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Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes  [PDF]
Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0005553
Abstract: We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call ‘Golden Gate shuffling’, is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.
A One Pot, One Step, Precision Cloning Method with High Throughput Capability  [PDF]
Carola Engler, Romy Kandzia, Sylvestre Marillonnet
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0003647
Abstract: Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called ‘Golden Gate’ cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation.
A Modular Cloning System for Standardized Assembly of Multigene Constructs  [PDF]
Ernst Weber,Carola Engler,Ramona Gruetzner,Stefan Werner,Sylvestre Marillonnet
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0016765
Abstract: The field of synthetic biology promises to revolutionize biotechnology through the design of organisms with novel phenotypes useful for medicine, agriculture and industry. However, a limiting factor is the ability of current methods to assemble complex DNA molecules encoding multiple genetic elements in various predefined arrangements. We present here a hierarchical modular cloning system that allows the creation at will and with high efficiency of any eukaryotic multigene construct, starting from libraries of defined and validated basic modules containing regulatory and coding sequences. This system is based on the ability of type IIS restriction enzymes to assemble multiple DNA fragments in a defined linear order. We constructed a 33 kb DNA molecule containing 11 transcription units made from 44 individual basic modules in only three successive cloning steps. This modular cloning (MoClo) system can be readily automated and will be extremely useful for applications such as gene stacking and metabolic engineering.
GoldenBraid: An Iterative Cloning System for Standardized Assembly of Reusable Genetic Modules  [PDF]
Alejandro Sarrion-Perdigones,Erica Elvira Falconi,Sara I. Zandalinas,Paloma Juárez,Asun Fernández-del-Carmen,Antonio Granell,Diego Orzaez
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0021622
Abstract: Synthetic Biology requires efficient and versatile DNA assembly systems to facilitate the building of new genetic modules/pathways from basic DNA parts in a standardized way. Here we present GoldenBraid (GB), a standardized assembly system based on type IIS restriction enzymes that allows the indefinite growth of reusable gene modules made of standardized DNA pieces. The GB system consists of a set of four destination plasmids (pDGBs) designed to incorporate multipartite assemblies made of standard DNA parts and to combine them binarily to build increasingly complex multigene constructs. The relative position of type IIS restriction sites inside pDGB vectors introduces a double loop (“braid”) topology in the cloning strategy that allows the indefinite growth of composite parts through the succession of iterative assembling steps, while the overall simplicity of the system is maintained. We propose the use of GoldenBraid as an assembly standard for Plant Synthetic Biology. For this purpose we have GB-adapted a set of binary plasmids for A. tumefaciens-mediated plant transformation. Fast GB-engineering of several multigene T-DNAs, including two alternative modules made of five reusable devices each, and comprising a total of 19 basic parts are also described.
Bifunctional TaqII restriction endonuclease: redefining the prototype DNA recognition site and establishing the Fidelity Index for partial cleaving
Agnieszka ?ylicz-Stachula, Olga ?o?nierkiewicz, Katarzyna ?liwińska, Joanna Je?ewska-Fr?ckowiak, Piotr M Skowron
BMC Biochemistry , 2011, DOI: 10.1186/1471-2091-12-62
Abstract: Barker et al. reported the Type IIS/IIC restriction endonuclease TaqII as recognizing two distinct cognate site variants (5'-GACCGA-3' and 5'-CACCCA-3') while cleaving 11/9 nucleotides downstream. We used four independent methods, namely, shotgun cloning and sequencing, restriction pattern analysis, digestion of particular custom substrates and GeneScan analysis, to demonstrate that the recombinant enzyme recognizes only 5'-GACCGA-3' sites and cleaves 11/9 nucleotides downstream. We did not observe any 5'-CACCCA-3' cleavage under a variety of conditions and site arrangements tested. We also characterized the enzyme biochemically and established new digestion conditions optimal for practical enzyme applications. Finally, we developed and propose a new version of the Fidelity Index - the Fidelity Index for Partial Cleavage (FI-PC).The DNA recognition sequence of the bifunctional prototype TaqII endonuclease-methyltransferase from Thermus aquaticus has been redefined as recognizing only 5'-GACCGA-3' cognate sites. The reaction