One-step generation of error-prone PCR libraries using Gateway<sup>® </sup>technology
<p>Abstract</p> <p>Background</p> <p>Error-prone PCR (epPCR) libraries are one of the tools used in directed evolution. The Gateway<sup>® </sup>technology allows constructing epPCR libraries virtually devoid of any background (<it>i.e</it>., of i...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2012-01-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | http://www.microbialcellfactories.com/content/11/1/14 |
| _version_ | 1818491373488177152 |
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| author | Gruet Antoine Longhi Sonia Bignon Christophe |
| author_facet | Gruet Antoine Longhi Sonia Bignon Christophe |
| author_sort | Gruet Antoine |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Error-prone PCR (epPCR) libraries are one of the tools used in directed evolution. The Gateway<sup>® </sup>technology allows constructing epPCR libraries virtually devoid of any background (<it>i.e</it>., of insert-free plasmid), but requires two steps: the BP and the LR reactions and the associated <it>E. coli </it>cell transformations and plasmid purifications.</p> <p>Results</p> <p>We describe a method for making epPCR libraries in Gateway<sup>® </sup>plasmids using an LR reaction without intermediate BP reaction. We also describe a BP-free and LR-free sub-cloning method for in-frame transferring the coding sequence of selected clones from the plasmid used to screen the library to another one devoid of tag used for screening (such as the green fluorescent protein). We report preliminary results of a directed evolution program using this method.</p> <p>Conclusions</p> <p>The one-step method enables producing epPCR libraries of as high complexity and quality as does the regular, two-step, protocol for half the amount of work. In addition, it contributes to preserve the original complexity of the epPCR product.</p> |
| first_indexed | 2024-12-10T17:29:58Z |
| format | Article |
| id | doaj.art-9c3cbe6b805844c3be784a24a0d977d3 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-10T17:29:58Z |
| publishDate | 2012-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-9c3cbe6b805844c3be784a24a0d977d32022-12-22T01:39:43ZengBMCMicrobial Cell Factories1475-28592012-01-011111410.1186/1475-2859-11-14One-step generation of error-prone PCR libraries using Gateway<sup>® </sup>technologyGruet AntoineLonghi SoniaBignon Christophe<p>Abstract</p> <p>Background</p> <p>Error-prone PCR (epPCR) libraries are one of the tools used in directed evolution. The Gateway<sup>® </sup>technology allows constructing epPCR libraries virtually devoid of any background (<it>i.e</it>., of insert-free plasmid), but requires two steps: the BP and the LR reactions and the associated <it>E. coli </it>cell transformations and plasmid purifications.</p> <p>Results</p> <p>We describe a method for making epPCR libraries in Gateway<sup>® </sup>plasmids using an LR reaction without intermediate BP reaction. We also describe a BP-free and LR-free sub-cloning method for in-frame transferring the coding sequence of selected clones from the plasmid used to screen the library to another one devoid of tag used for screening (such as the green fluorescent protein). We report preliminary results of a directed evolution program using this method.</p> <p>Conclusions</p> <p>The one-step method enables producing epPCR libraries of as high complexity and quality as does the regular, two-step, protocol for half the amount of work. In addition, it contributes to preserve the original complexity of the epPCR product.</p>http://www.microbialcellfactories.com/content/11/1/14CloningSub-cloningGateway<sup>®</sup>Directed evolutionGFPError-prone PCRepPCRLibraryScreening |
| spellingShingle | Gruet Antoine Longhi Sonia Bignon Christophe One-step generation of error-prone PCR libraries using Gateway<sup>® </sup>technology Microbial Cell Factories Cloning Sub-cloning Gateway<sup>®</sup> Directed evolution GFP Error-prone PCR epPCR Library Screening |
| title | One-step generation of error-prone PCR libraries using Gateway<sup>® </sup>technology |
| title_full | One-step generation of error-prone PCR libraries using Gateway<sup>® </sup>technology |
| title_fullStr | One-step generation of error-prone PCR libraries using Gateway<sup>® </sup>technology |
| title_full_unstemmed | One-step generation of error-prone PCR libraries using Gateway<sup>® </sup>technology |
| title_short | One-step generation of error-prone PCR libraries using Gateway<sup>® </sup>technology |
| title_sort | one step generation of error prone pcr libraries using gateway sup r sup technology |
| topic | Cloning Sub-cloning Gateway<sup>®</sup> Directed evolution GFP Error-prone PCR epPCR Library Screening |
| url | http://www.microbialcellfactories.com/content/11/1/14 |
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