The Design, Assembly, and Characterization of a New Library of Standardized Modular DNA Parts

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Traci Haddock, Monique De Freitas, Shawn Jin, Douglas Densmore

Boston University, United States

One of the major hurdles of synthetic biology is in the process of standardizing the assembly of DNA Parts into Devices. The first attempt at addressing this hurdle was in the form of BioBricks, a binary assembly technique with a standardized library of DNA Parts including, but not limited to, promoters, ribosomal binding sites, coding sequences, and terminators. The next phase of DNA assembly included multi-way techniques such as Golden Gate, Gibson, and Gateway. These techniques greatly increased the speed when making Devices, but they lack modularity in terms of interchangeability of Parts and, thus far, there are no standardized libraries of Parts available for these methods. Modular cloning (or MoClo) was introduced in 2011 as a modular, multi-way assembly technique that’s based on the Type IIS restriction enzyme strategy used in Golden Gate assembly. Here, we present a MoClo library of standardized DNA Parts that includes various promoters, ribosomal binding sites, coding sequences, fusion proteins, and transcriptional terminators. The majority of these parts has been converted from BioBricks and will be made available for use through the Registry of Standard Biological Parts. We also discuss our improvements to the reaction time and efficiency of the original MoClo protocol and highlight various logic gate Devices generated using our new library of Parts. In addition to the biological experiments, we demonstrate how the wet lab knowledge of MoClo is captured in the synthetic biology software tools that have been developed in our dry lab. These include the Clotho Apps Eugene Scripter and Raven, as well as a new MoClo format for use in Clotho. Finally, we will discuss the advantages of a collaborative research environment between computational and biological researchers and how this collaboration is leading to an automation pipeline for the design, assembly, and characterization of DNA Devices.