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Frances H Arnold - One of the best experts on this subject based on the ideXlab platform.
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staggered extension process step In Vitro recombInation
Methods of Molecular Biology, 2003Co-Authors: Anna Marie Aguinaldo, Frances H ArnoldAbstract:In Vitro polymerase chaIn reaction (PCR)-based recombInation methods are used to shuffle segments from various homologous DNA sequences to produce highly mosaic chimeric sequences. Genetic variations created In the laboratory or existIng In nature can be recombIned to generate libraries of molecules contaInIng novel combInations of sequence Information from any or all of the parent template sequences. Evolutionary proteIn design approaches, In which libraries created by In Vitro recombInation methods are coupled with screenIng (or selection) strategies, have successfully produced variant proteIns with a wide array of modified properties IncludIng Increased drug resistance (1,2), stability (3, 4, 5, 6), bIndIng affInity (6), improved foldIng and solubility (7), altered or expanded substrate specificity (8,9), and new catalytic activity (10).
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directed enzyme evolution screenIng and selection methods
2003Co-Authors: Frances H Arnold, George GeorgiouAbstract:Part I. Genetic Selections Genetic Complementation Protocols Jessica L. Sneeden and Lawrence A. Loeb Use of Pol I-Deficient E. coli for Functional Complementation of DNA Polymerase Manel Camps and Lawrence A. Loeb Selection of Novel Eukaryotic DNA Polymerases by Mutagenesis and Genetic Complementation of Yeast Ranga N. Venkatesan and Lawrence A. Loeb Autogene Selections Jijumon Chelliserrykattil and Andrew D. EllIngton Selection for Soluble ProteIns via Fusion with Chloramphenicol Acetyltransferase Volker Sieber Proside: A Phage-Based Method for SelectIng Thermostable ProteIns Andreas MartIn, Franz X. Schmid, and Volker Sieber MInimization of ProteIns by Random Fragmentation and Selection Gary W. Rudgers and Timothy Palzkill Part II. Screens for Enzymes EvaluatIng a Screen and Analysis of Mutant Libraries Oriana Salazar and Lianhong Sun ScreenIng Mutant Libraries In Saccharomyces cerevisiae Thomas Bulter, Volker Sieber, and Miguel Alcalde Solid-Phase ScreenIng UsIng Digital Image Analysis Alexander V. Tobias and John M. Joern ScreenIng for Thermostability Patrick C. CirIno and Radu Georgescu High-Throughput ScreenIng of Mutant a-Amylase Libraries for Increased Activity at 129 DegreesC Holger Berk and Robert J. LebbInk High-Throughput Carbon Monoxide BIndIng Assay for Cytochromes P450 Christopher R. Otey High-Throughput Screen for Aromatic Hydroxylation Christopher R. Otey and John M. Joern Colorimetric Screen for Aliphatic Hydroxylation by Cytochrome P450 UsIng p-Nitrophenyl-Substituted Alkanes Edgardo T. FarInas High-Throughput Screens Based on NAD(P)H Depletion Anton Glieder and Peter MeInhold High-Throughput TetramethylbenzidIne (TMB) Screen for Peroxidases Radu Georgescu Screenfor Oxidases by Detection of Hydrogen Peroxide with Horseradish Peroxidase Lianhong Sun and Makoto Yagasaki Colorimetric Dehydrogenase Screen Based on NAD(P)H Generation Kimberly M. Mayer Colorimetric Assays for ScreenIng Laccases Miguel Alcalde and Thomas Bulter pH SensIng Agar Plate Assays for Esterolytic Enzyme Activity Karl E. Griswold A pH-Indicator-Based Screen for Hydrolytic Haloalkane Dehalogenase HuimIn Zhao Detection of Aromatic a-Hydroxyketones with Tetrazolium Salts Michael Breuer and Bernhard Hauer Selection of Heat-Stable Clostridium cellulovorans Cellulases After In Vitro RecombInation Koichiro Murashima and Roy H. Doi ScreenIng and Selection Strategies for Disulfide Isomerase Activity Ronald Lafond, XiaomIng Zhan, and George Georgiou An Overview of High-Throughput ScreenIng Systems for Enantioselective Enzymatic Transformations Manfred T. Reetz Select Protocols of High-Throughput ee-ScreenIng Systems for AssayIng Enantioselective Enzymes Manfred T. Reetz Directed Evolution of the Substrate Specificities of a Site-Specific RecombInase and an AmInoacyl-tRNA Synthetase UsIng Fluorescence-Activated Cell SortIng (FACS) Stephen W. Santoro and Peter G. Schultz CalmodulIn-Tagged Phage and Two-Filter Sandwich Assays for the Identification of Enzymatic Activities Christian HeInis, Julian BertschInger, and Dario Neri High-Throughput FACS Method for Directed Evolution of Substrate Specificity Mark J. Olsen, Jongsik Gam, Brent L. Iverson, and George Georgiou ImprovIng ProteIn FoldIng Efficiency by Directed Evolution UsIng the GFP FoldIng Reporter Geoffrey S. Waldo Index
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methods for In Vitro dna recombInation and random chimeragenesis
Methods in Enzymology, 2000Co-Authors: Alexander Volkov, Frances H ArnoldAbstract:Publisher Summary In Vitro polymerase chaIn reaction (PCR)-based methods for recombInIng homologous deoxyribonucleic acid (DNA) sequences are capable of creatIng highly mosaic chimeric sequences. Several different methods have been reported for In Vitro recombInation or DNA shufflIng: for example, the origInal Stemmer method of DNase I fragmentation and reassembly, the staggered extension process (STEP), and random primIng recombInation. Slight variations In the shufflIng protocols can affect the outcome of the experiment. Different gene sequences recombIne most efficiently under different conditions. This chapter provides protocols that are designed to give a high likelihood of success. The protocols are known to work for recombInIng sequences of ∼>85% identity.
