A self-replicating artificial module-genome that generates bacterial chromosome replication system in vitro.

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• Autonomous self-reproduction is a key objective in bottom-up synthetic biology for creating artificial cells.
• Genome replication by self-encoded machinery is essential for this goal.
• The Escherichia coli Replication-Cycle Reaction (RCR) system is a promising platform for genome replication but has not been generated from genetic information.

Purpose of the Study:

• To achieve self-replication of a genome module from genetic information.
• To develop a system for genome-scale replication in a synthetic biology context.
• To advance the construction of self-reproducing artificial cells.

Main Methods:

• Construction of a 53 kb circular DNA module (RCR module-genome) encoding all 26 RCR proteins.
• Expression of the RCR module-genome using the protein synthesis using recombinant elements (PURE) system.
• Optimization of reaction conditions and resolution of expression bottlenecks for robust self-replication.

Main Results:

• Demonstrated robust self-replication of the RCR module-genome in a one-pot reaction.
• Achieved over 28 doublings of recursive self-replication.
• Established the PRIMES (PURE-driven RCR for In-vitro Module-gEnome Self-replication) system.

Conclusions:

• The PRIMES system enables self-replication of an artificial module-genome from genetic information.
• This represents a significant milestone toward the creation of self-reproducing artificial cells.
• The study provides a foundation for future bottom-up synthetic biology endeavors aiming for cellular autonomy.