Engineering Fellowships for Growth: Systems and control engineering framework for robust and efficient synthetic biology

Lead Research Organisation: Imperial College London
Department Name: Dept of Bioengineering

Abstract

Synthetic Biology is the engineering of biology. In this spirit, this Fellowship aims at combining control engineering methodology and expertise with synthetic biology current know-how to solve important real-world problems of high industrial and societal importance.

Anticipated high-impact applications of synthetic biology range from cell-based diagnostics and therapies for treating human diseases, to efficiently transforming feedstocks into fuels or biochemicals, to biosensing, bioremediation or production of advanced biomaterials. Central to tackling these problems is the development of in-cell automatic feedback control mechanisms ensuring robust functionality and performance of engineered cells that need to operate under uncertain and changing environments. The availability of methods for designing and implementing feedback control mechanisms that yield improved robustness, efficiency and performance is one of the key factors behind the tremendous advances in engineering fields such as transportation, industrial production and energy. As in these and other engineering disciplines, systems and control engineering will accelerate the development of high-impact synthetic biology applications of societal, commercial and industrial importance.

In particular, through this Fellowship, I propose a comprehensive engineering approach to push forward the robustness frontier in synthetic biology towards reliable cell-based biotechnology and biomedicine. This ambitious goal requires: (1) the development of feedback mechanisms to reduce the footprint of engineered metabolic pathways on their cell "chassis", (2) the development of system-level feedback mechanisms to robustly and efficiently manage one or more synthetic devices in the context of whole-cell fitness, and (3) the development of synthetic cell-based systems designed to restore and maintain the extra-cellular concentration of some biomolecules within tight homeostatic bounds.

These three aspects define three work packages in my Fellowship. Each work package on its own tackles important synthetic biology challenges for real-world applications, while their combination in WP4 aims towards robust cell-based biotechnology and biomedicine. The corresponding work packages are:

*WP1*: Automatic management of fluxes for robust and efficient metabolic pathways (through genetic-metabolic feedback control)

*WP2*: Automatic management of cellular burden for robust and efficient whole-cell behaviour (through host-circuit feedback control)

*WP3*: Automatic management of extra-cellular concentrations for robust homeostatic regulation of environmental conditions (through cell-environment feedback control)

*WP4*: System integration and combination of the feedback control mechanisms developed in WP 1-3

The first two work packages address device robustness to cellular context, while the third addresses robust adaptation to and control of changing environmental conditions. WP4 will use and further develop the systems and control engineering framework developed in WP 1-3 to explore the synergistic combination of the proposed feedback control mechanisms.

By providing systematic engineering solutions that endow engineered biosystems with robust functionalities, we will enable the enhancement of existing biotechnological processes and the reliable development of industrial applications to improve health and quality of life. Through the above, this Fellowship will foster strong and long-lasting economic and societal impact in the UK and globally and promote knowledge-based UK leadership.

Planned Impact

Synergistic research at the confluence of synthetic biology and systems and control engineering is key to enable the understanding of fundamental design principles of living systems, systematically create robust biological functionalities, deliver important new applications and improve existing industrial processes. These steps are recognised by major international bodies such as the OECD and the UK and US governments to be of crucial importance to create new synthetic biology industries, attract foreign investments, develop a thriving biotechnology and biomedicine sector, support existing and new SMEs, and create new jobs and a vibrant bioeconomy (and wider economy). This Fellowship, which is directly aligned with the EPSRC strategic priority in Synthetic Biology and the UK Synthetic Biology Roadmap, will directly contribute to the growing excellence of synthetic biology in the UK and facilitate its industrialisation through the creation of a much-needed systems and control engineering framework that will accelerate research and development, enable scale-up, and provide robust and efficient solutions for a large variety of applications.

*Impact on Society* Synthetic biology's considerable anticipated impact in biotechnology, biomedicine, energy production, food technology, and society in general has been acknowledged worldwide by the OECD and UK, EU and US policy makers.

The availability of feedback control mechanisms that ensure the robustness (to perturbations and uncertainties) and performance of systems is one of the key factors behind the tremendous advances in engineering fields like robotics and autonomous systems, aerospace, transportation, industrial production and energy. However, unlike for these traditional engineering disciplines, we are still ill-equipped to systematically design automatic feedback control systems within cells. This lack of systems and control engineering methods and tools is a major bottleneck in the development of synthetic biology as a true engineering discipline that can be used to solve important, real-world problems. This Fellowship will narrow this gap by engineering robustness in synthetic biology systems through feedback and thereby bring synthetic biology technologies closer to real-world applications. This will open up avenues for new research and world-leading developments in the UK lasting well beyond the Fellowship's five year duration.

*Impact on Knowledge* There is a recent trend in the Control Engineering community to focus on problems from the biosciences. This tendency is having an enormous impact in control theory and biology, by providing a new class of theoretical problems and practical solutions motivated by the unique features and constraints of cellular systems. This Fellowship is in line with this trend as it lies at the interface between synthetic biology and control engineering. It will stimulate the development of new in vivo feedback control methods and provide a major emphasis on the role of systems and control engineering in synthetic biology research and applications. The multidisciplinary knowledge developed through this Fellowship will strongly contribute to the excellence of control engineering and synthetic biology research in the UK and worldwide.

