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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>10(11)) mutant libraries...

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Detalles Bibliográficos
Autores principales: Troll, Chris, Alexander, David, Allen, Jennifer, Marquette, Jacob, Camps, Manel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3376869/
https://www.ncbi.nlm.nih.gov/pubmed/21445044
http://dx.doi.org/10.3791/2505
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author Troll, Chris
Alexander, David
Allen, Jennifer
Marquette, Jacob
Camps, Manel
author_facet Troll, Chris
Alexander, David
Allen, Jennifer
Marquette, Jacob
Camps, Manel
author_sort Troll, Chris
collection PubMed
description The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>10(11)) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.
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spelling pubmed-33768692012-06-20 Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli Troll, Chris Alexander, David Allen, Jennifer Marquette, Jacob Camps, Manel J Vis Exp Genetics The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>10(11)) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities. MyJove Corporation 2011-03-16 /pmc/articles/PMC3376869/ /pubmed/21445044 http://dx.doi.org/10.3791/2505 Text en Copyright © 2011, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/
spellingShingle Genetics
Troll, Chris
Alexander, David
Allen, Jennifer
Marquette, Jacob
Camps, Manel
Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
title Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
title_full Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
title_fullStr Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
title_full_unstemmed Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
title_short Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
title_sort mutagenesis and functional selection protocols for directed evolution of proteins in e. coli
topic Genetics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3376869/
https://www.ncbi.nlm.nih.gov/pubmed/21445044
http://dx.doi.org/10.3791/2505
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