Uracil Excision for Assembly of Complex Pathways

Abstract

Despite decreasing prices on synthetic DNA constructs, higher-order assembly of PCR-generated DNA continues to be an important exercise in molecular and synthetic biology. Simplicity and robustness are attractive features met by the uracil excision DNA assembly method, which is one of the most inexpensive technologies available. Here, we describe four different protocols for uracil excision-based DNA editing: one for simple manipulations such as site-directed mutagenesis, one for plasmid-based multigene assembly in Escherichia coli, one for one-step assembly and integration of single or multiple genes into the genome, and a standardized assembly pipeline using benchmarked oligonucleotides for pathway assembly and multigene expression optimization.

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“…crtEBIY is industrially relevant and a convenient model pathway mainly due to the simple product output (orange color), but also because the robustness of the phenotype seems sensitive to the cell factory design parameters . Indeed, we were unable to obtain surviving colonies by swapping the pathway into vectors with the high-copy pUC origin of replication and we obtained highly variable phenotypes when T7 polymerase was used to drive expression from the construct in comparison with the weaker K1F variant (Figure a). − Overall, we observed a 10-fold difference in the β-carotene product titers, from the lowest to the highest performing cell factories (Supporting Information Table S3) and several of the combinations showed clear toxicity and population bias effects (e.g., pBR322/ROP in combination with chloramphenicol or pSC101 in combination with tetracycline, Figure a). In contrast, the pBR322/ROP origin in combination with both ampicillin and tetracycline was highly performing in both expression strains.…”