Abstract
Advances in molecular and synthetic biology call for efficient assembly of multi-modular DNA constructs. We hereby present a novel modular cloning method that obviates the need for restriction endonucleases and significantly improves the efficiency in the design and construction of complex DNA molecules by standardizing all DNA elements and cloning reactions. Our system, named HomeRun Vector Assembly System (HVAS), employs a three-tiered vector series that utilizes both multisite gateway cloning and homing endonucleases, with the former building individual functional modules and the latter linking modules into the final construct. As a proof-of-principle, we first built a two-module construct that supported doxycycline-induced expression of green fluorescent protein (GFP). Further, with a three-module construct we showed quantitatively that there was minimal promoter leakage between neighbouring modules. Finally, we developed a method, in vitro Cre recombinase-mediated cassette exchange (RMCE) cloning, to regenerate a gateway destination vector from a previous multisite gateway cloning reaction, allowing access to existing DNA element libraries in conventional gateway entry clones, and simple creation of constructs ready for in vivo RMCE. We believe these methods constitute a useful addition to the standard molecular cloning techniques that could potentially support industrial scale synthesis of DNA constructs.
Highlights
Modern molecular biology owes much to the invention of molecular cloning which creates recombinant DNA molecules, and allows individual DNA elements to be studied in detail
To further improve the flexibility of modular cloning, we present a new cloning scheme, termed HomeRun Vector Assembly System (HVAS), to take advantage of both multisite gateway cloning and homing endonucleases, with the former building modules from DNA elements, while the latter assembling modules into a final construct
We show here that, starting from a DNA element library in the form of gateway entry clones, no restriction endonucleases or PCR are required for building a functional multi-modular DNA construct, and as such the entire cloning process could be standardized
Summary
Modern molecular biology owes much to the invention of molecular cloning which creates recombinant DNA molecules, and allows individual DNA elements to be studied in detail. Despite development of many new technologies, restriction endonuclease based methods remain the cornerstone of molecular cloning. For each cloning step they must be individually selected based on restriction analysis of both the insert and vector. The construct is often nearly impossible to modify. For these reasons, despite what was implied by ‘‘engineering’’ in its name, genetic engineering is still an art that requires advanced craftsmanship and thoughtful efforts, and, as such, is not amenable for automation or high-throughput production
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