The Experts below are selected from a list of 121815 Experts worldwide ranked by ideXlab platform
Ahmed H. Badran - One of the best experts on this subject based on the ideXlab platform.
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Continuous bioactivity dependent Evolution of an antibiotic biosynthetic pathway
Nature Communications, 2020Co-Authors: Chad W Johnston, Ahmed H. Badran, James J CollinsAbstract:Antibiotic biosynthetic gene clusters (BGCs) produce bioactive metabolites that impart a fitness advantage to their producer, providing a mechanism for natural selection. This selection drives antibiotic Evolution and adapts BGCs for expression in different organisms, potentially providing clues to improve heterologous expression of antibiotics. Here, we use phage-assisted Continuous Evolution (PACE) to achieve bioactivity-dependent adaptation of the BGC for the antibiotic bicyclomycin (BCM), facilitating improved production in a heterologous host. This proof-of-principle study demonstrates that features of natural bioactivity-dependent Evolution can be engineered to access unforeseen routes of improving metabolic pathways and product yields. Biosynthetic gene clusters (BGCs) make small molecules with fitness-enhancing activities that drive BGC Evolution. Here, the authors show that synthetic biology can leverage bioactivity to achieve Continuous Evolution of an antibiotic BGC in the lab and improve antibiotic production in a new host.
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Continuous Evolution of Bacillus thuringiensis toxins overcomes insect resistance
Nature, 2016Co-Authors: Ahmed H. Badran, Victo M Guzov, Qing Huai, Prashanth Vishwanath, Autum M Nance, Artem G Evdokimov, Farhad Moshiri, Melissa M. Kemp, Wendy Kain, Keith H. TurnerAbstract:The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted Continuous Evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd = 11-41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the Evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.
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Continuous directed Evolution of DNA-binding proteins to improve TALEN specificity
Nature methods, 2015Co-Authors: Basil P. Hubbard, Ahmed H. Badran, John A. Zuris, John Paul Guilinger, Kevin Davis, Chen Liwei, Shengdar Q. Tsai, Jeffry D. Sander, J. Keith Joung, David R. LiuAbstract:DNA-binding phage-assisted Continuous Evolution improves the specificity of DNA-binding proteins, as demonstrated with TALENs.
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a system for the Continuous directed Evolution of proteases rapidly reveals drug resistance mutations
Nature Communications, 2014Co-Authors: Bryan C Dickinson, Michael S. Packer, Ahmed H. BadranAbstract:Phage-assisted Continuous Evolution (PACE) has the potential to rapidly evolve drug-resistant mutations. Here, Dickinson et al. present a protease PACE system that identifies clinically relevant mutations conferring resistance to protease inhibitors in only a few days of Continuous Evolution.
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Negative selection and stringency modulation in phage-assisted Continuous Evolution.
Nature chemical biology, 2014Co-Authors: Jacob C. Carlson, Ahmed H. Badran, Drago Guggiana-nilo, David R. LiuAbstract:Phage-assisted Continuous Evolution (PACE) minimizes researcher intervention while maximizing rounds of protein Evolution. New strategies now eliminate the need for intermediate substrate analogs and promote altered selectivity instead of promiscuity, exemplified by a 10,000-fold switch in polymerase specificity while retaining wild-type activity.
Farhad Moshiri - One of the best experts on this subject based on the ideXlab platform.
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Continuous Evolution of bacillus thuringiensis toxins overcomes insect resistance
Nature, 2016Co-Authors: Ahmed H Adra, Victo M Guzov, Qing Huai, Melissa Kemp, Prashanth Vishwanath, Wendy Kai, Autum M Nance, Artem G Evdokimov, Farhad MoshiriAbstract:The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted Continuous Evolution selection that rapidly evolves high-affinity protein–protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd = 11–41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the Evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects. Phage-assisted Continuous Evolution (PACE) rapidly evolves Bacillus thuringiensis toxins through more than 500 generations of mutation, selection, and replication to bind a receptor expressed on the surface of insect-pest midgut cells. The emergence of insects resistant to Bacillus thuringiensis δ-endotoxins (Bt toxins) is threatening to reduce the effectiveness of this system in crops engineered to carry these insecticidal proteins. David Liu and colleagues have used phage-assisted Continuous Evolution (PACE) selection to rapidly evolve high-affinity protein–protein interactions, and applied the system to evolve Bt toxin variants that kill insects through binding a new insect gut cell protein target — a cadherin-like receptor from the insect pest Trichoplusia ni. The modified Bt toxins are shown to overcome Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.
