Periodic Reporting for period 1 - DropContEvo (A droplet microfluidic system for continuous in vivo evolution.) Reporting period: 2018-01-01 to 2019-12-31 Summary of the context and overall objectives of the project Directed evolution is now arguably the method of choice for improvement of proteins used in applications ranging from washing powders to fine chemicals and medical applications. Screening and selection of improved variants is widely used both in the research laboratories as well in industry towards improved activity, selectivity and stability of relevant biomolecules. Directed evolution has gained well-deserved recognition in 2018 when the Nobel Prize in Chemistry was awarded to researchers who developed laboratory approaches for discovery of enzymes and biotherapeutics. However, screening and selection of mutated variants of enzymes is currently performed mainly in low or moderate throughput and often uses liquid chromatography and mass spectrometry for detection of the reaction output. Implementation of these screening methods is costly and usually requires substantial investments both on laboratory and on industrial scales. Droplet microfluidics offers an unprecedented increase in the screening throughput of reactions that can be measured within a single day and using greatly reduced volumes of reagents. The analysis of the enzymatic activity in a microdroplet format requires capturing of the reaction product and the cell together in microcompartments to maintain the genotype-phenotype linkage. Recently it has become possible to generate pico-litre volume water-in-oil droplets containing single cells, incubate them and sort them in high throughput. However, a microfluidics approach is partially compromised by technical limitations of microfluidic modules, limited choice of methods of optical readouts of the reaction outcome and general low accessibility to non-specialized researchers. Microdroplet technologies so far have been used only for limited number of reactions, usually using non-natural bait substrates and with limited success. This project aimed at development of new microfluidic modules and strategies dedicated mainly for in-vivo directed evolution. Experimental laboratory evolution must comprise four key steps: mutation, gene expression, selection, and replication. The key to success is the ability to handle the enormous combinatorial diversity that randomization of the amino acids of protein brings about. From the point of view of development of microfluidic technology, the project aimed at development of novel and highly reliable microfluidic modules for long long-term and unsupervised experiments involving multiple operations on a single chip - e.g. generation of droplets, cultivation of microorganisms in hundreds of thousands of picoliter droplets, picoinjection of substrates or nutrients, splitting of droplets and high-throughput sorting. The second objective of the project was to test and develop new enzymatic assays and alternative formats of protein expression in microdroplet format. One of such alternative is to use whole cell microdroplet assays combined with microbial growth towards the in vivo continuous evolution. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The technical goal of the project was to develop new droplet microfluidic technologies for directed evolution of biocatalysts using bacteria as hosts expressing enzymes. In collaboration with co-workers, I developed several automated microfluidic modules, which were further validated and integrated to multiple directed evolution campaigns. Those microfluidic devices allowed for unsupervised and reliable selection of single bacteria or single cell lysates expressing variants of enzymes characterized by improved catalytic properties. With respect to the main objective of the project, the microfluidic devices support all steps of in vivo directed evolution protocol, such as: encapsulation of single cells, bacteria growth, splitting of microbial population into single cells and selection of the most active whole-cell biocatalysts displaying enzymes on the surface. Development of novel and superior microfluidic technologies also opened new research opportunities. The main research focus was implementation of Scatter Activated Droplet Sorter for discovery and evolution of enzymes degrading polyester plastic particles e.g. bottle PET. In this assay the PET plastic nanoparticles were encapsulated together with single bacteria expressing thousands of variants of enzyme degrading PET nanoplastics, in a way that only single variant of the enzyme was present in a single microdroplet. During the screening campaign, we have isolated and identified dozens of variants of enzymes with improved catalytic activity towards plastic degradation. The genes of new variants were sequenced and selected enzymes were further characterized via secondary screening using HPLC and microtiter plate assays. Currently, we are finalizing the project and preparing the manuscript describing the first successful ultra-high throughput assay applied to discovery of new polyester plastics-degrading enzymes. Devices developed for the purpose of the project are characterized by universal applicability and high reliability and suggesting that they can be quickly adopted by others. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) In this project, I developed series of universal microfluidic devices characterized by high reliability and superior performance. Novel and robust microfluidic modules will support further efforts towards integration into complete systems for continuous in vivo evolution. The development of a Scatter Activated Droplet Sorter (SADS), initially aimed to select quickly growing bacteria variants, has found a unique and breakthrough application in evolving of enzymes degrading PET plastics. This project has already proliferated into several subprojects in the host laboratory and brought interest of industrial partners. Plastic and micro-plastic pollution is one of the biggest socio-economic concern and currently there are extensive research efforts taking place towards a development of efficient and sustainable methods for plastic removal and recycling. We believe that novel microfluidic technologies developed during this project will help in the close future to discover and improve new highly specific and active biocatalysts that will be used in industrial process of enzymatic bio-recycling. Microfluidic screening workflows used in the DropContEvo project
