Book ChapterDOI
USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories.
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TLDR
This chapter presents a general protocol for converting any vector into a USER-compatible vector, together with protocols for both USER cloning and USER fusion.Abstract:
The explosive development of the field of molecular biology has led to the need for simpler and more efficient cloning techniques. These requirements are elegantly met by the ligation-free cloning technique called USER cloning. USER cloning is suitable not only for everyday and high-throughput cloning but also for the one-step construction of complex DNA constructs, which can be achieved in a variant called USER fusion. In this chapter, we present a general protocol for converting any vector into a USER-compatible vector, together with protocols for both USER cloning and USER fusion.read more
Citations
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Methods for the directed evolution of proteins
Michael S. Packer,David R. Liu +1 more
TL;DR: This Review describes some of the tools used to diversify genes, as well as informative examples of screening and selection methods that identify or isolate evolved proteins.
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A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi
TL;DR: A CRISPR-Cas9 based system adapted for use in filamentous fungi that performs RNA-guided mutagenesis in six species of which one has not previously been genetically engineered and demonstrates that the resulting strain can be used for iterative gene targeting.
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DNA assembly for synthetic biology: from parts to pathways and beyond
TL;DR: This review provides a critical examination of recent DNA assembly strategies and considers how this important facilitating aspect of synthetic biology may proceed in the future.
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An alternative route to cyclic terpenes by reductive cyclization in iridoid biosynthesis
Fernando Geu-Flores,Nathaniel H. Sherden,Vincent Courdavault,Vincent Burlat,Vincent Burlat,Weslee S. Glenn,Weslee S. Glenn,Cen Wu,Ezekiel Nims,Yuehua Cui,Sarah E. O'Connor,Sarah E. O'Connor +11 more
TL;DR: The discovery of iridoid synthase is reported, a plant-derived enzyme that generates the iridoids ring scaffold, as evidenced by biochemical assays, gene silencing, co-expression analysis and localization studies, and the prospects of using unrelated reductases to generate artificial cyclic scaffolds are highlighted.
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Microbial production of indolylglucosinolate through engineering of a multi-gene pathway in a versatile yeast expression platform
Michael Dalgaard Mikkelsen,Line Due Buron,Bo Salomonsen,Carl Erik Olsen,Bjarne Gram Hansen,Uffe Hasbro Mortensen,Barbara Ann Halkier +6 more
TL;DR: Production of indolylglucosinolate serves as a proof-of-concept for the expression platform, and provides a basis for large-scale microbial production of specific glucosinolates for the benefit of human health.
References
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Journal ArticleDOI
Gene silencing in plants using artificial microRNAs and other small RNAs
TL;DR: In this article, the authors review various strategies for small RNA-based gene silencing, and describe in detail the design and application of amiRNAs in many plant species.
Journal ArticleDOI
Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments
Hussam Hassan Nour-Eldin,Bjarne Gram Hansen,Morten H. H. Nørholm,Jacob Krüger Jensen,Barbara Ann Halkier +4 more
TL;DR: The largely unused uracil-excision molecular cloning technique is advanced by identifying PfuCx as a compatible proof-reading DNA polymerase and by developing an improved vector design strategy.
Journal ArticleDOI
USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products
TL;DR: The use of PCR primers that contain a single deoxyuridine residue near their 5′ end provides a simple, fast and very efficient method to simultaneously fuse and clone multiple PCR fragments into a vector of interest.
Journal ArticleDOI
A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering.
TL;DR: The addition of a highly efficient DNA polymerase and a low-background-, large-insertion- compatible site-directed mutagenesis protocol is described, largely expanding the versatility of uracil-excision DNA engineering.
Journal ArticleDOI
Structural basis for uracil recognition by archaeal family B DNA polymerases.
TL;DR: This work has identified a 'pocket' in the N-terminal domains of archaeal DNA polymerases that is positioned to interact with the template strand and provides interacting groups that discriminate uracil from the four normal DNA bases (including thymine).
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