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combInatorial proteIn design by In Vitro recombInation
Current Opinion in Chemical Biology, 1998Co-Authors: Lori Giver, Frances H ArnoldAbstract:DNA recombInation is a powerful engIne for the creation of new phenotypes. Recently, methods for In Vitro DNA recombInation (DNA shufflIng) have been developed and applied to the evolution of novel molecules In the laboratory. An excitIng new development is the shufflIng of homologous genes to create diversity for directed evolution.
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molecular evolution by staggered extension process step In Vitro recombInation
Nature Biotechnology, 1998Co-Authors: Huimin Zhao, Zhixin Shao, Lori Giver, Joseph A Affholter, Frances H ArnoldAbstract:We have developed a simple and efficient method for In Vitro mutagenesis and recombInation of polynu-cleotide sequences. The staggered extension process (StEP) consists of primIng the template sequence(s) followed by repeated cycles of denaturation and extremely abbreviated annealIng/polymerase-catalyzed extension. In each cycle the growIng fragments anneal to different templates based on sequence complementarity and extend further. This is repeated until full-length sequences form. Due to template switchIng, most of the polynucleotides contaIn sequence Information from different parental sequences. The method is demonstrated by the recombInation of two genes encodIng thermostable subtilisIns carryIng two phenotypic markers separated by 113 base pairs and eight other poInt mutation markers. To demonstrate its utility for directed evolution, we have used StEP to recombIne a set of five thermostabilized subtilisIn E variants identified durIng a sIngle round of error-prone PCR mutagenesis and screenIng. ScreenIng the StEP-recombIned library yielded an enzyme whose half-life at 65°C is 50 times that of wild-type subtilisIn E.
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.
Lori Giver - One of the best experts on this subject based on the ideXlab platform.
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combInatorial proteIn design by In Vitro recombInation
Current Opinion in Chemical Biology, 1998Co-Authors: Lori Giver, Frances H ArnoldAbstract:DNA recombInation is a powerful engIne for the creation of new phenotypes. Recently, methods for In Vitro DNA recombInation (DNA shufflIng) have been developed and applied to the evolution of novel molecules In the laboratory. An excitIng new development is the shufflIng of homologous genes to create diversity for directed evolution.
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molecular evolution by staggered extension process step In Vitro recombInation
Nature Biotechnology, 1998Co-Authors: Huimin Zhao, Zhixin Shao, Lori Giver, Joseph A Affholter, Frances H ArnoldAbstract:We have developed a simple and efficient method for In Vitro mutagenesis and recombInation of polynu-cleotide sequences. The staggered extension process (StEP) consists of primIng the template sequence(s) followed by repeated cycles of denaturation and extremely abbreviated annealIng/polymerase-catalyzed extension. In each cycle the growIng fragments anneal to different templates based on sequence complementarity and extend further. This is repeated until full-length sequences form. Due to template switchIng, most of the polynucleotides contaIn sequence Information from different parental sequences. The method is demonstrated by the recombInation of two genes encodIng thermostable subtilisIns carryIng two phenotypic markers separated by 113 base pairs and eight other poInt mutation markers. To demonstrate its utility for directed evolution, we have used StEP to recombIne a set of five thermostabilized subtilisIn E variants identified durIng a sIngle round of error-prone PCR mutagenesis and screenIng. ScreenIng the StEP-recombIned library yielded an enzyme whose half-life at 65°C is 50 times that of wild-type subtilisIn E.
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random primIng In Vitro recombInation an effective tool for directed evolution
Nucleic Acids Research, 1998Co-Authors: Zhixin Shao, Huimin Zhao, Lori Giver, Frances H ArnoldAbstract:A simple and efficient method for In Vitro mutagenesis and recombInation of polynucleotide sequences is reported. The method Involves primIng template polynucleotide(s) with random-sequence primers and extendIng to generate a pool of short DNA fragments which contaIn a controllable level of poInt mutations. The fragments are reassembled durIng cycles of denaturation, annealIng and further enzyme-catalyzed DNA polymerization to produce a library of full-length sequences. ScreenIng or selectIng the expressed gene products leads to new variants with improved functions, as demonstrated by the recombInation of genes encodIng different thermostable subtilisIns In order to obtaIn enzymes more stable than either parent.
Willem P. C. Stemmer - One of the best experts on this subject based on the ideXlab platform.
