RoboMoClo: A Robotics-Assisted Modular Cloning Framework for Multiple Gene Assembly in Biofoundry

Efficient and versatile DNA assembly frameworks have had an impact on promoting synthetic biology to build complex biological systems. To accelerate system development, laboratory automation (or biofoundry) provides an opportunity to construct organisms and DNA assemblies via computer-aided design. However, a modular doning (MoClo) system for multiple DNA assemblies limits the biofoundry workflow in terms of 10 . simplicity and feasibility by preparing the number of doning materials such as destination vectors prior to the automation process. Herein, we propose robot-assisted MoClo (RoboMoClo) to accelerate a synthetic biology project with multiple gene expressions at the biofoundry. The architecture of the RoboMoClo framework provides a hybrid strategy of hierarchical gene assembly and iterative gene assembly, and fewer destination vectors compared with other MoClo systems. An industrial bacterium, Corynebacteriunt glutamicunt, was used as a model host for RoboMoClo. After building a biopart library (promoter and terminator; level 0) and evaluating its features (level 1), various transcriptional directions in multiple gene assemblies (level 2) were studied using the RoboMoClo vectors. Among the constructs, the convergent construct exhibited potential transcriptional interference through the collision of RNA polymerases. To study design of experiment-guided lycopene biosynthesis in C. glutamicum (levels 1, 2, and 3), the biofoundry-assisted multiple gene assembly was demonstrated as a proof-of-concept by constructing various sub-pathway units (level 2) and pathway units (level 3) for C. glutamicum. The RoboMoClo framework provides an improved MoClo toolkit for laboratory automation in a synthetic biology application.

Automated summary

Efficient and versatile DNA assembly frameworks have had a significant impact on promoting synthetic biology. However, the traditional MoClo system for multiple DNA assemblies in a biofoundry workflow has limitations in terms of simplicity and feasibility. In this study, a robot-assisted MoClo (RoboMoClo) system was proposed to accelerate synthetic biology projects with multiple gene expressions. The RoboMoClo framework combines hierarchical gene assembly and iterative gene assembly strategies, and requires fewer destination vectors. The application of RoboMoClo in an industrial bacterium, C. glutamicum, demonstrated its potential in laboratory automation for synthetic biology.