified by the reconstruction, in the filamentous fungus Penicillium paxilli, of the metabolic pathway for production of paspaline and paxilline, key intermediates in the biosynthesis of a range of indole diterpenes-a class of secondary metabolites produced by several species of filamentous fungi. MIDAS was used to efficiently assemble a 25.2 kb plasmid from 21 different modules (seven genes, each composed of three basic parts). By using a parts library-based system for construction of complex assemblies, and a unique set of vectors, MIDAS can provide a flexible route to assembling tailored combinations of genes and other genetic elements, thereby supporting synthetic biology applications in a wide range of expression hosts.
ACS Synth Biol 2018 04 20
PMID:MIDAS: A Modular DNA Assembly System for Synthetic Biology. 2962 Aug 66

The ability to rapidly design, build, and test prototypes is of key importance to every engineering discipline. DNA assembly often serves as a rate limiting step of the prototyping cycle for synthetic biology. Recently developed DNA assembly methods such as isothermal assembly and type IIS restriction enzyme systems take different approaches to accelerate DNA construction. We introduce a hybrid method, Golden Gate-Gibson (3G), that takes advantage of modular part libraries introduced by type IIS restriction enzyme systems and isothermal assembly's ability to build large DNA constructs in single pot reactions. Our method is highly efficient and rapid, facilitating construction of entire multigene circuits in a single day. Additionally, 3G allows generation of variant libraries enabling efficient screening of different possible circuit constructions. We characterize the efficiency and accuracy of 3G assembly for various construct sizes, and demonstrate 3G by characterizing variants of an inducible cell-lysis circuit.
ACS Synth Biol 2018 05 18
PMID:Single Day Construction of Multigene Circuits with 3G Assembly. 2971 10

Synthetic biology relies on the manufacture of large and complex DNA constructs from libraries of genetic parts. Golden Gate and other Type IIS restriction enzyme-dependent DNA assembly methods enable rapid construction of genes and operons through one-pot, multifragment assembly, with the ordering of parts determined by the ligation of Watson-Crick base-paired overhangs. However, ligation of mismatched overhangs leads to erroneous assembly, and low-efficiency Watson Crick pairings can lead to truncated assemblies. Using sets of empirically vetted, high-accuracy junction pairs avoids this issue but limits the number of parts that can be joined in a single reaction. Here, we report the use of comprehensive end-joining ligation fidelity and bias data to predict high accuracy junction sets for Golden Gate assembly. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions and enabled accurate and efficient assembly of a lac cassette from up to 24-fragments in a single reaction.
ACS Synth Biol 2018 11 16
PMID:Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly. 3033 70

Re-engineering of transcriptional networks regulating secondary cell wall formation may allow the improvement of plant biomass in widely grown plantation crops such as Eucalyptus. However, there is currently a scarcity of freely available standardized biological parts (e.g., Phytobricks) compatible with Type IIS assembly approaches from forest trees, and there is a need to accelerate transcriptional network inference in nonmodel biomass crops. Here we describe the design and synthesis of a versatile three-panel biological parts collection of 221 secondary cell wall-related Eucalyptus grandis transcription factor coding sequences and 65 promoters that are compatible with GATEWAY, Golden Gate, MoClo, and GoldenBraid DNA assembly methods and generally conform to accepted Phytobrick syntaxes. This freely available resource is intended to accelerate synthetic biology applications in multiple plant biomass crops and enable reconstruction of secondary cell wall transcriptional networks using high-throughput assays such as DNA affinity purification sequencing (DAP-seq) and enhanced yeast one-hybrid (eY1H) screening.
ACS Synth Biol 2019 02 15
PMID:A Standardized Synthetic Eucalyptus Transcription Factor and Promoter Panel for Re-engineering Secondary Cell Wall Regulation in Biomass and Bioenergy Crops. 3060 15