Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening.
The Golden Gate strategy entails the use of type IIS restriction enzymes, which cut outside of their recognition sequence. It enables unrestricted design of unique DNA fragments that can be readily and seamlessly recombined. Successfully employed in other synthetic biology applications, we demonstra...
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ftdoajarticles:oai:doaj.org/article:22ccb32669e245bfbd118bd9a69d8e5b 2023-05-15T14:00:18+02:00 Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening. Daniela Quaglia Maximilian C C J C Ebert Paul F Mugford Joelle N Pelletier 2017-01-01T00:00:00Z https://doi.org/10.1371/journal.pone.0171741 https://doaj.org/article/22ccb32669e245bfbd118bd9a69d8e5b EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC5298319?pdf=render https://doaj.org/toc/1932-6203 1932-6203 doi:10.1371/journal.pone.0171741 https://doaj.org/article/22ccb32669e245bfbd118bd9a69d8e5b PLoS ONE, Vol 12, Iss 2, p e0171741 (2017) Medicine R Science Q article 2017 ftdoajarticles https://doi.org/10.1371/journal.pone.0171741 2022-12-30T23:22:56Z The Golden Gate strategy entails the use of type IIS restriction enzymes, which cut outside of their recognition sequence. It enables unrestricted design of unique DNA fragments that can be readily and seamlessly recombined. Successfully employed in other synthetic biology applications, we demonstrate its advantageous use to engineer a biocatalyst. Hot-spots for mutations were individuated in three distinct regions of Candida antarctica lipase A (Cal-A), the biocatalyst chosen as a target to demonstrate the versatility of this recombination method. The three corresponding gene segments were subjected to the most appropriate method of mutagenesis (targeted or random). Their straightforward reassembly allowed combining products of different mutagenesis methods in a single round for rapid production of a series of diverse libraries, thus facilitating directed evolution. Screening to improve discrimination of short-chain versus long-chain fatty acid substrates was aided by development of a general, automated method for visual discrimination of the hydrolysis of varied substrates by whole cells. Article in Journal/Newspaper Antarc* Antarctica Directory of Open Access Journals: DOAJ Articles Golden Gate ENVELOPE(-134.237,-134.237,59.616,59.616) PLOS ONE 12 2 e0171741 |
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Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
op_collection_id |
ftdoajarticles |
language |
English |
topic |
Medicine R Science Q |
spellingShingle |
Medicine R Science Q Daniela Quaglia Maximilian C C J C Ebert Paul F Mugford Joelle N Pelletier Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening. |
topic_facet |
Medicine R Science Q |
description |
The Golden Gate strategy entails the use of type IIS restriction enzymes, which cut outside of their recognition sequence. It enables unrestricted design of unique DNA fragments that can be readily and seamlessly recombined. Successfully employed in other synthetic biology applications, we demonstrate its advantageous use to engineer a biocatalyst. Hot-spots for mutations were individuated in three distinct regions of Candida antarctica lipase A (Cal-A), the biocatalyst chosen as a target to demonstrate the versatility of this recombination method. The three corresponding gene segments were subjected to the most appropriate method of mutagenesis (targeted or random). Their straightforward reassembly allowed combining products of different mutagenesis methods in a single round for rapid production of a series of diverse libraries, thus facilitating directed evolution. Screening to improve discrimination of short-chain versus long-chain fatty acid substrates was aided by development of a general, automated method for visual discrimination of the hydrolysis of varied substrates by whole cells. |
format |
Article in Journal/Newspaper |
author |
Daniela Quaglia Maximilian C C J C Ebert Paul F Mugford Joelle N Pelletier |
author_facet |
Daniela Quaglia Maximilian C C J C Ebert Paul F Mugford Joelle N Pelletier |
author_sort |
Daniela Quaglia |
title |
Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening. |
title_short |
Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening. |
title_full |
Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening. |
title_fullStr |
Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening. |
title_full_unstemmed |
Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening. |
title_sort |
enzyme engineering: a synthetic biology approach for more effective library generation and automated high-throughput screening. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2017 |
url |
https://doi.org/10.1371/journal.pone.0171741 https://doaj.org/article/22ccb32669e245bfbd118bd9a69d8e5b |
long_lat |
ENVELOPE(-134.237,-134.237,59.616,59.616) |
geographic |
Golden Gate |
geographic_facet |
Golden Gate |
genre |
Antarc* Antarctica |
genre_facet |
Antarc* Antarctica |
op_source |
PLoS ONE, Vol 12, Iss 2, p e0171741 (2017) |
op_relation |
http://europepmc.org/articles/PMC5298319?pdf=render https://doaj.org/toc/1932-6203 1932-6203 doi:10.1371/journal.pone.0171741 https://doaj.org/article/22ccb32669e245bfbd118bd9a69d8e5b |
op_doi |
https://doi.org/10.1371/journal.pone.0171741 |
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PLOS ONE |
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12 |
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2 |
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e0171741 |
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