Microfluidics – are tiny droplets the next big thing in directed evolution?

The quickly maturing technology of microfluidics is opening up new possibilities in enzyme engineering

TEV_protease_summary

Figure 1. Structure of a protease. Some are used in laundry detergents. (CC)  Source: wikimedia.org 

Over billions of years, evolution has created enzymes that enable biochemical reactions with remarkable catalytic power. Powerful catalysts such as enzymes also have many industrial or medical applications, for example enzymes in washing powder help clean your clothes. In order for us to use enzymes, we often have to engineer them to change properties like stability in unfriendly environments (i.e. washing machines) or we have to give enzymes completely new activities, such as the ability to synthesise a drug.

The challenge of engineering new proteins is that we do not yet understand enzymes well enough to design them on a computer. Instead, we use directed evolution techniques which mimic natural evolution. But unlike nature, researchers with the pressure to publish do not have the luxury of being able to wait for years. In addition, we often cannot select for the desired properties. Selection in a continuously evolving population can cause extremely fast evolution, for example using PACE, because the population is large.

In many directed evolution efforts however, mutated variants of an enzyme have to be laboriously screened. The enzyme has to be made for example in bacteria and then bacterial colonies, each containing a different variant of the enzyme, have to be tested for enzymatic activity. This limits the size of most academic projects to tens of thousands of mutants, much less than what can be selected from a population of many millions or even billions of mutants. In particularly difficult cases, for example when a completely novel activity is to be evolved, improved variants may be too rare to be found in such a small screen.

But what if we could screen millions of variants? Microfluidics now allows us to screen greater and greater numbers of variants and promises to significantly enhance our ability to evolve new proteins (Zinchenko et al, 2014): single cells, each expressing a different variant of an enzyme, are first encapsulated in oil to create picolitre droplets.

Enzyme variants

Figure 2.  Enzyme variants are encapsulated in droplets. Active enzymes cause the droplet to become fluorescent when an appropriate substrate is supplied. The droplets are then sorted using FACS and the most fluorescent ones selected as the winners. Source: (Kintses et al, 2012).

The cells are then lysed within the droplets and the substrate is supplied to them. A recent advance was to encapsulate the lysate-in-oil droplets themselves in water to create a cell-like structure (water surrounding a layer of oil, which surrounds proteins and DNA). This allows the droplets to be efficiently sorted using FACS machines. So far, the technique still requires the product of the enzymatic reaction to be fluorescent: all non-fluorescent droplets (with no product), which may contain an enzyme variant with a mutation that lowers activity, are discarded, while the fluorescent droplets containing enzymes with beneficial mutations are kept. As the DNA encoding the mutant enzyme is encapsulated along with the enzyme, it can easily be recovered to introduce more mutations in the best variants.

Amazingly, this new method allowed researchers to find a single active variant among a million of inactive ones. This demonstrates that even extremely rare but highly beneficial mutations can now be found by screening. In my own Masters’ research I attempted to use directed evolution to make an enzyme degrading organophosphate pesticides that pollute the environment. I started using traditional techniques of small screens with thousands of variants and achieved an increase in activity of only threefold. I then switched to using microfluidics and got more encouraging results, but, as in many cases, additional research is needed before we can definitively conclude that the new method is better than the old.

Lucas Kuhlen quadrat

 

Lucas Kuhlen, soon-to-be PhD student in structural biology, passionate procrastinator, interested in international relations and collaborator of WhatIf

 

 

References:

1. Zinchenko, A., Devenish, S., Kintses, B., Colin, P., Fischlechner, M. and Hollfelder, F. (2014). One in a Million: Flow Cytometric Sorting of Single Cell-Lysate Assays in Monodisperse Picolitre Double Emulsion Droplets for Directed Evolution. Analytical Chemistry, 86(5), pp.2526-2533.

2. Kintses, B., Hein, C., Mohamed, M., Fischlechner, M., Courtois, F., Lainé, C. and Hollfelder, F. (2012). Picoliter Cell Lysate Assays in Microfluidic Droplet Compartments for Directed Enzyme Evolution. Chemistry & Biology, 19(8), pp.1001-1009.

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