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.
