Codon-Restrained Method for Both Eliminating and Creating Intragenic Bacterial Promoters

Area of Science:

• Synthetic Biology
• Molecular Biology
• Genetic Engineering

Background:

• Synthetic biology applications require refactored genetic sequences free of internal regulatory elements.
• Intragenic promoters, including cryptic promoters, can constitute a significant portion of predicted promoters in organisms like *Escherichia coli*.
• Promoter activity relies on the structural interaction between DNA core bases and sigma (σ) factors.

Purpose of the Study:

• To present a novel system, codon-restrained promoter silencing (CORPSE), for the removal of intragenic promoters from coding sequences.
• To introduce an inverted system, iCORPSE, capable of generating highly active promoters within gene sequences without affecting gene function.

Main Methods:

• CORPSE system exploits the DNA-σ factor structural relationship to disrupt embedded σ⁷⁰ promoters.
• Minimal synonymous codon changes are utilized to maintain the integrity of the coding sequence.
• The iCORPSE system inverts the CORPSE strategy to engineer promoter activity.

Main Results:

• The CORPSE system effectively eliminates intragenic promoter activity by altering key nucleotides involved in σ factor interaction.
• The iCORPSE system successfully creates strong promoters within gene sequences while preserving the original gene's functional output.
• Both systems demonstrate precise control over transcriptional activity with minimal sequence modification.

Conclusions:

• CORPSE and iCORPSE offer powerful tools for genetic sequence refactoring in synthetic biology.
• These systems enable the precise removal or insertion of promoter elements, facilitating advanced genetic circuit design.
• The ability to engineer promoter activity without compromising gene function opens new avenues for biological engineering.