This technology enhances plasmid-based gene transfer by utilizing a leading region enriched with anti-defense genes. It enables more efficient overcoming of bacterial immune systems, improving applications in biotechnology and microbial engineering.

ABSTRACT
Our groundbreaking technology dramatically improves plasmid-based gene transfer by utilizing a strategically designed leading region enriched with anti-defense genes. We revealed that the leading region of plasmids, which is the first to enter recipient cells, is enriched with a diverse repertoire of “anti-defense” genes that can counteract bacterial defenses, and designing this region can enable overcoming bacterial immune systems, significantly enhancing conjugation, thus advancing applications in biotechnology, microbial engineering, and potentially revolutionizing fields from bioremediation to personalized medicine.

UNMET NEED
Plasmids have been explored as potential conjugative delivery systems for editing natural microbial communities and other biotechnological applications. Examples include targeting antibiotic-resistant bacteria using CRISPR-Cas, editing human microbiomes, and editing soil or aquatic microbial communities to provide them with new metabolic capabilities. However, current human-design conjugation systems remain mostly inefficient. Factors such as low transfer rates, inability of plasmids to establish, and competition with other mobile elements limit successful plasmid dissemination. Understanding the molecular mechanisms that allow plasmids to evade bacterial immunity is key for designing effective strategies to control and modulate gene dissemination via conjugation.

OUR SOLUTION
Our discoveries provide a novel set of genetic tools that facilitate the design of efficient conjugation-based delivery systems that can be applied for a wide range of medical and biotechnological applications. By leveraging the diverse repertoire of “anti-defense” genes and specialized promoters encoded in the leading regions of plasmids, we can engineer effective plasmid vectors capable of overcoming bacterial defenses during conjugation. This knowledge can guide the development of novel CRISPR-based antimicrobials, enhancement of bioreactors’ capabilities to degrade biomass for biofuel production, utilization of naturally-occurring bacteria for sequestration of greenhouse gases or degradation of contaminants, microbiome manipulation, and alteration of microbial communities for various other biotechnological and medical applications.

UNIQUE ADVANTAGES
• Early Expression of Anti-Defense and Selected Genes: Ensures rapid suppression of bacterial defense systems, such as CRISPR-Cas, restriction-modification, and SOS-response systems, during gene transfer.
• Superior Conjugation and Transfer Efficiency: Demonstrated more than 200 fold increase in transfer efficiency. Can be applied in various microbial communities, including those resistant to conventional plasmid technologies.
• Broad Spectrum of Defense Inhibition: Can target multiple bacterial defenses, offering a strategic approach to recipient counteract resistance mechanisms.
• Adaptable and Versatile Technology: Can be integrated into various plasmid systems, making it applicable for various bacterial hosts and across different fields such as biotechnology, pharmaceuticals, and microbial genetics, with substantial commercial potential.

POTENTIAL APPLICATIONS
• Antibiotic Resistance Management: Development of targeted tools to combat antibiotic-resistant bacteria by efficiently transferring resistance-neutralizing genes, potentially revolutionizing treatment of resistant infections.
• Bioremediation and Environmental Management: Engineer microbial strains for pollutant degradation, greenhouse gas sequestration, and ecosystem restoration, addressing critical environmental challenges.
• Industrial Biotechnology: Create highly efficient synthetic microbial strains for biofuel production, bioremediation, valuable biochemicals synthesis , and other industrial bioprocesses, potentially reducing costs and improving yields.
• Pharmaceutical Development: Use in microbial drug production and genetic manipulation for drug discovery.
• Agriculture and Food Technology: Develop crop protection strategies and enhance soil health through engineered beneficial microbes, potentially increasing crop yields and reducing chemical inputs.
• Medical Microbiome Modulation: Enable precise manipulation of human microbiomes for therapeutic purposes, opening new avenues in personalized medicine and treatment of microbiome-related disorders.”
This cutting edge project was developed by Bruria Samuel as a part of her Ph.D. studies

PATENT
PCT/IL2024/050132 METHODS AND CONSTRUCTS FOR IMPROVING CONJUGATION EFFICIENCY

REFERENCES
Samuel, B. & Burstein, D. Diverse anti-defence systems are encoded in the leading region of plasmids. Nature (2024). https://www.nature.com/articles/s41586-024-07994-w