A Clostridioides difficile cell-free gene expression system for prototyping and gene expression analysis

Area of Science:

• Microbiology and Molecular Biology
• Synthetic Biology

Background:

• Clostridioides difficile is an anaerobic, Gram-positive bacterium and an urgent threat due to antibiotic resistance.
• Current gene expression analysis of C. difficile is labor-intensive, requiring anaerobic conditions and inefficient genetic material introduction.
• Cell-free gene expression (CFE) systems offer a simplified approach for in vitro transcription and translation (TX-TL).

Purpose of the Study:

• To develop a simplified Clostridioides difficile cell-free gene expression (CFE) system for in vitro analysis.
• To optimize the CFE system for enhanced protein yield and assess its utility for studying gene regulation and toxin expression.

Main Methods:

• Development and optimization of a C. difficile cell extract preparation and reaction system.
• Evaluation of linear DNA templates versus circular plasmids for in vitro expression.
• Assessment of the CFE system's prototyping capability using synthetic Clostridium promoters.
• Testing expression of toxin genes (tcdB, tcdR) from clinical C. difficile isolates.

Main Results:

• A functional C. difficile CFE system was established, enabling in vitro TX-TL in the presence of oxygen.
• Optimized conditions significantly increased protein yield, with linear DNA templates outperforming plasmids.
• The CFE system demonstrated a good correlation between in vivo and in vitro promoter activity.
• Higher toxin expression was confirmed for the hypervirulent R20291 strain using the CFE system.

Conclusions:

• The developed C. difficile CFE system provides a simplified platform for in vitro protein synthesis and genetic part prototyping.
• This system facilitates accelerated study of gene expression and function in C. difficile, even in the presence of oxygen.
• The CFE system holds potential as a simplified model for investigating metabolic regulations in C. difficile.