Automation and Expansion of EMMA Assembly for Fast-Tracking Mammalian System Engineering.

With applications from functional genomics to the production of therapeutic biologics, libraries of mammalian expression vectors have become a cornerstone of modern biological investigation and engineering. Multiple modular vector platforms facilitate the rapid design and assembly of vectors. However, such systems approach a technical bottleneck when a library of bespoke vectors is required. Utilizing the flexibility and robustness of the Extensible Mammalian Modular Assembly (EMMA) toolkit, we present an automated workflow for the library-scale design, assembly, and verification of mammalian expression vectors. Vector design is simplified using our EMMA computer-aided design tool (EMMA-CAD), while the precision and speed of acoustic droplet ejection technology are applied in vector assembly. Our pipeline facilitates significant reductions in both reagent usage and researcher hands-on time compared with manual assembly, as shown by system Q-metrics. To demonstrate automated EMMA performance, we compiled a library of 48 distinct plasmid vectors encoding either CRISPR interference or activation modalities. Characterization of the workflow parameters shows that high assembly efficiency is maintained across vectors of various sizes and design complexities. Our system also performs strongly compared with manual assembly efficiency benchmarks. Alongside our automated pipeline, we present a straightforward strategy for integrating gRNA and Cas modules into the EMMA platform, enabling the design and manufacture of valuable genome editing resources.

EMMA-CAD: Design Automation for Synthetic Mammalian Constructs.

Computational design tools are the cornerstone of synthetic biology and have underpinned its rapid development over the past two decades. As the field has matured, the scale of biological investigation has expanded dramatically, and researchers often must rely on computational tools to operate in the high-throughput investigational space. This is especially apparent in the modular design of DNA expression circuits, where complexity is accumulated rapidly. Alongside our automated pipeline for the high-throughput construction of Extensible Modular Mammalian Assembly (EMMA) expression vectors, we recognized the need for an integrated software solution for EMMA vector design. Here we present EMMA-CAD (https://emma.cailab.org), a powerful web-based computer-aided design tool for the rapid design of bespoke mammalian expression vectors. EMMA-CAD features a variety of functionalities, including a user-friendly design interface, automated connector selection underpinned by rigorous computer optimization algorithms, customization of part libraries, and personalized design spaces. Capable of translating vector assembly designs into human- and machine-readable protocols for vector construction, EMMA-CAD integrates seamlessly into our automated EMMA pipeline, hence completing an end-to-end design to production workflow.