A sigma factor toolbox for orthogonal gene expression in Escherichia coli

Area of Science:

• Synthetic biology
• Molecular biology
• Microbial genetics

Background:

• Synthetic genetic circuits offer programmable control over gene expression for complex pathways.
• Current limitations include a shortage of non-interfering regulatory parts, hindering circuit complexity.
• Orthogonal expression systems are crucial to minimize crosstalk and enable dynamic control of separate genetic modules.

Purpose of the Study:

• To develop a set of orthogonal expression systems for Escherichia coli.
• To enable independent and precise control of multiple genetic circuits.
• To expand the synthetic biology toolbox for assembling complex genetic systems.

Main Methods:

• Utilized heterologous sigma factors from Bacillus subtilis that recognize specific promoter sequences in E. coli.
• Combined up to four sigma factors to achieve orthogonal function between each other and the host.
• Developed promoter libraries for three sigma factors without compromising orthogonality.

Main Results:

• Demonstrated the orthogonal function of multiple sigma factors in E. coli.
• Created diverse promoter libraries enabling a wide range of transcription initiation frequencies.
• Showcased the ability to tune multiple circuit outputs orthogonally in response to sensory signals.

Conclusions:

• The developed sigma factor toolbox provides a robust set of orthogonal expression systems.
• This toolbox facilitates the dynamic control of separate genetic modules within complex synthetic systems.
• It represents a significant expansion of the synthetic biology toolbox for future applications.