Automation Assisted Anaerobic Phenotyping for Metabolic Engineering

Microorganisms can be metabolically engineered to produce a wide range of commercially important chemicals. Advancements in computational strategies for strain design and synthetic biological techniques to construct the designed strains have facilitated the generation of large libraries of potential candidates for chemical production. Consequently, there is a need for high-throughput, laboratory scale methods to characterize and screen these candidates to select strains for further investigation in large scale fermentation processes. Several small scale fermentation techniques, in conjunction with laboratory automation have enhanced the throughput of enzyme and strain phenotyping experiments. However, such high throughput experimentation typically entails large operational costs and generate massive amounts of laboratory plastic waste. In this work, we present an eco-friendly automation work-flow that effectively calibrates and decontaminates fixed-tip liquid handling systems to reduce tip waste. We also investigate inexpensive methods to establish anaerobic conditions in microplates for high-throughput anaerobic phenotyping. To validate our phenotyping platform, we perform two case studies - an anaerobic enzyme screen, and a microbial phenotypic screen. We used our automation platform to investigate conditions under which several strains of E. coli exhibit the same phenotypes in 0.5 L bioreactors and in our scaled-down fermentation platform. Further, we use dimensionality reduction through t-distributed stochastic neighbours embedding in conjunction with our phenotyping platform to serve as an effective scale-down model for bioreactor phenotypes. By integrating an in-house data-analysis pipeline, we were able to accelerate the 'test' phase of the design-build-test-learn cycle of metabolic engineering.