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Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

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Design of a tunable bacterial gene expression system using engineered σ factors.

Twinkal Patel¹, Amit Dinda¹, Sankar Mahesh¹

¹Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, India.

Applied and Environmental Microbiology

|April 12, 2024

Summary

Engineered bacterial transcription factors (σ factors) can control gene expression by altering flexible loops. This method enhances biosynthetic pathways in E. coli without genetic modification.

Keywords:

ECF σ factors promoter specificity transcription initiation

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Area of Science:

Bacterial transcription regulation
Synthetic microbiology
Protein engineering

Background:

Extracytoplasmic function (ECF) σ factors regulate specific gene expression in bacteria.
Promoter specificity and transcription initiation efficiency are governed by dynamic polypeptide segments (L3 loop and linker) within σ factors.
Limited conservation of these segments hinders correlation between protein-DNA interactions and transcription efficiency.

Purpose of the Study:

To characterize features governing gene expression dynamic range using chimeric Escherichia coli σ^E.
To engineer σ factors for bespoke gene expression levels.
To improve efficiency of microbial biosynthetic pathways.

Main Methods:

Created chimeric σ^E by replacing L3 loop and linker with segments from 10 Mycobacterium tuberculosis ECF σ factors.
Performed in vitro and in vivo measurements to assess effects of polypeptide replacements on gene expression.
Utilized the chimeric σ^E library to modulate enzyme levels in a two-enzyme biosynthetic pathway in E. coli.

Main Results:

Generated an experimentally validated dataset of gene expression levels for σ^E chimeras.
Demonstrated increased desired product levels in a biosynthetic pathway by altering intracellular enzyme concentrations.
Showed that modifying flexible loops of σ^E modulates target gene expression without altering the genomic makeup.

Conclusions:

Engineering σ factors via modification of flexible loops offers a route to control gene expression.
Chimeric σ^E library enables pre-determined conditional gene expression for synthetic microbiology applications.
This approach facilitates pathway optimization without significant genetic engineering, improving microbial cell fitness.