The Experts below are selected from a list of 171735 Experts worldwide ranked by ideXlab platform
Hironori Niki - One of the best experts on this subject based on the ideXlab platform.
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exonuclease iii xtha enforces in vivo dna Cloning of escherichia coli to create cohesive ends
Journal of Bacteriology, 2018Co-Authors: Shingo Nozaki, Hironori NikiAbstract:ABSTRACT Escherichia coli has an ability to assemble DNA fragments with homologous overlapping sequences of 15 to 40 bp at each end. Several modified protocols have already been reported to improve this simple and useful DNA Cloning technology. However, the molecular mechanism by which E. coli accomplishes such Cloning is still unknown. In this study, we provide evidence that the in vivo Cloning of E. coli is independent of both RecA and RecET recombinases but is dependent on XthA, a 3′ to 5′ exonuclease. Here, in vivo Cloning of E. coli by XthA is referred to as in vivoE. coli Cloning (iVEC). We also show that iVEC activity is reduced by deletion of the C-terminal domain of DNA polymerase I (PolA). Collectively, these results suggest the following mechanism of iVEC. First, XthA resects the 3′ ends of linear DNA fragments that are introduced into E. coli cells, resulting in exposure of the single-stranded 5′ overhangs. Then, the complementary single-stranded DNA ends hybridize each other, and gaps are filled by DNA polymerase I. Elucidation of the iVEC mechanism at the molecular level would further advance the development of in vivo DNA Cloning technology. Already we have successfully demonstrated multiple-fragment assembly of up to seven fragments in combination with an effortless transformation procedure using a modified host strain for iVEC. IMPORTANCE Cloning of a DNA fragment into a vector is one of the fundamental techniques in recombinant DNA technology. Recently, an in vitro recombination system for DNA Cloning was shown to enable the joining of multiple DNA fragments at once. Interestingly, E. coli potentially assembles multiple linear DNA fragments that are introduced into the cell. Improved protocols for this in vivo Cloning have realized a high level of usability, comparable to that by in vitro recombination reactions. However, the mechanism of in vivo Cloning is highly controversial. Here, we clarified the fundamental mechanism underlying in vivo Cloning and also constructed a strain that was optimized for in vivo Cloning. Additionally, we streamlined the procedure of in vivo Cloning by using a single microcentrifuge tube.
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exonuclease iii xtha enforces in vivo dna Cloning of escherichia coli to create cohesive ends
bioRxiv, 2018Co-Authors: Shingo Nozaki, Hironori NikiAbstract:Abstract Escherichia coli has an ability to assemble DNA fragments with homologous overlapping sequences of 15-40 bp at each end. Several modified protocols have already been reported to improve this simple and useful DNA-Cloning technology. However, the molecular mechanism by which E. coli accomplishes such Cloning is still unknown. In this study, we provide evidence that the in vivo Cloning of E. coli is independent of both RecA and RecET recombinase, but is dependent on XthA, a 3’ to 5’ exonuclease. Here, in vivo Cloning of E. coli by XthA is referred to as iVEC (in vivo E. coli Cloning). Next, we show that the iVEC activity is reduced by deletion of the C-terminal domain of DNA polymerase I (PolA). Collectively, these results suggest the following mechanism of iVEC. First, XthA resects the 3′ ends of linear DNA fragments that are introduced into E. coli cells, resulting in exposure of the single-stranded 5′ overhangs. Then, the complementary single-stranded DNA ends hybridize each other, and gaps are filled by DNA polymerase I. Elucidation of the iVEC mechanism at the molecular level would further advance the development of in vivo DNA-Cloning technology. Already we have successfully demonstrated multiple-fragment assembly of up to seven fragments in combination with an effortless transformation procedure using a modified host strain for iVEC. Importance Cloning of a DNA fragment into a vector is one of the fundamental techniques in recombinant DNA technology. Recently, in vitro recombination of DNA fragments effectively joins multiple DNA fragments in place of the canonical method. Interestingly, E. coli can take up linear double-stranded vectors, insert DNA fragments and assemble them in vivo. The in vivo Cloning have realized a high level of usability comparable to that by in vitro recombination reaction, since now it is only necessary to introduce PCR products into E. coli for the in vivo Cloning. However, the mechanism of in vivo Cloning is highly controversial. Here we clarified the fundamental mechanism underlying in vivo Cloning of E. coli and also constructed an E. coli strain that was optimized for in vivo Cloning.
