1,721,480 research outputs found
Multi-fragment DNA assembly of biochemical pathways via automated Ligase Cycling Reaction
No full textThe microbial production of commodity, fine and specialty chemicals and biofuels is a driving force in biotechnology. To do this, biochemical pathways to the target compound(s) must be deduced, suitable enzymes selected and then the genetic pathways must be designed and built for in vivo activity. The genetic design is crucial for balancing the pathway in vivo through regulation of transcription and translation but the possible permutations quickly generates a vast design space. Traditionally pathway assembly has been time-consuming and laborious but the advent of multi-fragment DNA assembly technologies has facilitated the possibility of multiplexed pathway construction allowing an increased capability to sample the design space. Furthermore, the implementation of laboratory automation allows error-reduced, high-throughput (HTP) construction of pathways. In this chapter we present an automated workflow that combines in silico design of DNA parts followed by pathway assembly using the Ligase Cycling Reaction (LCR) on robotics platforms, to allow multiplexed assembly of plasmid-borne gene pathways with high efficiency. The workflow begins with the design of DNA part sequences considering biological issues and ensuring compatibility with DNA synthesis and LCR assembly. Subsequently a laboratory protocol for HTP pathway assembly and screening is detailed allowing the production of over 96 plasmids simultaneously with a success rate of over 40 %. This workflow is easy to modify for other laboratories and will help to accelerate synthetic biology for diverse application
The causative role and therapeutic potential of the kynurenine pathway in neurodegenerative disease
No full textMetabolites of the kynurenine pathway (KP), which arise from the degradation of tryptophan, have been studied in detail for over a century and garnered the interest of the neuroscience community in the late 1970s and early 1980s with work uncovering the neuromodulatory potential of this pathway. Much research in the following decades has found that perturbations in the levels of KP metabolites likely contribute to the pathogenesis of several neurodegenerative diseases. More recently, it has become apparent that targeting KP enzymes, in particular kynurenine 3-monooxygenase (KMO), may hold substantial therapeutic potential for these disorders. Here we provide an overview of the KP, the neuroactive properties of KP metabolites and their role in neurodegeneration. We also discuss KMO as a therapeutic target for these disorders, and our recent resolution of the crystallographic structure of KMO, which will permit the development of new and improved KMO inhibitors which may ultimately expedite clinical application of these compounds. © 2013 Springer-Verlag Berlin Heidelberg
YeastFab:High-Throughput Genetic Parts Construction, Measurement, and Pathway Engineering in Yeast
No full textFor 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.</p
The dimeric form of flavocytochrome P450 BM3 is catalytically functional as a fatty acid hydroxylase
No full textIn the model P450 BM3 system, the P450 is fused to its diflavin reductase partner in a single polypeptide. BM3 dimerizes in solution, but the catalytic relevance of the phenomenon was hitherto unknown. We show that BM3 fatty acid hydroxylase specific activity decreases sharply at low enzyme concentrations, consistent with separation of active dimer into inactive monomer. Reductase-dependent specific activities are maintained or enhanced at low concentration, suggesting inter-flavin electron transfer is unaffected. Fatty acid oxidation is reconstituted by mixing inactive oxygenase (A264H) and FMN-depleted (G570D) mutants, demonstrating that inter-monomer (FMN1-to-heme2) electron transfer supports oxygenase activity in the BM3 dimer
Flavocytochrome P450 BM3 substrate selectivity and electron transfer in a model cytochrome P450
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Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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