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Jensen TØ, Pogrebnyakov I, Falkenberg KB, et al. Application of the thermostable β-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions. AMB Express. 2017;7(1):169doi: 10.1186/s13568-017-0469-z.
Jensen, T. �. �., Pogrebnyakov, I., Falkenberg, K. B., Redl, S., & Nielsen, A. T. (2017). Application of the thermostable β-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions. AMB Express, 7(1), 169. https://doi.org/10.1186/s13568-017-0469-z
Jensen, Torbjørn Ølshøj, et al. "Application of the thermostable β-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions." AMB Express vol. 7,1 (2017): 169. doi: https://doi.org/10.1186/s13568-017-0469-z
Jensen TØ, Pogrebnyakov I, Falkenberg KB, Redl S, Nielsen AT. Application of the thermostable β-galactosidase, BgaB, from Geobacillus stearothermophilus as a versatile reporter under anaerobic and aerobic conditions. AMB Express. 2017 Sep 06;7(1):169. doi: 10.1186/s13568-017-0469-z. PMID: 28875485; PMCID: PMC5585113.
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AMB Express. 2017 Sep 06;7(1):169. doi: 10.1186/s13568-017-0469-z.

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

AMB Express

Torbjørn Ølshøj Jensen, Ivan Pogrebnyakov, Kristoffer Bach Falkenberg, Stephanie Redl, Alex Toftgaard Nielsen

Affiliations

  1. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet Building 220, 2800, Kongens Lyngby, Denmark.
  2. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet Building 220, 2800, Kongens Lyngby, Denmark. [email protected].

PMID: 28875485 PMCID: PMC5585113 DOI: 10.1186/s13568-017-0469-z

Abstract

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.

Keywords: Anaerobic genetics; Genetic reporter; Thermophile; Thermostable enzyme

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