EMMA-CAD: Design Automation for Synthetic Mammalian Constructs

Abstract

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 highthroughput 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 webbased 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.

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

“…We further detail EMMA-CAD functionalities in the companion paper. 27 During the vector assembly stage, Golden Gate master mix and water are transferred into a reaction plate using a Certus Flex bulk dispensing robot (Gyger) (Figure 1B). Plasmid parts are then added using an Echo 550 acoustic dispenser (Labcyte) according to the script generated by EMMA-CAD (Figure 1C).…”

“…This plate was then transferred to an Echo 550 acoustic dispenser (Labcyte), where plasmid parts were dispensed using the 384LDV_AQ_BP2 plate setting calibration according to an uploaded .csv file generated by EMMA-CAD. 27 The plates were sealed, and a plate spinner was used to pool the reagents at the bottoms of the reaction wells prior to thermocycling via the following program: step 1, 37 °C for 5 min; step 2, 37 °C for 5 min; step 3, 16 °C for 10 min; repeat steps 2 and 3 for 25 cycles; step 4, 16 °C for 20 min; step 5, 37 °C for 30 min; step 6, 75 °C for 6 min; step 7, 4 °C, hold. Subsequently, 4 μL of DNase master mix (3.625 μL of T4 DNA ligase buffer (1×) (NEB), 0.25 μL of 25 mM ATP solution (Epicenter), and 0.125 μL of PlasmidSafe DNase (Epicenter)) was added to each assembly reaction using a Certus Flex bulk dispenser (Gyger), and the reactions were incubated further at 37 °C for 15 min.…”

“…27 EMMA-CAD guides users through the design process, reducing the time and effort expended during the vector design phase, and translates the designed vectors into machineexecutable protocols that can be fed directly into the assembly stage of our pipeline. 27…”

“…We calculate the scale of cost and time savings of the automated system using Q-metrics . To complement our automated pipeline and streamline vector design, we have also developed a novel computer-aided design tool, EMMA-CAD, which is described in the companion paper in this issue . EMMA-CAD guides users through the design process, reducing the time and effort expended during the vector design phase, and translates the designed vectors into machine-executable protocols that can be fed directly into the assembly stage of our pipeline …”

FRAGLER: A Fragment Recycler Application Enabling Rapid and Scalable Modular DNA Assembly

“…Mammalian synthetic biology toolkits have a higher level of complexity, in which EMMA is the most comprehensive to date supporting up to three transcription units (TUs) over a 25-module assembly reaction . The EMMA toolkit also has an associated design tool to facilitate building of complex circuits . The Mammalian ToolKit (MTK) supports up to 8 modules that can be used in subsequent assembly reactions to build up to nine TUs .…”