Fluorescent riboswitch-controlled biosensors for the genome scale analysis of metabolic pathways.
Sci Rep
; 14(1): 12555, 2024 05 31.
Article
en En
| MEDLINE
| ID: mdl-38821978
ABSTRACT
Fluorescent detection in cells has been tremendously developed over the years and now benefits from a large array of reporters that can provide sensitive and specific detection in real time. However, the intracellular monitoring of metabolite levels still poses great challenges due to the often complex nature of detected metabolites. Here, we provide a systematic analysis of thiamin pyrophosphate (TPP) metabolism in Escherichia coli by using a TPP-sensing riboswitch that controls the expression of the fluorescent gfp reporter. By comparing different combinations of reporter fusions and TPP-sensing riboswitches, we determine key elements that are associated with strong TPP-dependent sensing. Furthermore, by using the Keio collection as a proxy for growth conditions differing in TPP levels, we perform a high-throughput screen analysis using high-density solid agar plates. Our study reveals several genes whose deletion leads to increased or decreased TPP levels. The approach developed here could be applicable to other riboswitches and reporter genes, thus representing a framework onto which further development could lead to highly sophisticated detection platforms allowing metabolic screens and identification of orphan riboswitches.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Tiamina Pirofosfato
/
Técnicas Biosensibles
/
Escherichia coli
/
Redes y Vías Metabólicas
/
Riboswitch
Idioma:
En
Revista:
Sci Rep
Año:
2024
Tipo del documento:
Article
País de afiliación:
Canadá
Pais de publicación:
Reino Unido