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molecular evolution of an arsenate detoxification pathway by dna shufflIng
Nature Biotechnology, 1997Co-Authors: Andreas Crameri, Glenn Dawes, Emilio Rodriguez, Simon Silver, Willem P. C. StemmerAbstract:Functional evolution of an arsenic resistance operon has been accomplished by DNA shufflIng, InvolvIng multiple rounds of In Vitro recombInation and mutation of a pool of related sequences, followed by selection for Increased resistance In vivo. Homologous recombInation is achieved by random fragmentation of the PCR templates and reassembly by primerless PCR. Plasmid-determIned arsenate resistance from plasmid pI258 encoded by genes arsR, arsE, and arsC was evolved In Escherichia coli. Three rounds of shufflIng and selection resulted In cells that grew In up to 0.5 M arsenate, a 40-fold Increase In resistance. Whereas the native plasmid remaIned episomal, the evolved operon reproducibly Integrated Into the bacterial chromosome. In the absence of shufflIng, no Increase In resistance was observed after four selection cycles, and the control plasmid remaIned episomal. The Integrated ars operon had 13 mutations. Ten mutations were located In arsB, encodIng the arsenite membrane pump, resultIng In a fourfold to sixfold Increase In arsenite resistance. While arsC, the arsenate reductase gene, contaIned no mutations, its expression level was Increased, and the rate of arsenate reduction was Increased 12–fold. These results show that DNA shufflIng can improve the function of pathways by complex and unexpected mutational mechanisms that may be activated by poInt mutation. These mechanisms may be difficult to explaIn and are likely to be overlooked by rational design.
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dna shufflIng by random fragmentation and reassembly In Vitro recombInation for molecular evolution
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Willem P. C. StemmerAbstract:Abstract Computer simulations of the evolution of lInear sequences have demonstrated the importance of recombInation of blocks of sequence rather than poInt mutagenesis alone. Repeated cycles of poInt mutagenesis, recombInation, and selection should allow In Vitro molecular evolution of complex sequences, such as proteIns. A method for the reassembly of genes from their random DNA fragments, resultIng In In Vitro recombInation is reported. A 1-kb gene, after DNase I digestion and purification of 10- to 50-bp random fragments, was reassembled to its origInal size and function. Similarly, a 2.7-kb plasmid could be efficiently reassembled. Complete recombInation was obtaIned between two markers separated by 75 bp; each marker was located on a separate gene. Oligonucleotides with 3' and 5' ends that are homologous to the gene can be added to the fragment mixture and Incorporated Into the reassembled gene. Thus, mixtures of synthetic oligonucleotides and PCR fragments can be mixed Into a gene at defIned positions based on homology. As an example, a library of chimeras of the human and murIne genes for InterleukIn 1 beta has been prepared. ShufflIng can also be used for the In Vitro equivalent of some standard genetic manipulations, such as a backcross with parental DNA. The advantages of recombInation over existIng mutagenesis methods are likely to Increase with the numbers of cycles of molecular evolution.
Huimin Zhao - One of the best experts on this subject based on the ideXlab platform.
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molecular evolution by staggered extension process step In Vitro recombInation
Nature Biotechnology, 1998Co-Authors: Huimin Zhao, Zhixin Shao, Lori Giver, Joseph A Affholter, Frances H ArnoldAbstract:We have developed a simple and efficient method for In Vitro mutagenesis and recombInation of polynu-cleotide sequences. The staggered extension process (StEP) consists of primIng the template sequence(s) followed by repeated cycles of denaturation and extremely abbreviated annealIng/polymerase-catalyzed extension. In each cycle the growIng fragments anneal to different templates based on sequence complementarity and extend further. This is repeated until full-length sequences form. Due to template switchIng, most of the polynucleotides contaIn sequence Information from different parental sequences. The method is demonstrated by the recombInation of two genes encodIng thermostable subtilisIns carryIng two phenotypic markers separated by 113 base pairs and eight other poInt mutation markers. To demonstrate its utility for directed evolution, we have used StEP to recombIne a set of five thermostabilized subtilisIn E variants identified durIng a sIngle round of error-prone PCR mutagenesis and screenIng. ScreenIng the StEP-recombIned library yielded an enzyme whose half-life at 65°C is 50 times that of wild-type subtilisIn E.
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random primIng In Vitro recombInation an effective tool for directed evolution
Nucleic Acids Research, 1998Co-Authors: Zhixin Shao, Huimin Zhao, Lori Giver, Frances H ArnoldAbstract:A simple and efficient method for In Vitro mutagenesis and recombInation of polynucleotide sequences is reported. The method Involves primIng template polynucleotide(s) with random-sequence primers and extendIng to generate a pool of short DNA fragments which contaIn a controllable level of poInt mutations. The fragments are reassembled durIng cycles of denaturation, annealIng and further enzyme-catalyzed DNA polymerization to produce a library of full-length sequences. ScreenIng or selectIng the expressed gene products leads to new variants with improved functions, as demonstrated by the recombInation of genes encodIng different thermostable subtilisIns In order to obtaIn enzymes more stable than either parent.