Adam Miranowicz - One of the best experts on this subject based on the ideXlab platform.
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Cloning of arbitrary mirror-symmetric distributions on Bloch sphere: Optimality proof and proposal for practical photonic realization
2020Co-Authors: Karol Bartkiewicz, Adam MiranowiczAbstract:We study state-dependent quantum Cloning which can outperform universal Cloning. This is possible by using some a priori information on a given quantum state to be cloned. Specifically, we propose a generalization and optical implementation of quantum optimal mirror phase-covariant Cloning, which refers to optimal Cloning of sets of qubits of known modulus of expectation value of Pauli's Z operator. Our results can be applied for Cloning of an arbitrary mirror-symmetric distribution of qubits on Bloch sphere including in special cases the universal Cloning and phasecovariant Cloning. We show that the Cloning is optimal by adapting our former optimality proof for axisymmetric Cloning [Phys. Rev. 82, 042330 (2010)]. Moreover, we propose an optical realization of the optimal mirror phase-covariant 1 → 2 Cloning of a qubit, for which the mean probability of successful Cloning varies from 1/6 to 1/3 depending on prior information on the set of qubits to be cloned. The qubits are represented by polarization states of photons generated by the type-I spontaneous parametric down-conversion. The scheme is based on the interference of two photons on an unbalanced polarization-dependent beam splitter with different splitting ratios for vertical and horizontal polarization components and the additional application of feedforward by means of Pockels cells. The experimental feasibility of the proposed setup is carefully studied including various kinds of imperfections and losses including: (i) finite efficiency of generating a pair of entangled photons in the type-I spontaneous parametric down conversion, (ii) the influence of choosing various splitting ratios of the unbalanced beam splitter, (iii) the application of conventional and singlephoton discriminating detectors, (iv) dark counts and finite efficiency of the detectors
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Optimal Cloning of arbitrary mirror-symmetric distributions on the Bloch sphere: a proposal for practical photonic realization
Physica Scripta, 2012Co-Authors: Karol Bartkiewicz, Adam MiranowiczAbstract:We study state-dependent quantum Cloning that can outperform universal\nCloning (UC). This is possible by using some a priori information on a\ngiven quantum state to be cloned. Specifically, we propose a\ngeneralization and optical implementation of quantum optimal mirror\nphase-covariant Cloning, which refers to optimal Cloning of sets of\nqubits of known modulus of the expectation value of Pauli's Z operator.\nOur results can be applied to Cloning of an arbitrary mirror-symmetric\ndistribution of qubits on the Bloch sphere including in special cases UC\nand phase-covariant Cloning. We show that the Cloning is optimal by\nadapting our former optimality proof for axisymmetric Cloning\n(Bartkiewicz and Miranowicz 2010 Phys. Rev. A 82 042330). Moreover, we\npropose an optical realization of the optimal mirror phase-covariant 1\n-> 2 Cloning of a qubit, for which the mean probability of successful\nCloning varies from 1/6 to 1/3 depending on prior information on the set\nof qubits to be cloned. The qubits are represented by polarization\nstates of photons generated by the type-I spontaneous parametric\ndown-conversion. The scheme is based on the interference of two photons\non an unbalanced polarization-dependent beam splitter with different\nsplitting ratios for vertical and horizontal polarization components and\nthe additional application of feed forward by means of Pockels cells.\nThe experimental feasibility of the proposed setup is carefully studied\nincluding various kinds of imperfections and losses. Moreover, we\nbriefly describe two possible cryptographic applications of the optimal\nmirror phase-covariant Cloning corresponding to state discrimination (or\nestimation) and secure quantum teleportation.
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Optimal Cloning of qubits given by an arbitrary axisymmetric distribution on the Bloch sphere
Physical Review A - Atomic, Molecular, and Optical Physics, 2010Co-Authors: Karol Bartkiewicz, Adam MiranowiczAbstract:We find an optimal quantum Cloning machine, which clones qubits of arbitrary symmetrical distribution around the Bloch vector with the highest fidelity. The process is referred to as phase-independent Cloning in contrast to the standard phase-covariant Cloning for which an input qubit state is a priori better known. We assume that the information about the input state is encoded in an arbitrary axisymmetric distribution (phase function) on the Bloch sphere of the cloned qubits. We find analytical expressions describing the optimal Cloning transformation and fidelity of the clones. As an illustration, we analyze Cloning of qubit state described by the von Mises-Fisher and Brosseau distributions. Moreover, we show that the optimal phase-independent Cloning machine can be implemented by modifying the mirror phase-covariant Cloning machine for which quantum circuits are known.
