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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 Enzyme Body Doubles and PCR Cloning: On the General Use of Type IIS Restriction Enzymes for Cloning  [PDF]
Eszter Tóth, Krisztina Huszár, Petra Bencsura, Péter István Kulcsár, Barbara Vodicska, Antal Nyeste, Zsombor Welker, Szilvia Tóth, Ervin Welker
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0090896
Abstract: The procedure described here allows the cloning of PCR fragments containing a recognition site of the restriction endonuclease (Type IIP) used for cloning in the sequence of the insert. A Type IIS endonuclease - a Body Double of the Type IIP enzyme - is used to generate the same protruding palindrome. Thus, the insert can be cloned to the Type IIP site of the vector without digesting the PCR product with the same Type IIP enzyme. We achieve this by incorporating the recognition site of a Type IIS restriction enzyme that cleaves the DNA outside of its recognition site in the PCR primer in such a way that the cutting positions straddle the desired overhang sequence. Digestion of the PCR product by the Body Double generates the required overhang. Hitherto the use of Type IIS restriction enzymes in cloning reactions has only been used for special applications, the approach presented here makes Type IIS enzymes as useful as Type IIP enzymes for general cloning purposes. To assist in finding Body Double enzymes, we summarised the available Type IIS enzymes which are potentially useful for Body Double cloning and created an online program (http://group.szbk.u-szeged.hu/welkergr/b?ody_double/index.html) for the selection of suitable Body Double enzymes and the design of the appropriate primers.
The vegitation of the Golden Gate Highlands National Park  [cached]
B. R. Roberts
Koedoe : African Protected Area Conservation and Science , 1969, DOI: 10.4102/koedoe.v12i1.744
Abstract: The vegitation of the Golden Gate Highlands National Park
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.
Additions to the bird list of the Golden Gate Highlands National Park
Mieke S. Hutsebaut,O.B. Kok,J-H. Daneel
Koedoe : African Protected Area Conservation and Science , 1992, DOI: 10.4102/koedoe.v35i1.392
Abstract: Additions to the bird list of the Golden Gate Highlands National Park
Shuffling and Unshuffling  [PDF]
D. Henshall,N. Rampersad,J. Shallit
Computer Science , 2011,
Abstract: We consider various shuffling and unshuffling operations on languages and words, and examine their closure properties. Although the main goal is to provide some good and novel exercises and examples for undergraduate formal language theory classes, we also provide some new results and some open problems.
Assembly of Designer TAL Effectors by Golden Gate Cloning  [PDF]
Ernst Weber,Ramona Gruetzner,Stefan Werner,Carola Engler,Sylvestre Marillonnet
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0019722
Abstract: Generation of customized DNA binding domains targeting unique sequences in complex genomes is crucial for many biotechnological applications. The recently described DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas consists of a series of repeats arranged in tandem, each repeat binding a nucleotide of the target sequence. We present here a strategy for engineering of TALE proteins with novel DNA binding specificities based on the 17.5 repeat-containing AvrBs3 TALE as a scaffold. For each of the 17 full repeats, four module types were generated, each with a distinct base preference. Using this set of 68 repeat modules, recognition domains for any 17 nucleotide DNA target sequence of choice can be constructed by assembling selected modules in a defined linear order. Assembly is performed in two successive one-pot cloning steps using the Golden Gate cloning method that allows seamless fusion of multiple DNA fragments. Applying this strategy, we assembled designer TALEs with new target specificities and tested their function in vivo.
Die Geologiese en Geomorfologiese geskiedenis van Golden Gate-Hoogland Nasionale Park  [cached]
J. J. Spies
Koedoe : African Protected Area Conservation and Science , 1969, DOI: 10.4102/koedoe.v12i1.754
Abstract: Die Geologiese en Geomorfologiese geskiedenis van Golden Gate-Hoogland Nasionale Park
Shuffling with ordered cards  [PDF]
Steve Butler,Ron Graham
Mathematics , 2010,
Abstract: We consider a problem of shuffling a deck of cards with ordered labels. Namely we split the deck of N=k^tq cards (where t>=1 is maximal) into k equally sized stacks and then take the top card off of each stack and sort them by the order of their labels and add them to the shuffled stack. We show how to find stacks of cards invariant and periodic under the shuffling. We also show when gcd(q,k)=1 the possible periods of this shuffling are all divisors of order_k(N-q).
Carries, Shuffling and An Amazing Matrix  [PDF]
Persi Diaconis,Jason Fulman
Mathematics , 2008,
Abstract: The number of ``carries'' when $n$ random integers are added forms a Markov chain [23]. We show that this Markov chain has the same transition matrix as the descent process when a deck of $n$ cards is repeatedly riffle shuffled. This gives new results for the statistics of carries and shuffling.
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