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

Abstract

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 cloning (MoClo) system for multiple DNA assemblies limits the biofoundry workflow in terms of simplicity and feasibility by preparing the number of cloning 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, Corynebacterium glutamicum, 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.

Application of Multipopulation Genetic Algorithm in Industrial Special Clothing Design

“…To this end, a multiuser interactive genetic algorithm for population design is proposed. In practical optimization problems, the optimization objectives of some problems cannot be expressed by explicit mathematical functions [15]. Such optimization objectives are called implicit objectives, and the corresponding optimization problems are called implicit objective optimization problems [16].…”

Biofoundry Palette: Planning-Assistant Software for Liquid Handler-Based Experimentation and Operation in the Biofoundry Workflow

“…Automated gene cloning workflow requires continuous execution of the steps in biofoundry. SKy BioFoundry has the capability of semiautomated gene cloning and multiple gene assembly for synthetic biology . The biofoundry workflow involves five steps (Step 1, PCR; Step 2, golden-gate assembly or restriction enzyme reaction; Step 3, E.…”

“…SKy BioFoundry has the capability of semiautomated gene cloning and multiple gene assembly for synthetic biology. 7 The biofoundry workflow involves five steps (Step 1, PCR; Step 2, golden-gate assembly or restriction enzyme reaction; Step 3, E. coli cell transformation; Step 4, colony selection and inoculation; Step 5, plasmid isolation) (Figure 1B). To perform each step, robotic units must handle not only the input materials (i.e., PCR DNA template or destination vector) but also the ready-to-use materials (e.g., buffers, enzyme mix) and supplies (e.g., tips, Petri dishes, and others) as a BEU.…”

“…1 Globally, biofoundries 2,3 have established automated laboratories with integrated molecular biology facilities (MIT-Broad Foundry, MA; iBioFAB, 4 IL; DOE Agile Biofoundry, 5 CA; London DNA foundry, 6 SKy BioFoundry, 7 South Korea, other Global Biofoundry Alliance members 3 ). Lab automation enables full or semiautomated biofoundry workflows to accelerate the DBTL cycle to perform gene cloning, 8 pathway construction, 7 genome engineering, 4 library construction and screening, 9 and others. Biofoundry workflows consist of multiple steps (or process modules) in a biological procedure, such as PCR, golden-gate assembly, cell transformation, colony selection on the Petri dish, incubation of either a Petri dish or test tube, or DNA purification and isolation (Figure 1A).…”

“…The workflow requires multiple before-experiment units (BEUs) to be ready beforehand, prior to initiating the workflow. (B) The BEUs (top panel) included tip racks (200 μL tips and 1000 μL; eight racks), tube racks (up to 2 mL; 32 different racks), troughs (up to 100 mL; three units), plate racks (96-well plates; 24 racks), and stackers for Petri dishes (4 different partitions; 16 floors) designated in either EVO-Cell or EVO-DNA platform 7. Consumables (tips, kits, plates, Petri dishes, etc.)…”