Karol Bartkiewicz - One of the best experts on this subject based on the ideXlab platform.
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Cloning of arbitrary mirror-symmetric distributions on Bloch sphere: Optimality proof and proposal for practical photonic realization
2020Co-Authors: Karol Bartkiewicz, Adam MiranowiczAbstract:We study state-dependent quantum Cloning which can outperform universal Cloning. This is possible by using some a priori information on a given quantum state to be cloned. Specifically, we propose a generalization and optical implementation of quantum optimal mirror phase-covariant Cloning, which refers to optimal Cloning of sets of qubits of known modulus of expectation value of Pauli's Z operator. Our results can be applied for Cloning of an arbitrary mirror-symmetric distribution of qubits on Bloch sphere including in special cases the universal Cloning and phasecovariant Cloning. We show that the Cloning is optimal by adapting our former optimality proof for axisymmetric Cloning [Phys. Rev. 82, 042330 (2010)]. Moreover, we propose an optical realization of the optimal mirror phase-covariant 1 → 2 Cloning of a qubit, for which the mean probability of successful Cloning varies from 1/6 to 1/3 depending on prior information on the set of qubits to be cloned. The qubits are represented by polarization states of photons generated by the type-I spontaneous parametric down-conversion. The scheme is based on the interference of two photons on an unbalanced polarization-dependent beam splitter with different splitting ratios for vertical and horizontal polarization components and the additional application of feedforward by means of Pockels cells. The experimental feasibility of the proposed setup is carefully studied including various kinds of imperfections and losses including: (i) finite efficiency of generating a pair of entangled photons in the type-I spontaneous parametric down conversion, (ii) the influence of choosing various splitting ratios of the unbalanced beam splitter, (iii) the application of conventional and singlephoton discriminating detectors, (iv) dark counts and finite efficiency of the detectors
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Optimal Cloning of arbitrary mirror-symmetric distributions on the Bloch sphere: a proposal for practical photonic realization
Physica Scripta, 2012Co-Authors: Karol Bartkiewicz, Adam MiranowiczAbstract:We study state-dependent quantum Cloning that can outperform universal\nCloning (UC). This is possible by using some a priori information on a\ngiven quantum state to be cloned. Specifically, we propose a\ngeneralization and optical implementation of quantum optimal mirror\nphase-covariant Cloning, which refers to optimal Cloning of sets of\nqubits of known modulus of the expectation value of Pauli's Z operator.\nOur results can be applied to Cloning of an arbitrary mirror-symmetric\ndistribution of qubits on the Bloch sphere including in special cases UC\nand phase-covariant Cloning. We show that the Cloning is optimal by\nadapting our former optimality proof for axisymmetric Cloning\n(Bartkiewicz and Miranowicz 2010 Phys. Rev. A 82 042330). Moreover, we\npropose an optical realization of the optimal mirror phase-covariant 1\n-> 2 Cloning of a qubit, for which the mean probability of successful\nCloning varies from 1/6 to 1/3 depending on prior information on the set\nof qubits to be cloned. The qubits are represented by polarization\nstates of photons generated by the type-I spontaneous parametric\ndown-conversion. The scheme is based on the interference of two photons\non an unbalanced polarization-dependent beam splitter with different\nsplitting ratios for vertical and horizontal polarization components and\nthe additional application of feed forward by means of Pockels cells.\nThe experimental feasibility of the proposed setup is carefully studied\nincluding various kinds of imperfections and losses. Moreover, we\nbriefly describe two possible cryptographic applications of the optimal\nmirror phase-covariant Cloning corresponding to state discrimination (or\nestimation) and secure quantum teleportation.
