Development of a Multiplex Modular Cloning Assembly System

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Sonya Iverson, Traci Haddock, Douglas Densmore

Boston University, United States

Assembling larger biological systems from primitive Parts is a key design paradigm in synthetic biology. While DNA synthesis technology continues to improve, piecewise assembly will remain crucial for rapid combinatorial assembly of biological Devices. This approach can develop and expand only as quickly as DNA assembly systems allow. Most efficient assembly systems currently either require one-off planning and oligo synthesis or depend upon slower assembly methods but allow more modularity. Utilizing a variation of modular cloning (MoClo, Weber et al., 2011) and a growing library of interchangeable DNA Parts, we have a developed a multiplex modular cloning (MMC) methodology which rapidly increases the efficiency of characterization efforts and construction of related Devices. Standard MoClo assembly allows for the rapid assembly of up to six DNA Parts in a single digestion-ligation reaction by utilizing restriction type IIS enzymes. By adjusting the reaction conditions, Multiplex Modular Cloning provides the ability to construct multiple Devices by adding a library of any one Part type rather than single Parts with only a modest decrease in ligation efficiency. Additionally, MMC can be designed as a high throughput assembly and screening system where in a variety of design candidates are assembled and functionally screened for activity by transforming into cells along with a reporter cassette such that only cells in which the variable construct functions correctly produces antibiotic resistance or a visible reporter. Further efforts are underway to integrate this assembly and screening methodology with a combination of liquid handling robotics and flow cytometry. This variation of assembly methodology and integration with computational design and analysis tools will rapidly increase the rate at which related devices can be synthesized and characterized.