conditions (pH and salt concentrations) were designed either to minimize (pH = 8.0 and 10 mM ammonium sulphate) or to enhance star activity (pH = 6.0 and no salt). Redefinition of the recognition site and reaction conditions makes this prototype endonuclease a useful tool for DNA manipulation; as yet, this enzyme has no practical applications. The extension of the Fidelity Index will be helpful for DNA manipulation with enzymes only partially cleaving DNA.Restriction endonucleases (REases) are indispensable as molecular scissors in the analysis, rearrangement, cloning and sequencing of DNA [1-3]. Restriction-modification (RM) systems have been classified into four major types on the basis of their genetic and polypeptide organization, cofactor requirements, and their modes of recognition and cleavage: I, II, III, and IV [4]. Of the 3945 biochemically or genetically characterized restriction enzymes [5] currently known, 3834 Type II RM systems mak
Alteration of Sequence Specificity of the Type IIS Restriction Endonuclease BtsI  [PDF]
Shengxi Guan,Aine Blanchard,Penghua Zhang,Zhenyu Zhu
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0011787
Abstract: The Type IIS restriction endonuclease BtsI recognizes and digests at GCAGTG(2/0). It comprises two subunits: BtsIA and BtsIB. The BtsIB subunit contains the recognition domain, one catalytic domain for bottom strand nicking and part of the catalytic domain for the top strand nicking. BtsIA has the rest of the catalytic domain that is responsible for the DNA top strand nicking. BtsIA alone has no activity unless it mixes with BtsIB to reconstitute the BtsI activity. During characterization of the enzyme, we identified a BtsIB mutant R119A found to have a different digestion pattern from the wild type BtsI. After characterization, we found that BtsIB(R119A) is a novel restriction enzyme with a previously unreported recognition sequence CAGTG(2/0), which is named as BtsI-1. Compared with wild type BtsI, BtsI-1 showed different relative activities in NEB restriction enzyme reaction buffers NEB1, NEB2, NEB3 and NEB4 and less star activity. Similar to the wild type BtsIB subunit, the BtsI-1 B subunit alone can act as a bottom nicking enzyme recognizing CAGTG(-/0). This is the first successful case of a specificity change among this restriction endonuclease type.
Restriction Enzymes in Microbiology, Biotechnology and Biochemistry
Geoffrey G. Wilson,Hua Wang,Daniel F. Heiter,Keith D. Lunnen
Encuentro , 2012,
Abstract: Since their discovery in the nineteen-seventies, a collection of simple enzymes termed Type II restriction endonucleases, made by microbes to ward off viral infections, have transformed molecular biology, spawned the multi-billion dollar Biotechnology industry, and yielded fundamental insights into the biochemistry of life, health and disease. In this article we describe how these enzymes were discovered, and we review their properties, organizations and genetics. We summarize current ideas about the mechanism underlying their remarkable ability to recognize and bind to specific base pair sequences in DNA, and we discuss why these ideas might not be correct. We conclude by proposing an alternative explanation for sequence-recognition that resolves certain inconsistencies and provides, in our view, a more satisfactory account of the mechanism.
GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis  [PDF]
Athanasios Lampropoulos, Zoran Sutikovic, Christian Wenzl, Ira Maegele, Jan U. Lohmann, Joachim Forner
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0083043
Abstract: Building expression constructs for transgenesis is one of the fundamental day-to-day tasks in modern biology. Traditionally it is based on a multitude of type II restriction endonucleases and T4 DNA ligase. Especially in case of long inserts and applications requiring high-throughput, this approach is limited by the number of available unique restriction sites and the need for designing individual cloning strategies for each project. Several alternative cloning systems have been developed in recent years to overcome these issues, including the type IIS enzyme based Golden Gate technique. Here we introduce our GreenGate system for rapidly assembling plant transformation constructs, which is based on the Golden Gate method. GreenGate cloning is simple and efficient since it uses only one type IIS restriction endonuclease, depends on only six types of insert modules (plant promoter, N-terminal tag, coding sequence, C-terminal tag, plant terminator and plant resistance cassette), but at the same time allows assembling several expression cassettes in one binary destination vector from a collection of pre-cloned building blocks. The system is cheap and reliable and when combined with a library of modules considerably speeds up cloning and transgene stacking for plant transformation.
