Abstract:Synthetic biology aims to improve human health and the environment by repurposing biological enzymes for use in practical applications. However, natural enzymes often function with suboptimal activity when engineered into biological pathways or challenged to recognize unnatural substrates. Overcoming this problem requires efficient directed evolution methods for discovering new enzyme variants that function with a desired activity. Here, we describe the construction, validation, and application of a fluorescen… Show more
“…However, unlike CSR and CST, microfluidic devices are used to generate a uniform population of droplets. The latest microfluidic designs are capable of generating 18 μm droplets at a rate of 30 000 per second, which allows for the production of >10 8 droplets in 1 h (Vallejo et al ., 2019). Following droplet production, the polymerase and encoding plasmid are released into the droplet by lysing the E. coli with heat.…”
Section: Engineering Polymerases By Directed Evolutionmentioning
confidence: 99%
“…Polymerases that successfully copy the template into full-length product produce a fluorescent signal by disrupting a donor–quencher pair located at the 5′-end of the template strand. The population of droplets can then either be sorted directly using a custom microfluidic fluorescence-activated droplet sorting (FADS) device or converted to double emulsion droplets that are compatible with a traditional fluorescence-activated cell sorting instrument (Vallejo et al ., 2019). Fig.…”
Section: Engineering Polymerases By Directed Evolutionmentioning
DNA polymerases play a central role in biology by transferring genetic information from one generation to the next during cell division. Harnessing the power of these enzymes in the laboratory has fueled an increase in biomedical applications that involve the synthesis, amplification, and sequencing of DNA. However, the high substrate specificity exhibited by most naturally occurring DNA polymerases often precludes their use in practical applications that require modified substrates. Moving beyond natural genetic polymers requires sophisticated enzyme-engineering technologies that can be used to direct the evolution of engineered polymerases that function with tailor-made activities. Such efforts are expected to uniquely drive emerging applications in synthetic biology by enabling the synthesis, replication, and evolution of synthetic genetic polymers with new physicochemical properties.
“…However, unlike CSR and CST, microfluidic devices are used to generate a uniform population of droplets. The latest microfluidic designs are capable of generating 18 μm droplets at a rate of 30 000 per second, which allows for the production of >10 8 droplets in 1 h (Vallejo et al ., 2019). Following droplet production, the polymerase and encoding plasmid are released into the droplet by lysing the E. coli with heat.…”
Section: Engineering Polymerases By Directed Evolutionmentioning
confidence: 99%
“…Polymerases that successfully copy the template into full-length product produce a fluorescent signal by disrupting a donor–quencher pair located at the 5′-end of the template strand. The population of droplets can then either be sorted directly using a custom microfluidic fluorescence-activated droplet sorting (FADS) device or converted to double emulsion droplets that are compatible with a traditional fluorescence-activated cell sorting instrument (Vallejo et al ., 2019). Fig.…”
Section: Engineering Polymerases By Directed Evolutionmentioning
DNA polymerases play a central role in biology by transferring genetic information from one generation to the next during cell division. Harnessing the power of these enzymes in the laboratory has fueled an increase in biomedical applications that involve the synthesis, amplification, and sequencing of DNA. However, the high substrate specificity exhibited by most naturally occurring DNA polymerases often precludes their use in practical applications that require modified substrates. Moving beyond natural genetic polymers requires sophisticated enzyme-engineering technologies that can be used to direct the evolution of engineered polymerases that function with tailor-made activities. Such efforts are expected to uniquely drive emerging applications in synthetic biology by enabling the synthesis, replication, and evolution of synthetic genetic polymers with new physicochemical properties.
“…Moreover, the breadth of enzyme classes that can be engineered has also been expanded, owing to the rapid increase in the number of chemistries made applicable in droplet formats. Recent examples of library screening campaign performed in microfluidic workflows involve enzyme classes including aldolases , hydrolases (e.g., sulfatases, amylases) , amino acid dehydrogenase , as well as polymerases . For more detailed discussions of the relevant reactions, interested readers are kindly asked to refer to review articles drafted by Mair et al.…”
Section: Biology In Droplets: Current Droplet‐based Platforms For Thementioning
Natural enzymes have evolved over millions of years to allow for their effective operation within specific environments. However, it is significant to note that despite their wide structural and chemical diversity, relatively few natural enzymes have been successfully applied to industrial processes. To address this limitation, directed evolution (DE) (a method that mimics the process of natural selection to evolve proteins toward a user-defined goal) coupled with droplet-based microfluidics allows the detailed analysis of millions of enzyme variants on ultra-short timescales, and thus the design of novel enzymes with bespoke properties. In this review, we aim at presenting the development of DE over the last years and highlighting the most important advancements in droplet-based microfluidics, made in this context towards the high-throughput demands of enzyme optimization. Specifically, an overview of the range of microfluidic unit operations available for the construction of DE platforms is provided, focusing on their suitability and benefits for cell-based assays, as in the case of directed evolution experimentations.
“…Surfactant systems enable the stabilization of droplets such that they can be incubated off-chip and reintroduced intact into subsequent microfluidic device(s) for sorting and analysis. However, it was not until the most recent decade that the HTS capacity of droplet microfluidics had been demonstrated for protein engineering, especially directed evolution [19,21,22,29,30,31].…”
Protein engineering—the process of developing useful or valuable proteins—has successfully created a wide range of proteins tailored to specific agricultural, industrial, and biomedical applications. Protein engineering may rely on rational techniques informed by structural models, phylogenic information, or computational methods or it may rely upon random techniques such as chemical mutation, DNA shuffling, error prone polymerase chain reaction (PCR), etc. The increasing capabilities of rational protein design coupled to the rapid production of large variant libraries have seriously challenged the capacity of traditional screening and selection techniques. Similarly, random approaches based on directed evolution, which relies on the Darwinian principles of mutation and selection to steer proteins toward desired traits, also requires the screening of very large libraries of mutants to be truly effective. For either rational or random approaches, the highest possible screening throughput facilitates efficient protein engineering strategies. In the last decade, high-throughput screening (HTS) for protein engineering has been leveraging the emerging technologies of droplet microfluidics. Droplet microfluidics, featuring controlled formation and manipulation of nano- to femtoliter droplets of one fluid phase in another, has presented a new paradigm for screening, providing increased throughput, reduced reagent volume, and scalability. We review here the recent droplet microfluidics-based HTS systems developed for protein engineering, particularly directed evolution. The current review can also serve as a tutorial guide for protein engineers and molecular biologists who need a droplet microfluidics-based HTS system for their specific applications but may not have prior knowledge about microfluidics. In the end, several challenges and opportunities are identified to motivate the continued innovation of microfluidics with implications for protein engineering.
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