Multilayered safety framework for living diagnostics in the colon.

Area of Science:

• Synthetic biology
• Microbial diagnostics
• Genetic engineering

Background:

• Colorectal cancer is a leading cause of cancer death globally.
• Current screening methods for colorectal cancer have limitations in detection rates and specificity.
• There is a need for innovative diagnostic tools to improve early detection and patient outcomes.

Purpose of the Study:

• To develop a novel living diagnostic tool for colorectal cancer using synthetic biology.
• To engineer a strain of *Escherichia coli* Nissle 1917 for detecting cancer biomarkers and reporting results in stool.
• To implement a multi-layered biosafety strategy for the safe *in vivo* application of genetically modified bacteria.

Main Methods:

• Development of an engineered *Escherichia coli* Nissle 1917 strain.
• Design of a synthetic chimeric receptor for mucin-dependent gene expression, confining bacteria to the colon.
• Implementation of a temperature-sensitive kill switch for viability control outside the human body.
• Integration of a CRISPR-Cas-mediated emergency kill switch for rapid bacterial inactivation.

Main Results:

• The engineered bacteria successfully bind to tumor cells and detect two distinct colorectal cancer biomarkers.
• A colored protein is secreted by the bacteria, making the diagnostic signal observable in stool.
• The implemented biosafety measures include auxotrophy to mucin, a temperature-sensitive kill switch, and an emergency CRISPR-Cas kill switch.
• The safe-by-design strategy ensures bacterial confinement to the colon, limits viability to the human body, and provides an emergency shutdown mechanism.

Conclusions:

• The Colourectal project presents a viable synthetic biology approach for a living diagnostic tool for colorectal cancer.
• The multi-layered biosafety strategy provides a robust framework for the safe development and potential future application of engineered microorganisms in human diagnostics.
• This research contributes to advancing the field of microbial diagnostics and offers a promising direction for improving colorectal cancer screening.