Directed evolution of the transcriptional regulator DntR: isolation of mutants with improved DNT-response.
The transcriptional regulator DntR, which previously has been isolated from bacterial strains capable of degrading 2,4-dinitrotoluene (DNT), was engineered in order to improve the ability to detect DNT. A directed evolution strategy was employed, where sequence diversity first was created by random...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2012-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3261848?pdf=render |
| _version_ | 1828353808917331968 |
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| author | Rosa Lönneborg Edina Varga Peter Brzezinski |
| author_facet | Rosa Lönneborg Edina Varga Peter Brzezinski |
| author_sort | Rosa Lönneborg |
| collection | DOAJ |
| description | The transcriptional regulator DntR, which previously has been isolated from bacterial strains capable of degrading 2,4-dinitrotoluene (DNT), was engineered in order to improve the ability to detect DNT. A directed evolution strategy was employed, where sequence diversity first was created by random mutagenesis in three subsequent rounds, followed by recombination of previously selected mutants. A gfp gene was used as a reporter for transcriptional activity mediated by DntR and cells with higher GFP expression after addition of DNT were sorted out using fluorescence-activated cell sorting (FACS). A DntR mutant, which displayed 10 times higher induction levels than wild-type DntR in response to DNT was isolated. This mutant still maintained low levels of gfp expression in the absence of DNT. The detection limit was ∼10 µM, a 25-fold improvement compared to wild-type DntR. The functional role of some substitutions found in this clone is discussed in the framework of the structural changes observed when comparing the recently determined structures of DntR with and without bound inducer ligand. |
| first_indexed | 2024-04-14T02:14:46Z |
| format | Article |
| id | doaj.art-d766fc73bfd843909bccd57396e9a0b5 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-04-14T02:14:46Z |
| publishDate | 2012-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-d766fc73bfd843909bccd57396e9a0b52022-12-22T02:18:16ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-0171e2999410.1371/journal.pone.0029994Directed evolution of the transcriptional regulator DntR: isolation of mutants with improved DNT-response.Rosa LönneborgEdina VargaPeter BrzezinskiThe transcriptional regulator DntR, which previously has been isolated from bacterial strains capable of degrading 2,4-dinitrotoluene (DNT), was engineered in order to improve the ability to detect DNT. A directed evolution strategy was employed, where sequence diversity first was created by random mutagenesis in three subsequent rounds, followed by recombination of previously selected mutants. A gfp gene was used as a reporter for transcriptional activity mediated by DntR and cells with higher GFP expression after addition of DNT were sorted out using fluorescence-activated cell sorting (FACS). A DntR mutant, which displayed 10 times higher induction levels than wild-type DntR in response to DNT was isolated. This mutant still maintained low levels of gfp expression in the absence of DNT. The detection limit was ∼10 µM, a 25-fold improvement compared to wild-type DntR. The functional role of some substitutions found in this clone is discussed in the framework of the structural changes observed when comparing the recently determined structures of DntR with and without bound inducer ligand.http://europepmc.org/articles/PMC3261848?pdf=render |
| spellingShingle | Rosa Lönneborg Edina Varga Peter Brzezinski Directed evolution of the transcriptional regulator DntR: isolation of mutants with improved DNT-response. PLoS ONE |
| title | Directed evolution of the transcriptional regulator DntR: isolation of mutants with improved DNT-response. |
| title_full | Directed evolution of the transcriptional regulator DntR: isolation of mutants with improved DNT-response. |
| title_fullStr | Directed evolution of the transcriptional regulator DntR: isolation of mutants with improved DNT-response. |
| title_full_unstemmed | Directed evolution of the transcriptional regulator DntR: isolation of mutants with improved DNT-response. |
| title_short | Directed evolution of the transcriptional regulator DntR: isolation of mutants with improved DNT-response. |
| title_sort | directed evolution of the transcriptional regulator dntr isolation of mutants with improved dnt response |
| url | http://europepmc.org/articles/PMC3261848?pdf=render |
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