Cloning and analysis of a bifunctional methyltransferase/restriction endonuclease TspGWI, the prototype of a Thermus sp. enzyme family
Agnieszka Zylicz-Stachula, Janusz M Bujnicki, Piotr M Skowron
BMC Molecular Biology , 2009, DOI: 10.1186/1471-2199-10-52
Abstract: Here we describe cloning, mutagenesis and analysis of the prototype TspGWI enzyme that recognises the 5'-ACGGA-3' site and cleaves 11/9 nt downstream. We cloned, expressed, and mutagenised the tspgwi gene and investigated the properties of its product, the bifunctional TspGWI restriction/modification enzyme. Since TspGWI does not cleave DNA completely, a cloning method was devised, based on amino acid sequencing of internal proteolytic fragments. The deduced amino acid sequence of the enzyme shares significant sequence similarity with another representative of the Thermus sp. family – TaqII. Interestingly, these enzymes recognise similar, yet different sequences in the DNA. Both enzymes cleave DNA at the same distance, but differ in their ability to cleave single sites and in the requirement of S-adenosylmethionine as an allosteric activator for cleavage. Both the restriction endonuclease (REase) and methyltransferase (MTase) activities of wild type (wt) TspGWI (either recombinant or isolated from Thermus sp.) are dependent on the presence of divalent cations.TspGWI is a bifunctional protein comprising a tandem arrangement of Type I-like domains; particularly noticeable is the central HsdM-like module comprising a helical domain and a highly conserved S-adenosylmethionine-binding/catalytic MTase domain, containing DPAVGTG and NPPY motifs. TspGWI also possesses an N-terminal PD-(D/E)XK nuclease domain related to the corresponding domains in HsdR subunits, but lacks the ATP-dependent translocase module of the HsdR subunit and the additional domains that are involved in subunit-subunit interactions in Type I systems. The MTase and REase activities of TspGWI are autonomous and can be uncoupled. Structurally and functionally, the TspGWI protomer appears to be a streamlined 'half' of a Type I enzyme.Restriction-modification systems (RM) usually consist of at least two enzymatic activities: a restriction endonuclease (REase), which recognises and cleaves a specific DNA seq
Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase
Tobias Fromme, Martin Klingenspor
Journal of Biological Engineering , 2007, DOI: 10.1186/1754-1611-1-7
Abstract: We here present a novel benchtop procedure to achieve rapid recombination into any destination vector of choice with the sole requirement of an endonuclease recognition site. The method relies on a specifically designed entry vector and the combined action of type II and type IIs endonucleases with ligase. The formulation leads to accumulation of a single stable cloning product representing the desired insert carrying destination vector.The described method provides a fast single step procedure for routine subcloning from an entry vector into a series of destination vectors with the same restriction enzyme recognition site.One of the most routinely performed tasks in molecular biology labs around the world undoubtedly is the subcloning of a given DNA fragment from one plasmid vector into a different one. The reasons to do so are as numerous and diverse as are the applied methods. We here describe a further such technique involving the orchestrated action of a typeII and a typeIIs endonuclease ("outside cutter") with ligase. This procedure achieves the speed of recombinase based methods without the need for unusual recognition elements in the target vector.In an example experiment we subcloned a 1166 bp long DNA fragment* from an entry vector (modified pGEM-T easy, Promega) into a NheI site of a destination vector (pEGFP-N1, Clontech). The method relies on a specifically engineered entry vector which comprises two key elements flanking the insert to be subcloned (Figure 1A). To test our method we accordingly modified the plasmid pGEM-T easy and included a BglII site to be able to insert a DNA fragment between these elements. A blunt end cutting enzyme to Taq-generate T overhangs for TA cloning of PCR products or any other means to insert a fragment of choice are of course feasible as well. Both of the two identical key elements comprise a recognition sequence for a type IIs restriction endonuclease – Esp3I in our case – and a restriction site (Figure 1B). The latter
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