Reconstruction of a robust bacterial replication module

Chromosomal DNA replication is a fundamental process of life, involving the assembly of complex machinery and dynamic regulation. In this study, we reconstructed a bacterial replication module (pRC) by artificially clustering 23 genes involved in DNA replication and sequentially deleting these genes from their naturally scattered loci on the chromosome of Escherichia coli. The integration of pRC into the chromosome, moving from positions farther away to close to the replication origin, leads to an enhanced efficiency in DNA synthesis, varying from lower to higher. Strains containing replication modules exhibited increased DNA replication by accelerating the replication fork movement and initiating chromosomal replication earlier in the replication cycle. The minimized module pRC16, containing only replisome and elongation encoding genes, exhibited chromosomal DNA replication efficiency comparable to that of pRC. The replication module demonstrated robust and rapid DNA replication, regardless of growth conditions. Moreover, the replication module is plug-and-play, and integrating it into Mb-sized extrachromosomal plasmids improves their genetic stability. Our findings indicate that DNA replication, being a fundamental life process, can be artificially reconstructed into replication functional modules. This suggests potential applications in DNA replication and the construction of synthetic modular genomes.