Related Experiment Video
Updated: Jun 25, 2026
Standardized Modular Assembly of Polycistronic Operons with Modular Cloning (MoClo) using the In-Cloning toolkit
Published on: September 2, 2025
Cloning and expression of the sco2127 gene from Streptomyces coelicolor M145
Adán Chávez1, Yolanda García-Huante, Beatriz Ruiz
1Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico DF, Mexico.
Abstract:
It is known that Streptomyces peucetius var. caesius mutants resistant to 2-deoxyglucose (Dog(R)) exhibit glucose transport deficiency, low glucose kinase (Glk) activity and insensitivity to carbon catabolite repression (CCR). This phenotype can be pleiotropically complemented by a 576-bp gene encoding SCO2127 from Streptomyces coelicolor, suggesting the participation of this protein in the CCR process. In the present work, the sco2127 region was subcloned into pQE30 and its transcription product (SCO2127-His(6)) overexpressed. This procedure allowed purification of SCO2127 (with a Ni-sepharose resin) and production of polyclonal antibodies. In western blot assays, the antibodies gave a positive reaction against protein extracts from both S. coelicolor and S. peucetius var. caesius, appearing as a single band of 34 kDa. No protein was detected using extracts from a S. coelicolor mutant lacking the sco2127 gene (Deltasco2127). In agreement with its possible involvement in the CCR process, SCO2127 was detected during the logarithmic growth phase of S. coelicolor grown in minimal medium supplemented with 50 and 100 mM glucose. In addition, when 50 mM glucose was utilized, SCO2127 and residual glucose concentration simultaneously decreased at later stages of the microbial growth.
Insights
SCO2127 protein from Streptomyces coelicolor complements glucose transport deficiency in Streptomyces peucetius mutants. This protein is involved in carbon catabolite repression (CCR) and is detected during active growth phases when glucose is present.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Mutants of Streptomyces peucetius var. caesius resistant to 2-deoxyglucose (Dog(R)) display impaired glucose transport, reduced glucose kinase (Glk) activity, and resistance to carbon catabolite repression (CCR).
- A gene, SCO2127, from Streptomyces coelicolor was found to complement this phenotype, suggesting its role in CCR.
Purpose of the Study:
- To investigate the role of SCO2127 in carbon catabolite repression (CCR) in Streptomyces species.
- To characterize the SCO2127 protein and determine its expression pattern in relation to glucose availability.
Main Methods:
- Subcloning and overexpression of the sco2127 gene to produce SCO2127-His(6) for purification.
- Purification of SCO2127 using Ni-sepharose chromatography and generation of polyclonal antibodies.
- Western blot analysis to detect SCO2127 protein in Streptomyces extracts and a knockout mutant.
Main Results:
- SCO2127 protein was successfully purified and antibodies confirmed its presence in both S. coelicolor and S. peucetius var. caesius, with a molecular weight of 34 kDa.
- No SCO2127 protein was detected in a Streptomyces coelicolor mutant lacking the sco2127 gene (Deltasco2127).
- SCO2127 expression was observed during the logarithmic growth phase in minimal medium with glucose, and its levels decreased along with residual glucose concentration during later growth stages.
Conclusions:
- The SCO2127 protein is confirmed to be involved in the carbon catabolite repression (CCR) process in Streptomyces.
- The expression of SCO2127 is regulated by glucose availability, indicating its role in glucose metabolism and transport regulation.
