Sentinel cells programmed to respond to environmental DNA including human sequences

Area of Science:

• Synthetic Biology
• Environmental DNA Monitoring
• Molecular Diagnostics

Background:

• Environmental DNA (eDNA) monitoring enables tracking of diverse organisms but faces challenges with continuous sampling and transient sequences.
• Detecting specific DNA in complex environmental samples requires highly sensitive and robust methods.

Purpose of the Study:

• To engineer a living sentinel system for detecting specific DNA sequences in complex environments.
• To enhance DNA uptake efficiency and enable sensitive detection of target sequences, including human SNPs.

Main Methods:

• Designed Bacillus subtilis living sentinels to report DNA sequence uptake upon matching a pre-encoded target.
• Utilized ComK overexpression to achieve a 3,000-fold increase in DNA uptake, enabling femtomolar detection.
• Employed CRISPR interference for in vivo SNP determination by modulating reporter gene expression.

Main Results:

• Demonstrated femtomolar detection limits in samples with high background DNA concentrations.
• Successfully detected human sequences with single-nucleotide polymorphisms (SNPs) associated with facial features.
• Achieved efficient sequence recording, nuclease protection for weeks, and multiplexed detection using single cells or consortia.

Conclusions:

• Engineered Bacillus subtilis living sentinels offer a powerful tool for sensitive and stable DNA sequence detection.
• The system demonstrates potential for long-term surveillance in applications like forensics, ecology, and epidemiology.
• This approach advances the field of environmental monitoring through synthetic biology innovations.