Award details

Towards designing synthetic molecular motors: in situ visualization of the progressive evolution of molecular gearing by bacteria

Reference	BB/L023091/1
Principal Investigator / Supervisor	Dr Morgan Beeby
Co-Investigators / Co-Supervisors
Institution	Imperial College London
Department	Life Sciences
Funding type	Research
Value (£)	400,159
Status	Completed
Type	Research Grant
Start date	02/01/2015
End date	01/01/2018
Duration	36 months

Abstract

Designed nanomachinery holds promise to transport nanocargoes, seek cancer cells, power microscale mechanics, and mix microfluidics. Yet we have been restricted to using pre-existing machinery instead of adapting or designing our own because we lack techniques to visualize and understand function and evolution. The bacterial flagellar motor is a model nanomachine. It is tantalising to note that different motors produce different torques and if we could understand their evolutionary fine-tuning we might capitalize on this knowledge to fine-tune our own through directed evolution. We recently identified three motors that may represent descendants of an evolutionary path to a high-torque motor. We used electron cryo-tomography to image motors in situ in Escherichia coli, Vibrio fischeri, and Campylobacter jejuni. C. jejuni can push through very viscous fluids using a high-torque motor. Strikingly, the diameter of the "C-ring" responsible for torque transmission is wider in C. jejuni. C. jejuni also has a large extra motor-associated structure, one protein of which is also found in V. fischeri. To understand its function we obtained results locating component proteins, and saw that the torque-generation stator protein MotB is spaced at a wider radius than E. coli, in correspondence with the wider C-ring. We hypothesize that C. jejuni evolved from a Vibrio-esque motor that enabled evolution of a wider C-ring to generate higher torque. It would be of high impact to understand this evolution to mimic it to evolve our own machinery. I propose to: 1. Determine locations and numbers of novel components of the C. jejuni disk complex using gene deletions, truncations, and fusion of designed peptide tags. 2. Perform a similar analysis of the "missing link", V. fischeri. 3. Contextualize results and identify other intermediary bacteria by calculating phylogenies for each protein family, infer their order of recruitment, and what conditions predisposed them to recruitment.

Summary

We are entering a new era in which we will make our own tiny 'nanoscale' machinery. We hope that this machinery will produce food, fuel, and clean water, help fight disease, accurately read and write information to DNA, and perform emission-free mechanical tasks. Recent successes have come from using machines that have evolved inside microbes in making chemicals using living cells and storing information in DNA, but we haven't done much work to make our own "molecular motors" that perform physical tasks inside cells. This is mainly because we've been limited by our techniques: we haven't been able to see the machines that have naturally evolved inside cells over the billenia, so we've found it difficult to understand how they work or evolved - and how to do it ourselves (instead we've had to break open cells and purify the machines to study them, a process that often badly affects the machinery that we're trying to study). Thankfully recently we've developed ways to to image machines in 3-D inside cells by freezing the bacteria and putting them in an electron microscope. I recently trained to use this new technology. I've used it to study probably the most captivating example of molecular motors, a motor that spins a spiral-shaped propellor (the "flagellum") to push bacteria in good directions. But the flagellar motor is more than just a fascinating example of the wonders of life on earth, and studying it promises many useful things to us. One use would be to work out how to harness its emission-less power. A single motor from E. coli (a very well studied bacterium), if it were the man-sized, would be as powerful as an airplane turboprop engine! It has been known for some time that different motors are stronger or weaker than the E. coli motor, but we don't know why because we haven't been able to see them. This all changed when we used the new technique for imaging whole cells to look at a wide range of bacteria to see their motors. A bacterium called Campylobacter jejuni, or just "Campy", struck us as interesting: not only was it known to swim through very sticky fluids better than other bacteria, but the part that generates rotation is bigger than other bacteria, which we think makes a stronger motor that helps Campy swim. What makes this particularly interesting is that Campy has evolved this adaptation. If we could understand how it did this, we might try to copy it to modify motors to our own specifications. Then we might control bacteria to ferry cargoes around, selectively seek and destroy cancers cells, push miniature rotors, or mix fluids together. I aim to collect data to fully understand how the Campy motor evolved. To do this I'll image Campy, and simultaneously alter the DNA to change the appearance of the motor. In this way we'll be able to work out where parts of the motor are. At the same time I'll image a bacterium that looks like it's halfway between Campy and E. coli to see if this "half-way" cousin tells us anything about the evolutionary pathway that Campy had to follow. Finally we'll take each part of the motors from all of these bacteria and use their DNA sequences to work out their ancestry. This will help us to see where the Campy motor recruited the additional parts that it uses to be more powerful. As well as these aims I'm planning on developing methods to work out which parts of the motor go where. To do this I'm going to develop computer programs to design better ways to 'tag' components of the motor with extra bits. These extra bits will be easily visible in the 3-D structures that we collect, enabling us to directly visualize where the tagged components are. Indeed, if this works out it will open the next chapter: instead of using these tags to understand the motor, we'll use them as modifications that we can improve by directing evolution. Maybe someday we'll have a motor that we can gear as we wish.