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Continuous Evolution of Bacillus thuringiensis toxins overcomes insect resistance
Nature, 2016Co-Authors: Ahmed H. Badran, Victo M Guzov, Qing Huai, Prashanth Vishwanath, Autum M Nance, Artem G Evdokimov, Farhad Moshiri, Melissa M. Kemp, Wendy Kain, Keith H. TurnerAbstract:The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted Continuous Evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd = 11-41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the Evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.
Ahmed H Adra - One of the best experts on this subject based on the ideXlab platform.
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Continuous Evolution of bacillus thuringiensis toxins overcomes insect resistance
Nature, 2016Co-Authors: Ahmed H Adra, Victo M Guzov, Qing Huai, Melissa Kemp, Prashanth Vishwanath, Wendy Kai, Autum M Nance, Artem G Evdokimov, Farhad MoshiriAbstract:The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted Continuous Evolution selection that rapidly evolves high-affinity protein–protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd = 11–41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the Evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects. Phage-assisted Continuous Evolution (PACE) rapidly evolves Bacillus thuringiensis toxins through more than 500 generations of mutation, selection, and replication to bind a receptor expressed on the surface of insect-pest midgut cells. The emergence of insects resistant to Bacillus thuringiensis δ-endotoxins (Bt toxins) is threatening to reduce the effectiveness of this system in crops engineered to carry these insecticidal proteins. David Liu and colleagues have used phage-assisted Continuous Evolution (PACE) selection to rapidly evolve high-affinity protein–protein interactions, and applied the system to evolve Bt toxin variants that kill insects through binding a new insect gut cell protein target — a cadherin-like receptor from the insect pest Trichoplusia ni. The modified Bt toxins are shown to overcome Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.
David R. Liu - One of the best experts on this subject based on the ideXlab platform.
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Phage-assisted Continuous and non-Continuous Evolution
Nature Protocols, 2020Co-Authors: Shannon M. Miller, Tina Wang, David R. LiuAbstract:Directed Evolution, which applies the principles of Darwinian Evolution to a laboratory setting, is a powerful strategy for generating biomolecules with diverse and tailored properties. This technique can be implemented in a highly efficient manner using Continuous Evolution, which enables the steps of directed Evolution to proceed seamlessly over many successive generations with minimal researcher intervention. Phage-assisted Continuous Evolution (PACE) enables Continuous directed Evolution in bacteria by mapping the steps of Darwinian Evolution onto the bacteriophage life cycle and allows directed Evolution to occur on much faster timescales compared to conventional methods. This protocol provides detailed instructions on evolving proteins using PACE and phage-assisted non-Continuous Evolution (PANCE) and includes information on the preparation of selection phage and host cells, the assembly of a Continuous flow apparatus and the performance and analysis of Evolution experiments. This protocol can be performed in as little as 2 weeks to complete more than 100 rounds of Evolution (complete cycles of mutation, selection and replication) in a single PACE experiment. This phage-assisted Continuous Evolution protocol enables directed protein Evolution. It includes preparation of selection phage and host cells, assembly of a Continuous flow apparatus and performance and analysis of Evolution experiments.
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Phage-assisted Continuous and non-Continuous Evolution.
Nature protocols, 2020Co-Authors: Shannon M. Miller, Tina Wang, David R. LiuAbstract:Directed Evolution, which applies the principles of Darwinian Evolution to a laboratory setting, is a powerful strategy for generating biomolecules with diverse and tailored properties. This technique can be implemented in a highly efficient manner using Continuous Evolution, which enables the steps of directed Evolution to proceed seamlessly over many successive generations with minimal researcher intervention. Phage-assisted Continuous Evolution (PACE) enables Continuous directed Evolution in bacteria by mapping the steps of Darwinian Evolution onto the bacteriophage life cycle and allows directed Evolution to occur on much faster timescales compared to conventional methods. This protocol provides detailed instructions on evolving proteins using PACE and phage-assisted non-Continuous Evolution (PANCE) and includes information on the preparation of selection phage and host cells, the assembly of a Continuous flow apparatus and the performance and analysis of Evolution experiments. This protocol can be performed in as little as 2 weeks to complete more than 100 rounds of Evolution (complete cycles of mutation, selection and replication) in a single PACE experiment.
