Abstract
We present a Bacterial Expression Vector Archive (BEVA) for the modular assembly of bacterial vectors compatible with both traditional and Golden Gate cloning, utilizing the Type IIS restriction enzyme Esp3I, and have demonstrated its use for Golden Gate cloning in Escherichia coli. Ideal for synthetic biology and other applications, this modular system allows a rapid, low-cost assembly of new vectors tailored to specific tasks. Using the principles outlined here, new modules (e.g., origin of replication for plasmids in other bacteria) can easily be designed for specific applications. It is hoped that this vector construction system will be expanded by the scientific community over time by creation of novel modules through an open source approach. To demonstrate the potential of the system, three example vectors were constructed and tested. The Golden Gate level 1 vector pOGG024 (pBBR1-based broad-host range and medium copy number) was used for gene expression in laboratory-cultured Rhizobium leguminosarum. The Golden Gate level 1 vector pOGG026 (RK2-based broad-host range, lower copy number and stable in the absence of antibiotic selection) was used to demonstrate bacterial gene expression in nitrogen-fixing nodules on pea plant roots formed by R. leguminosarum. Finally, the level 2 cloning vector pOGG216 (RK2-based broad-host range, medium copy number) was used to construct a dual reporter plasmid expressing green and red fluorescent proteins.
Highlights
IntroductionSynthetic biology has emerged as a powerful tool for biotechnology and basic science research (Church et al, 2014)
Over the last decade, synthetic biology has emerged as a powerful tool for biotechnology and basic science research (Church et al, 2014)
We demonstrate the efficacy of this system by constructing several vectors, analogous to useful traditional cloning broad-host range vectors but that are Golden Gate-compatible: level 1 cloning broad-host range vectors pOGG024, a medium copy plasmid for bacterial gene, and pOGG026, a low copy number vector for gene expression in environmental samples where no antibiotic selection is applied
Summary
Synthetic biology has emerged as a powerful tool for biotechnology and basic science research (Church et al, 2014). Coupled with rapidly improving DNA synthesis technologies, one of the key advances that has potentiated the rapid expansion of synthetic biology is new DNA assembly technologies (Kosuri and Church, 2014). The ability for synthetic biologists to rapidly assemble and test new designs at low cost is invaluable to progress in a wide-range of life sciences research. Applications for this range from investigating new antibody production with higher success rates, to increased food production minimizing the carbon footprint with the effective transfer of nitrogen fixation between bacterial species (Smanski et al, 2014).
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