Efficient assembly of large DNA constructs is a key technology in synthetic biology. One of the most popular assembly systems is the MoClo standard in which restriction and ligation of multiple fragments occurs in a one-pot reaction. The system is based on a smart vector design and type IIs restriction enzymes, which cut outside their recognition site. While the initial MoClo vectors had been developed for the assembly of multiple transcription units of plants, some derivatives of the vectors have been developed over the last years. Here we present a new set of MoClo vectors for the assembly of fragment libraries and insertion of constructs into bacterial chromosomes. The vectors are accompanied by a computer program that generates a degenerate synthetic DNA sequence that excludes "forbidden" DNA motifs. We demonstrate the usability of the new approach by construction of a stable fluorescence repressor operator system (FROS).

The standard outline of bacterial genomes is a single circular chromosome with a single replication origin. From the bioengineering perspective, it appears attractive to extend this basic setup. Bacteria with split chromosomes or multiple replication origins have been successfully constructed in the last few years. The characteristics of these engineered strains will largely depend on the respective DNA replication patterns. However, the DNA replication has not been investigated systematically in engineered bacteria with multiple origins or split replicons. Here we fill this gap by studying a set of strains consisting of (i) E. coli strains with an extra copy of the native replication origin (oriC), (ii) E. coli strains with an extra copy of the replication origin from the secondary chromosome of Vibrio cholerae (oriII), and (iii) a strain in which the E. coli chromosome is split into two linear replicons. A combination of flow cytometry, microarray-based comparative genomic hybridization (CGH), and modeling revealed silencing of extra oriC copies and differential timing of ectopic oriII copies compared to the native oriC. The results were used to derive construction rules for future multiorigin and multireplicon projects.

Regulators of DNA replication in bacteria are an attractive target for new antibiotics, as not only is replication essential for cell viability, but its underlying mechanisms also differ from those operating in eukaryotes. The genetic information of most bacteria is encoded on a single chromosome, but about 10% of species carry a split genome spanning multiple chromosomes. The best studied bacterium in this context is the human pathogen Vibrio cholerae, with a primary chromosome (Chr1) of 3 M bps, and a secondary one (Chr2) of about 1 M bps. Replication of Chr2 is under control of a unique mechanism, presenting a potential target in the development of V. cholerae-specific antibiotics. A common challenge in such endeavors is whether the effects of candidate chemicals can be focused on specific mechanisms, such as DNA replication. To test the specificity of antimicrobial substances independent of other features of the V. cholerae cell for the replication mechanism of the V. cholerae secondary chromosome, we establish the replication machinery in the heterologous E. coli system. We characterize an E. coli strain in which chromosomal replication is driven by the replication origin of V. cholerae Chr2. Surprisingly, the E. coli ori2 strain was not inhibited by vibrepin, previously found to inhibit ori2-based replication.