Phage-mediated intercellular CRISPRi for biocomputation in bacterial consortia

Area of Science:

• Synthetic biology
• Microbial engineering
• Genetic circuits

Background:

• Cellular coordination is essential for complex functions in microbial communities and multicellular organisms.
• Biological engineers are developing synthetic cellular consortia with communication systems for enhanced functionalities.

Purpose of the Study:

• To investigate the signaling dynamics of a phage-mediated synthetic DNA messaging system.
• To couple this system with CRISPR interference (CRISPRi) for distributed logic gate operations in bacterial consortia.

Main Methods:

• Utilized a phage-mediated synthetic DNA messaging system for intercellular communication.
• Integrated CRISPR interference (CRISPRi) to regulate gene expression across cells.
• Developed and multiplexed an intercellular CRISPRi (i-CRISPRi) system to implement multicellular logic gates.

Main Results:

• Identified that cell growth phases and resource competition influence communication outcomes.
• Successfully engineered eight orthogonal DNA signals for programmable communication.
• Demonstrated i-CRISPRi's ability to regulate gene expression in a distributed manner across bacterial cells.
• Implemented multicellular logic gates (NOT, YES, AND, AND-AND-NOT) with up to seven cells and three inputs using single- and dual-rail encoding.

Conclusions:

• The developed phage-mediated DNA communication system provides a programmable platform for building complex biological circuits in engineered bacterial communities.
• Intercellular CRISPRi offers a robust mechanism for distributed gene regulation and logic operations in synthetic microbial consortia.
• This work lays the foundation for advanced synthetic biology applications in multicellular systems.