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Optimal Cloning of qubits given by an arbitrary axisymmetric distribution on the Bloch sphere
Physical Review A - Atomic, Molecular, and Optical Physics, 2010Co-Authors: Karol Bartkiewicz, Adam MiranowiczAbstract:We find an optimal quantum Cloning machine, which clones qubits of arbitrary symmetrical distribution around the Bloch vector with the highest fidelity. The process is referred to as phase-independent Cloning in contrast to the standard phase-covariant Cloning for which an input qubit state is a priori better known. We assume that the information about the input state is encoded in an arbitrary axisymmetric distribution (phase function) on the Bloch sphere of the cloned qubits. We find analytical expressions describing the optimal Cloning transformation and fidelity of the clones. As an illustration, we analyze Cloning of qubit state described by the von Mises-Fisher and Brosseau distributions. Moreover, we show that the optimal phase-independent Cloning machine can be implemented by modifying the mirror phase-covariant Cloning machine for which quantum circuits are known.
Darren J Hart - One of the best experts on this subject based on the ideXlab platform.
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Recent advances in universal TA Cloning methods for use in function studies.
Protein Engineering Design and Selection, 2016Co-Authors: Shuo Yao, Darren J HartAbstract:As one of the simplest and most efficient Cloning methods, T-vector-based TA Cloning has been widely used for Cloning of single genes and construction of DNA libraries. This approach is especially suitable for high-throughput Cloning of diverse DNA fragments since inserts can be cloned without knowledge of their sequence; it is therefore an ideal tool for high-throughput analysis of protein structure and function. Although most of the currently available T-vectors can only be used for Cloning purposes, some novel variants with improved functions have be developed. This review focuses on recent developments of universal TA Cloning methods and T-vectors constructed for function studies.
Ken Motohashi - One of the best experts on this subject based on the ideXlab platform.
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A novel series of high-efficiency vectors for TA Cloning and blunt-end Cloning of PCR products
Scientific reports, 2019Co-Authors: Ken MotohashiAbstract:An efficient PCR Cloning method is indispensable in modern molecular biology, as it can greatly improve the efficiency of DNA Cloning processes. Here, I describe the development of three vectors for TA Cloning and blunt-end Cloning. Specifically, pCRT and pCRZeroT were designed to improve the efficiency of TA Cloning. pCRZeroT can also be used with pCRZero to facilitate blunt-end Cloning using the ccdB gene. Using pCRZero and pCRZeroT and applying the Golden Gate reaction, I developed a direct PCR Cloning protocol with non-digested circular vectors and PCR products. This direct PCR Cloning protocol yielded colony-formation rates and Cloning efficiencies that are comparable with those obtained by conventional PCR Cloning with pre-digested vectors and PCR products. The three plasmids I designed are available from Addgene ( https://www.addgene.org/ ).
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Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA Cloning methods
Biochemistry and biophysics reports, 2017Co-Authors: Ken MotohashiAbstract:Abstract Simple and low-cost recombinant enzyme-free seamless DNA Cloning methods have recently become available. In vivo Escherichia coli Cloning (iVEC) can directly transform a mixture of insert and vector DNA fragments into E. coli , which are ligated by endogenous homologous recombination activity in the cells. Seamless ligation Cloning extract (SLiCE) Cloning uses the endogenous recombination activity of E. coli cellular extracts in vitro to ligate insert and vector DNA fragments. An evaluation of the efficiency and utility of these methods is important in deciding the adoption of a seamless Cloning method as a useful tool. In this study, both seamless Cloning methods incorporated inserting DNA fragments into linearized DNA vectors through short (15–39 bp) end homology regions. However, colony formation was 30–60-fold higher with SLiCE Cloning in end homology regions between 15 and 29 bp than with the iVEC method using DH5α competent cells. E. coli AQ3625 strains, which harbor a sbc A gene mutation that activates the RecE homologous recombination pathway, can be used to efficiently ligate insert and vector DNA fragments with short-end homology regions in vivo . Using AQ3625 competent cells in the iVEC method improved the rate of colony formation, but the efficiency and accuracy of SLiCE Cloning were still higher. In addition, the efficiency of seamless Cloning methods depends on the intrinsic competency of E. coli cells. The competency of chemically competent AQ3625 cells was lower than that of competent DH5α cells, in all cases of chemically competent cell preparations using the three different methods. Moreover, SLiCE Cloning permits the use of both homemade and commercially available competent cells because it can use general E. coli rec A − strains such as DH5α as host cells for transformation. Therefore, between the two methods, SLiCE Cloning provides both higher efficiency and better utility than the iVEC method for seamless DNA plasmid engineering.
