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Design and Characterization of Auxotrophy-Based Amino Acid Biosensors
Efficient and inexpensive methods are required for the high-throughput quantification of amino acids in physiological fluids or microbial cell cultures. Here we develop an array of Escherichia coli biosensors to sensitively quantify eleven different amino acids. By using online databases, genes invo...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3400592/ https://www.ncbi.nlm.nih.gov/pubmed/22829942 http://dx.doi.org/10.1371/journal.pone.0041349 |
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author | Bertels, Felix Merker, Holger Kost, Christian |
author_facet | Bertels, Felix Merker, Holger Kost, Christian |
author_sort | Bertels, Felix |
collection | PubMed |
description | Efficient and inexpensive methods are required for the high-throughput quantification of amino acids in physiological fluids or microbial cell cultures. Here we develop an array of Escherichia coli biosensors to sensitively quantify eleven different amino acids. By using online databases, genes involved in amino acid biosynthesis were identified that – upon deletion – should render the corresponding mutant auxotrophic for one particular amino acid. This rational design strategy suggested genes involved in the biosynthesis of arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, and tyrosine as potential genetic targets. A detailed phenotypic characterization of the corresponding single-gene deletion mutants indeed confirmed that these strains could neither grow on a minimal medium lacking amino acids nor transform any other proteinogenic amino acid into the focal one. Site-specific integration of the egfp gene into the chromosome of each biosensor decreased the detection limit of the GFP-labeled cells by 30% relative to turbidometric measurements. Finally, using the biosensors to determine the amino acid concentration in the supernatants of two amino acid overproducing E. coli strains (i.e. ΔhisL and ΔtdcC) both turbidometrically and via GFP fluorescence emission and comparing the results to conventional HPLC measurements confirmed the utility of the developed biosensor system. Taken together, our study provides not only a genotypically and phenotypically well-characterized set of publicly available amino acid biosensors, but also demonstrates the feasibility of the rational design strategy used. |
format | Online Article Text |
id | pubmed-3400592 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-34005922012-07-24 Design and Characterization of Auxotrophy-Based Amino Acid Biosensors Bertels, Felix Merker, Holger Kost, Christian PLoS One Research Article Efficient and inexpensive methods are required for the high-throughput quantification of amino acids in physiological fluids or microbial cell cultures. Here we develop an array of Escherichia coli biosensors to sensitively quantify eleven different amino acids. By using online databases, genes involved in amino acid biosynthesis were identified that – upon deletion – should render the corresponding mutant auxotrophic for one particular amino acid. This rational design strategy suggested genes involved in the biosynthesis of arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, and tyrosine as potential genetic targets. A detailed phenotypic characterization of the corresponding single-gene deletion mutants indeed confirmed that these strains could neither grow on a minimal medium lacking amino acids nor transform any other proteinogenic amino acid into the focal one. Site-specific integration of the egfp gene into the chromosome of each biosensor decreased the detection limit of the GFP-labeled cells by 30% relative to turbidometric measurements. Finally, using the biosensors to determine the amino acid concentration in the supernatants of two amino acid overproducing E. coli strains (i.e. ΔhisL and ΔtdcC) both turbidometrically and via GFP fluorescence emission and comparing the results to conventional HPLC measurements confirmed the utility of the developed biosensor system. Taken together, our study provides not only a genotypically and phenotypically well-characterized set of publicly available amino acid biosensors, but also demonstrates the feasibility of the rational design strategy used. Public Library of Science 2012-07-19 /pmc/articles/PMC3400592/ /pubmed/22829942 http://dx.doi.org/10.1371/journal.pone.0041349 Text en Bertels et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Bertels, Felix Merker, Holger Kost, Christian Design and Characterization of Auxotrophy-Based Amino Acid Biosensors |
title | Design and Characterization of Auxotrophy-Based Amino Acid Biosensors |
title_full | Design and Characterization of Auxotrophy-Based Amino Acid Biosensors |
title_fullStr | Design and Characterization of Auxotrophy-Based Amino Acid Biosensors |
title_full_unstemmed | Design and Characterization of Auxotrophy-Based Amino Acid Biosensors |
title_short | Design and Characterization of Auxotrophy-Based Amino Acid Biosensors |
title_sort | design and characterization of auxotrophy-based amino acid biosensors |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3400592/ https://www.ncbi.nlm.nih.gov/pubmed/22829942 http://dx.doi.org/10.1371/journal.pone.0041349 |
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