Innovations in Droplet Microfluidics for Enzyme Evolution
Creators: name: Holland-Moritz, Daniel
Publisher: name: My University
Year: 2020
Subjects: subject: Microfluidics
subject: Mass Spectrometry
subject: Directed Evolution
subject: Protein Engineering
subject: High Throughput Screening
subject: Biological Chemistry
subject: Chemical Engineering
subject: FOS: Chemical engineering
subject: Chemistry
subject: Science

description: High throughput screening (HTS) is a critical technology in discovery and process chemistry. Typically, HTS has relied on robotic handling of well plates of samples to rapidly perform and assess reactions, but novel technologies that take advantage of microfluidic sample processing hold potential as alternatives to typical well plate based assays. Droplet based microfluidics has opened the door to the execution of millions of reactions on the nanoliter scale but has lagged in application to high throughput screening due to limitations in the options for analysis of nanoliter samples, which has largely been performed using optical detection. The work described here addresses this analytical challenge, detailing the design and development of an integrated microfluidic system for mass activated droplet sorting (MADS). The MADS system uses electrospray ionization mass spectrometry (ESI-MS) to analyze the contents of nanoliter scale reactions, and microfluidics to both perform these reactions at nanoliter scale and sort and recover the droplets in which they were performed. MADS expands the toolbox for droplet detection and recovery, broadening the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows. To demonstrate the utility of the MADS system, it is applied to the process of Directed Evolution. Directed evolution of biocatalytic enzymes enables the design of enzymes that perform new catalytic functions, but requires screening thousands of modified enzymes to identify mutations favorable to new catalytic functions. In the pursuit of applying MADS sorting to biocatalytic reactions in droplets, this work details the development of the microfluidic devices and molecular biology workflow for the expression of a library of enzyme variants in vitro, each in its own 30 nL droplet. Digital droplet polymerase chain reaction (ddPCR) is applied to amplify individual DNA copies in microdroplets, overcoming statistical limitations in droplet loading using a high throughput fluorescence activated droplet sorting (FADS) system to enrich for individual droplets containing amplified DNA. PCR amplified droplet samples are paired with droplets of in vitro transcription and translation (ivTT) matrix to create a final library of droplets expressing enzymes in vitro. These samples are screened using MADS to demonstrate the capability of the system to enrich the best performing enzymes from a pool of library variants. As proof of concept, a deleterious mutation is introduced into an existing transaminase enzyme and this enzyme is then used as the backbone for a 2000 variant enzyme library. Proof of concept is demonstrated by enrichment of the original wildtype enzyme from this library, and recovery of the original enzyme activity. The work completed demonstrates the capability of the MADS system to screen tens of thousands of enzyme variants, using just a few milliliters of reagent and at significantly improved throughputs relative to traditional screening.