Ultra-Sensitive and Ultra-Fast Absorption Spectrometer for Micro-Droplet-based Enzyme Evolution Experiments

Most biological processes are driven by the function of enzymes, these are proteins that bind to a chemical compound and through this interaction alter the chemical compound into a different chemical substance. Our cells are driven by a huge diversity of enzyme functions that dictate everything from how our cells move and grow to how cells convert energy into a usable form. Much of biological, medical and industrial science is focused on understanding how enzymes work and discovering new enzymes that can provide us with new functions for medical and industrial applications. However, our ability to study enzyme function is often limited to focused studies encompassing a handful of minor changes in the gene/protein and, furthermore, is focused on a relatively small pool of previously characterised enzymes. What we need now is new technologies that allow us to study and compare the function of enzymes and which allows us to make huge numbers of changes to that enzyme. Furthermore, such technologies should also allow us to screen huge numbers of protein in order to search a wide diversity of unknown genes for new types of enzyme. This can only be achieved by developing new technologies that allow us to simultaneously sample and investigate millions of genes and then conduct millions of enzyme function experiments in short time frames. The aim of this project is to develop new technologies that allow us to do this.

Enzymes are formed of proteins which are encoded by genes on DNA, the nature of the genetic code recorded in DNA ultimately governs the nature of an enzyme and how it works. We will develop a new technology that will allow us to express thousands of fragments of DNA into protein all at once. We will then use cutting edge micro-plumbing to separate single cells each with different types of DNA/protein into a separate compartment as droplets. Using state of the art methods for studying chemical reactions we will build a new way of detecting chemical changes within each micro-compartment. This innovative and challenging combination of technology will allow us to screen huge numbers of variant enzymes and search DNA of unknown function for new gene functions.

The power of the optofluidic technology proposed will allow us to take highly sensitive measurements surpassing current approaches. Specifically, the new optofluidic technology that will be utilised for detecting chemical changes will be around 1000 times more sensitive than current technologies used. By combining it with the droplet based compartmentation we will be able to screen 1000 independent experiments a second. Our proposed project will unlock the door to a range of new approaches for both investigating how enzyme evolution relates to enzyme function, discovery of new enzymes and down-stream the development of new antimicrobial drugs.

High-throughput, ultrafast and ultrasensitive absorption measurements in micro-droplets can be achieved by integrating whispering-gallery mode optical sensors with state-of-the-art micro-droplet fluidics. Our idea is to monitor the optical losses as the light propagates around the circumference of a micro-drop by total internal reflection, allowing us to measure absorption and therefore study chemical changes within the droplet. The speed, throughput and sensitivity of this droplet-based absorption sensor can surpass currently available droplet-based spectroscopies: the sensitivity as compared to a single pass droplet absorption spectrometer is greatly enhanced, approximately by three orders of magnitude due to the more than ~1000 roundtrips of the light inside the drop. By combining this novel technology with synthetic biology approaches for expressing diverse DNA templates we will build a new tool for high throughput investigation of enzyme function, mutational permutations of enzyme function and screening meta R/DNA templates for discovery of novel enzyme functions.

This project will enable advances in several fields including healthcare, biotechnology and microfluidic engineering. The key potential beneficiaries are therefore biotech firms using biocatalysts and looking to evolve enzymes as synthons, for the use in workflows aiming to produce value-added product. This proposal will provide them with a tool to evolve enzymes. Enzymes are highly relevant biotechnological targets with applications across several fields from protein engineering, drug development to food industry.

Likewise, the discovery of novel and/or amended (hemi)cellulases will provide springboards for the directed evolution of efficient catalysts of lignocellulosic biomass, ultimately leading to increased bioethanol production. This will feed into a growing industry seeking to develop sustainable energy sources.

The second beneficiaries are engineering companies looking to commercialize hardware kits enabling screening of a vast number of enzymatic reactions. In particular microfluidic hardware innovations will be of interest for several biotechnology companies, who are keenly interested in using droplets for high-throughput selection within microfluidic devices.

Such approaches, with further development, will also have knock on benefits for how we approach studying pharmacological interactions with animal cell lines, providing new platform technologies that are likely to reduce either the amount of animal cell lines used or how animals are used to investigate pharmacological interactions.

We anticipate that these technologies will ultimately be complementary to classical plate screens and therefore represent a step change in throughput. As such, we will try to identify market niches and opportunities for licensing and commercialization of the technology coordinated by the department of Innovation, Impact and Business at Exeter. This may create jobs and new streams of research. If adopted, potential exists for huge savings in solutions and materials commonly utilised in more traditional, less efficient applications, indicating potential market value and representing an important ecological benefit and further advancement for sustainable biochemistry.

The skills, methods and results generated in this project will be directly important for companies and research institutions that engage in high-throughput screening technologies coupled to protein engineering by directed evolution. Staff development is also an objective of the project: companies will look for skilled staff to introduce the novel approach of evolution in droplets using microfluidic devices. Thus, the trained personnel including the recruited PDRA will acquire new skills in microfluidic technologies. The crossing of boundaries between optics, micro-engineering and directed evolution is one of the unique features of this training experience. The skills and capabilities achieved as part of this project will increase the availability of highly skilled workers in the UK that will undoubtedly be an advantage in a knowledge-based economy. The researcher trained in this project will be in a position to make a valuable and practical contribution to the continued growth of the biotechnology sector in the UK.

Protein analysis is crucial to numerous scientific subfields of biology, chemistry, medicine, and pharmacology. A multitude of industries will benefit from the advances in protein sensing, including analytical sensing ($48.4 billion), cell analysis ($23 billion), chiral sensing ($9.2 billion), analysis instrumentation ($10.2 billion) and more (see below for details). This project aims to revolutionize existing applications of protein sensing, instigating new domains with sensitivities beyond current limits. We anticipate high impact in several areas, especially protein analysis, analytical sensing, and protein structural studies.