Abstract
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.
Highlights
New DNA-assembly methods have been developed in recent years, and costs of DNA synthesis are constantly decreasing (Kosuri and Church, 2014; Chao et al, 2015)
The same is true for their gene organization and expression mechanisms leading to the important question of how the genome as operating system fits to a specific chassis (Danchin, 2012)
The efficiency of transplantation decreases with increasing evolutionary distance between chromosome donor and recipient (Labroussaa et al, 2016). Such chromosome transplantation is fundamentally different from other hosts of synthetic chromosomes where the extra DNA is maintained within the Optimization and Characterization of the Synthetic Secondary Chromosome synVicII
Summary
New DNA-assembly methods have been developed in recent years, and costs of DNA synthesis are constantly decreasing (Kosuri and Church, 2014; Chao et al, 2015). On the basis of oriII from V. cholerae we previously constructed a prototype of the synthetic secondary chromosome synVicII in E. coli (Messerschmidt et al, 2015). To test if the synVicII-integration frequency is dependent on homologous sequences on the replicon, we inserted two different genetic regions that occur on the E. coli chromosome.
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