Bacterial Cells Engineered with Synthetic Genetic Materials for Blind Testing of Random Mutagenesis

Synthetic genetic materials, particularly those in genetically modified organisms (GMOs) deployed into complex environments, necessitate robust postmarket surveillance for continuous monitoring of both the materials and their applications throughout their lifecycle. Here, we introduce novel-coded genomic material for a blind mutation test that evaluates mutagenesis in synthetic genomic sequences without requiring direct sequence comparison. This test utilizes a Genome-Digest, which is embedded within essential genes, establishing mathematical correlation between the nucleotide sequence and codon order. This novel design allows for independent assessment of mutations by decoding the nucleotide sequence, thereby eliminating the need for reference sequences or extensive bioinformatic analysis. Furthermore, the test has the capability to analyze mixed genomic materials from a single sample and can be extended to the pooled testing of multiple samples as well. Building on this framework, we propose the ‘Genome-ShockWatch’ methodology. In proof-of-concept trials, it successfully detected mutations that exceeded a predefined threshold in long-read sequencing data from a yogurt sample containing Genome-Digest encoded Nissle 1917 E. coli cells and naturally occurring probiotic bacteria. Consequently, the Genome-Digest system provides a robust foundation for the routine surveillance and management of GMOs and related synthetic products, ensuring their safety and efficacy in diverse environmental contexts.