Impact Summary

BBSRC Strategic Priorities. This project has relevance to multiple strategic priorities. Campylobacter is found in two-thirds of commercially available chicken, causing 460 000 cases of food poisoning, 22 000 hospital admissions, and 110 deaths per year. Campylobacter requires a working flagellar motor for virulence, making this work directly relevant to BBSRC Research Priority "Food Security" ("Healthy and Safe food"). This work also meets the BBSRC "Energy" priority: although harnessing nanomachinery is still underdeveloped, this project could lead to specifically designed motors for future application. Finally, this research meets all sub-priorities of the "Exploiting new ways of working" priority. It is "Data driven biology" (electron cryo-tomography produces terabytes of 3-D images on whole cells, and our tagging methodology mines PDB data to identify ideal tags for visualizing protein location). "Synthetic biology" is at the core of this proposal and the ultimate construction of a toolkit of parts to build motors to our own specification via directed evolution. Electron cryo-tomography is inherently a "Systems approach to the biosciences", imaging whole cells without stains or fixatives, and macromolecular machinery being imaged in situ instead of the standard reductionist approach of purifiying components. Finally, this work is "Technology development for the biosciences" to develop techniques for in situ dissection by method development in generation of minimally-perturbatory tags. The following people beyond my academic peers may also benefit: Industry. Results from this research may lead to commercial ventures to develop novel antibiotics that specifically target the Campylobacter or Vibrio flagellar motor. This benefit would be realized through Imperial Innovations, the Imperial College London technology commercialization company. In addition, novel methods to structurally adapt macromolecular machinery may stem from this work, and in this eventualitya patent will be sought through Imperial Innovations. Basic life scientists. The synthetic biology, evolutionary biology, biophysics, microbiology, C. jejuni and Vibrio communities will learn about the structure, function, and mechanisms and evolution of molecular machinery. This information will be communicated by myself and the unnamed postdoctoral research assistant through peer-reviewed publications, and invited and informal talks at conferences and research institutes. The public. I am promoting my work to the public in a number of ways. I will exhibit at the yearly Imperial Festival (I already presented my work at a stall in the 2013 Festival, which attracted 10 000 visitors over a weekend). I will also apply for future Royal Society Summer Science Exhibition to present my work. I am also engaging with artist colleagues to disseminate concepts on biological self-assembly and self-organization. For example I am developing plans for a sculpture series with Los Angeles-based artist Michael Parker (http://michaelparker.org) to illustrate the concept of biological self-assembly (we plan to submit an application to the Wellcome Trust Small Arts Awards program), and in spring 2013 delivered a talk on self-organisation and it's relation to bacterial multiprotein machinery at another artist's gallery opening in Cologne, Joel Kyack (http://ghebaly.com/artists/joel-kyack). I anticipate similar collaborations in the future. Despite being international, all work will prominently feature the Imperial and BBSRC brand identities. All of these projects will also focus my communication abilities with a non-specialist audience. Students. As with non-scientists, students are excited to learn about molecular machinery, particularly when the methods used for study incorporate new, exciting methods such as electron cryo-tomography. I will incorporate this research in my lectures to inspire undergraduates. PhD students who I will mentor will also benefit.

Committee	Research Committee D (Molecules, cells and industrial biotechnology)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search