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The developing toolkit of Continuous directed Evolution.
Nature chemical biology, 2020Co-Authors: Mary S Morrison, Christopher J Podracky, David R. LiuAbstract:Continuous directed Evolution methods allow the key steps of Evolution-gene diversification, selection, and replication-to proceed in the laboratory with minimal researcher intervention. As a result, Continuous Evolution can find solutions much more quickly than traditional discrete Evolution methods. Continuous Evolution also enables the exploration of longer and more numerous Evolutionary trajectories, increasing the likelihood of accessing solutions that require many steps through sequence space and greatly facilitating the iterative refinement of selection conditions and targeted mutagenesis strategies. Here we review the historical advances that have expanded Continuous Evolution from its earliest days as an experimental curiosity to its present state as a powerful and surprisingly general strategy for generating tailor-made biomolecules, and discuss more recent improvements with an eye to the future.
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Continuous Evolution of SpCas9 variants compatible with non-G PAMs.
Nature biotechnology, 2020Co-Authors: Shannon M. Miller, Tina Wang, Holly A. Rees, Peyton B. Randolph, Mandana Arbab, Max W. Shen, Tony P. Huang, Zaneta Matuszek, Gregory A. Newby, David R. LiuAbstract:The targeting scope of Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants is largely restricted to protospacer-adjacent motif (PAM) sequences containing G bases. Here we report the Evolution of three new SpCas9 variants that collectively recognize NRNH PAMs (where R is A or G and H is A, C or T) using phage-assisted non-Continuous Evolution, three new phage-assisted Continuous Evolution strategies for DNA binding and a secondary selection for DNA cleavage. The targeting capabilities of these evolved variants and SpCas9-NG were characterized in HEK293T cells using a library of 11,776 genomically integrated protospacer–sgRNA pairs containing all possible NNNN PAMs. The evolved variants mediated indel formation and base editing in human cells and enabled A•T-to-G•C base editing of a sickle cell anemia mutation using a previously inaccessible CACC PAM. These new evolved SpCas9 variants, together with previously reported variants, in principle enable targeting of most NR PAM sequences and substantially reduce the fraction of genomic sites that are inaccessible by Cas9-based methods. PAM sequences without G bases can be edited with SpCas9 variants that were Continuously evolved in the laboratory.
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Phage-assisted Continuous Evolution of proteases with altered substrate specificity
Nature Communications, 2017Co-Authors: Michael S. Packer, Holly A. Rees, David R. LiuAbstract:Proteases are promising therapeutics to treat diseases such as hemophilia which are due to endogenous protease deficiency. Here the authors use phage-assisted Continuous Evolution to evolve a variant TEV protease with altered target peptide sequence specificities. Here we perform phage-assisted Continuous Evolution (PACE) of TEV protease, which canonically cleaves ENLYFQS, to cleave a very different target sequence, HPLVGHM, that is present in human IL-23. A protease emerging from ∼2500 generations of PACE contains 20 non-silent mutations, cleaves human IL-23 at the target peptide bond, and when pre-mixed with IL-23 in primary cultures of murine splenocytes inhibits IL-23-mediated immune signaling. We characterize the substrate specificity of this evolved enzyme, revealing shifted and broadened specificity changes at the six positions in which the target amino acid sequence differed. Mutational dissection and additional protease specificity profiling reveal the molecular basis of some of these changes. This work establishes the capability of changing the substrate specificity of a protease at many positions in a practical time scale and provides a foundation for the development of custom proteases that catalytically alter or destroy target proteins for biotechnological and therapeutic applications.
Keith H. Turner - One of the best experts on this subject based on the ideXlab platform.
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Continuous Evolution of Bacillus thuringiensis toxins overcomes insect resistance
Nature, 2016Co-Authors: Ahmed H. Badran, Victo M Guzov, Qing Huai, Prashanth Vishwanath, Autum M Nance, Artem G Evdokimov, Farhad Moshiri, Melissa M. Kemp, Wendy Kain, Keith H. TurnerAbstract:The Bacillus thuringiensis δ-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted Continuous Evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd = 11-41 nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the Evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.
