Cargando…

Application of the thermostable β-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions

Use of thermophilic organisms has a range of advantages, but the significant lack of engineering tools limits their applications. Here we show that β-galactosidase from Geobacillus stearothermophilus (BgaB) can be applicable in a range of conditions, including different temperatures and oxygen conce...

Descripción completa

Detalles Bibliográficos
Autores principales: Jensen, Torbjørn Ølshøj, Pogrebnyakov, Ivan, Falkenberg, Kristoffer Bach, Redl, Stephanie, Nielsen, Alex Toftgaard
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Berlin Heidelberg 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585113/
https://www.ncbi.nlm.nih.gov/pubmed/28875485
http://dx.doi.org/10.1186/s13568-017-0469-z
_version_ 1783261550634598400
author Jensen, Torbjørn Ølshøj
Pogrebnyakov, Ivan
Falkenberg, Kristoffer Bach
Redl, Stephanie
Nielsen, Alex Toftgaard
author_facet Jensen, Torbjørn Ølshøj
Pogrebnyakov, Ivan
Falkenberg, Kristoffer Bach
Redl, Stephanie
Nielsen, Alex Toftgaard
author_sort Jensen, Torbjørn Ølshøj
collection PubMed
description Use of thermophilic organisms has a range of advantages, but the significant lack of engineering tools limits their applications. Here we show that β-galactosidase from Geobacillus stearothermophilus (BgaB) can be applicable in a range of conditions, including different temperatures and oxygen concentrations. This protein functions both as a marker, promoting colony color development in the presence of a lactose analogue S-gal, and as a reporter enabling quantitative measurement by a simple colorimetric assay. Optimal performance was observed at 70 °C and pH 6.4. The gene was introduced into G. thermoglucosidans. The combination of BgaB expressed from promoters of varying strength with S-gal produced distinct black colonies in aerobic and anaerobic conditions at temperatures ranging from 37 to 60 °C. It showed an important advantage over the conventional β-galactosidase (LacZ) and substrate X-gal, which were inactive at high temperature and under anaerobic conditions. To demonstrate the versatility of the reporter, a promoter library was constructed by randomizing sequences around −35 and −10 regions in a wild type groES promoter from Geobacillus sp. GHH01. The library contained 28 promoter variants and encompassed fivefold variation. The experimental pipeline allowed construction and measurement of expression levels of the library in just 4 days. This β-galactosidase provides a promising tool for engineering of aerobic, anaerobic, and thermophilic production organisms such as Geobacillus species. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13568-017-0469-z) contains supplementary material, which is available to authorized users.
format Online
Article
Text
id pubmed-5585113
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher Springer Berlin Heidelberg
record_format MEDLINE/PubMed
spelling pubmed-55851132017-09-22 Application of the thermostable β-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions Jensen, Torbjørn Ølshøj Pogrebnyakov, Ivan Falkenberg, Kristoffer Bach Redl, Stephanie Nielsen, Alex Toftgaard AMB Express Original Article Use of thermophilic organisms has a range of advantages, but the significant lack of engineering tools limits their applications. Here we show that β-galactosidase from Geobacillus stearothermophilus (BgaB) can be applicable in a range of conditions, including different temperatures and oxygen concentrations. This protein functions both as a marker, promoting colony color development in the presence of a lactose analogue S-gal, and as a reporter enabling quantitative measurement by a simple colorimetric assay. Optimal performance was observed at 70 °C and pH 6.4. The gene was introduced into G. thermoglucosidans. The combination of BgaB expressed from promoters of varying strength with S-gal produced distinct black colonies in aerobic and anaerobic conditions at temperatures ranging from 37 to 60 °C. It showed an important advantage over the conventional β-galactosidase (LacZ) and substrate X-gal, which were inactive at high temperature and under anaerobic conditions. To demonstrate the versatility of the reporter, a promoter library was constructed by randomizing sequences around −35 and −10 regions in a wild type groES promoter from Geobacillus sp. GHH01. The library contained 28 promoter variants and encompassed fivefold variation. The experimental pipeline allowed construction and measurement of expression levels of the library in just 4 days. This β-galactosidase provides a promising tool for engineering of aerobic, anaerobic, and thermophilic production organisms such as Geobacillus species. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13568-017-0469-z) contains supplementary material, which is available to authorized users. Springer Berlin Heidelberg 2017-09-06 /pmc/articles/PMC5585113/ /pubmed/28875485 http://dx.doi.org/10.1186/s13568-017-0469-z Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
spellingShingle Original Article
Jensen, Torbjørn Ølshøj
Pogrebnyakov, Ivan
Falkenberg, Kristoffer Bach
Redl, Stephanie
Nielsen, Alex Toftgaard
Application of the thermostable β-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions
title Application of the thermostable β-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions
title_full Application of the thermostable β-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions
title_fullStr Application of the thermostable β-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions
title_full_unstemmed Application of the thermostable β-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions
title_short Application of the thermostable β-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions
title_sort application of the thermostable β-galactosidase, bgab, from geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585113/
https://www.ncbi.nlm.nih.gov/pubmed/28875485
http://dx.doi.org/10.1186/s13568-017-0469-z
work_keys_str_mv AT jensentorbjørnølshøj applicationofthethermostablebgalactosidasebgabfromgeobacillusstearothermophilusasaversatilereporterunderanaerobicandaerobicconditions
AT pogrebnyakovivan applicationofthethermostablebgalactosidasebgabfromgeobacillusstearothermophilusasaversatilereporterunderanaerobicandaerobicconditions
AT falkenbergkristofferbach applicationofthethermostablebgalactosidasebgabfromgeobacillusstearothermophilusasaversatilereporterunderanaerobicandaerobicconditions
AT redlstephanie applicationofthethermostablebgalactosidasebgabfromgeobacillusstearothermophilusasaversatilereporterunderanaerobicandaerobicconditions
AT nielsenalextoftgaard applicationofthethermostablebgalactosidasebgabfromgeobacillusstearothermophilusasaversatilereporterunderanaerobicandaerobicconditions