Advanced and Safe Synthetic Microbial Chassis with Orthogonal Translation System Integration

Through the use of CRISPR-assisted transposition, we have engineered a safe <i>Escherichia coli</i> chassis that integrates an orthogonal translation system (OTS) directly into the chromosome. This approach circumvents the limitations and genetic instability associated with conventional plasmid vectors. Precision in genome modification is crucial for the top-down creation of synthetic cells, especially in the orthogonalization of vital cellular processes, such as metabolism and protein translation. Here, we targeted multiple loci in the <i>E. coli</i> chromosome to integrate the OTS simultaneously, creating a synthetic auxotrophic chassis with an altered genetic code to establish a reliable, robust, and safe synthetic protein producer. Our OTS-integrated chassis enabled the site-specific incorporation of <i>m</i>-oNB-Dopa through in-frame amber stop codon readthrough. This allowed for the expression of advanced underwater bioglues containing Dopa-Lysine motifs, which are crucial for wound healing and tissue regeneration. Additionally, we have enhanced the expression process by incorporating scaffold-stabilizing fluoroprolines into bioglues, utilizing our chassis, which has been modified through metabolic engineering (i.e., by introducing proline auxotrophy). We also engineered a synthetic auxotroph reliant on caged Dopa, creating a genetic barrier (genetic firewall) between the synthetic cells and their surroundings, thereby boosting their stability and safety.