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Learning by building is one of the core ideas of synthetic biology research. Consequently, building synthetic chromosomes is the way to fully understand chromosome characteristics. The last years have seen exciting synthetic chromosome studies. We had previously introduced the synthetic secondary chromosome synVicII in  Escherichia coli . It is based on the replication mechanism of the secondary chromosome in  Vibrio cholerae . Here, we present a detailed analysis of its genetic characteristics and a selection approach to optimize replicon stability. We probe the origin diversity of secondary chromosomes from  Vibrionaceae  by construction of several new respective replicons. Finally, we present a synVicII version 2.0 with several innovations including its full compatibility with the popular modular cloning (MoClo) assembly system.

Optimization and Characterization of the Synthetic Secondary Chromosome synVicII in Escherichia coli