biostudies-literatureZeng JMOST | National Natural Science Foundation of China (NSFC)e0156624https://www.ebi.ac.uk/biostudies/studies/S-EPMC11784378biostudies-literatureUnknown91(1)Clostridioides difficile is an obligate anaerobic, Gram-positive bacterium that produces toxins. Despite technological progress, conducting gene expression analysis of C. difficile under different conditions continues to be labor-intensive. Therefore, there is a demand for simplified tools to investigate the transcriptional and translational regulation of C. difficile. The cell-free gene expression (CFE) system has demonstrated utility in various applications, including prototyping, protein production, and in vitro screening. In this study, we developed a C. difficile CFE system capable of in vitro transcription and translation (TX-TL) in the presence of oxygen. Through optimization of cell extract preparation and reaction systems, we increased theApplied and environmental microbiologyA Clostridioides difficile cell-free gene expression system for prototyping and gene expression analysis.PMC117843783237002982061128001Li YLiu ZLi DZhang XZeng JWu CDong MWen STian G-BWang HHan JXu YfalseA Clostridioides difficile cell-free gene expression system for prototyping and gene expression analysis.Clostridioides difficile is an obligate anaerobic, Gram-positive bacterium that produces toxins. Despite technological progress, conducting gene expression analysis of C. difficile under different conditions continues to be labor-intensive. Therefore, there is a demand for simplified tools to investigate the transcriptional and translational regulation of C. difficile. The cell-free gene expression (CFE) system has demonstrated utility in various applications, including prototyping, protein production, and in vitro screening. In this study, we developed a C. difficile CFE system capable of in vitro transcription and translation (TX-TL) in the presence of oxygen. Through optimization of cell extract preparation and reaction systems, we increased the2025-01-01T00:00:00Z2025 Jan2025-04-04T14:04:56.567Z2025-04-04T14:04:56.567ZS-EPMC117843783974546710.1128/aem.01566-24