*Impact on Economy and Industry* The biotechnology and healthcare industries constitute the main target for economic impact. With large amounts of money spent on the efficient development of cells capable of robustly and optimally producing biofuels, commodity chemicals, pharmaceuticals, etc., the economic impact of robust in vivo feedback control solutions can be very large. I anticipate that the results of this Fellowship will have a very important impact on cell-based applications such as bioreactor biosynthesis and cell-based therapeutics.

Publications

10 25 50

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Tomazou M (2018) Portable gene expression guaranteed. in Nature biotechnology

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Tusk SE (2018) Subunit Exchange in Protein Complexes. in Journal of molecular biology

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Walker BJ (2017) Intracellular delivery of biologic therapeutics by bacterial secretion systems. in Expert reviews in molecular medicine

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Wright O (2015) GeneGuard: A modular plasmid system designed for biosafety. in ACS synthetic biology

 
Description We have developed the first burden-based biomolecular feedback regulation mechanism in living cells (E. coli MG1655 and DH10 strains). The results of this findings, will be published in March 2018 in Nature Methods, and demonstrate for the first time that a generic and modular, biomolecular feedback control system implemented using dCas9 can be used to improve the robust performance of engineered E. coli cells to increase production yield of recombinant proteins by dynamically balancing the allocation of shared cellular resources between growth and recombinant protein production. (Abstract of the Nature Methods 2018 paper: Cells use feedback regulation to ensure robust growth despite fluctuating demands on resources and different environmental conditions. Yet the expression of foreign proteins from engineered constructs is an unnatural burden that cells are not adapted for. Here we combined RNAseq with an in vivo assay to reveal the major transcriptional changes in E. coli when inducible synthetic constructs are expressed. We identified that native promoters related to the heat-shock response activate expression rapidly in response to synthetic expression, regardless of the construct. Using these promoters, we built a dCas9-based feedback regulation system that automatically adjusts synthetic construct expression in response to burden. Cells equipped with this general-use controller maintain capacity for native gene expression to ensure robust growth and as such outperform unregulated cells at protein yields in batch production. This engineered feedback is the first example of a universal, burden-based biomolecular control system and is modular, tunable and portable.) Following up on these exciting results, we are also developing new biomolecular feedback control mechanisms based on RNA-based regulation of gene expression so as to reduce the cellular cost of the feedback control system itself and improve further the robust performance of our novel class of our in vivo biomolecular feedback control systems. Furthermore, we have theoretically characterised the interplay between the two major mechanisms used in biology for homeostatic regulation of cellular processes, i.e. biomolecular negative feedback and buffering. The results of these findings have been published in Cell Systems in 2017 (Abstract of the Cell Systems 2017 paper: Buffering, the use of reservoirs of molecules to maintain concentrations of key molecular species, and negative feedback are the primary known mechanisms for robust homeostatic regulation. To our knowledge, however, the fundamental principles behind their combined effect have not been elucidated. Here, we study the interplay between buffering and negative feedback in the context of cellular homeostasis. We show that negative feedback counter- acts slow-changing disturbances, whereas buffering counteracts fast-changing disturbances. Furthermore, feedback and buffering have limitations that create trade-offs for regulation: instability in the case of feedback and molecular noise in the case of buffering. However, because buffering stabilizes feedback and feedback attenuates noise from slower-acting buffering, their combined effect on homeostasis can be synergistic. These effects can be explained within a traditional control theory framework and are consistent with experimental observations of both ATP homeostasis and pH regulation in vivo. These principles are critical for studying robustness and homeostasis in biology and biotechnology.). Finally, using a systems and control theoretic approach, we have proposed for the first time general design principles and associated biomolecular architectures for the design and implementation in bacterial cells of robust oscillators whose amplitude and frequency can be modified independently and over enlarged ranges. The findings of these results have been presented in Cell Systems 2018. (Abstract of the Cell Systems 2018 paper: To perform well in biotechnology applications, synthetic genetic oscillators must be engineered to allow independent modulation of amplitude and period. This need is currently unmet. Here, we demonstrate computationally how two classic genetic oscillators - the dual-feedback oscillator and the repressilator - can be re-designed to provide independent control of amplitude and period and improve tuneability, that is, a broad dynamic range of periods and amplitudes accessible through the input "dials". Our approach decouples frequency and amplitude modulation by incorporating an orthogonal "sink module" where the key molecular species are channelled for enzymatic degradation. This "sink module" maintains fast oscillation cycles while alleviating the translational coupling between the oscillator's transcription factors and output. We characterise the behaviour of our re-designed oscillators over a broad range of physiologically reasonable parameters, explain why this facilitates broader function and control, and provide general design principles for building synthetic genetic oscillators that are more precisely controllable.). We are developing a novel self-regulating protease biomolecular feedback system aiming to keep constant degradation rates despite perturbations of proteins competing for the finite amount of the protease, and even changes in growth rate of the host cell. This is expected to be concluded and published in 2018. Additional key findings that arose from the self-regulating protease work came from the fact that recycling amino acids through enzymatic degradation were shown in preliminary experiments to reduce burden on the host and increase the yield of the recombinant proteins. This research avenue was funded for further research (EP/P009352/1).
Exploitation Route The use of a biomolecular feedback system implemented in living cells offers robustness to perturbations such as change in growth conditions (e.g. temperature, nutrient source) and could open the door to streamlined designs that are more easily portable "as is" (i.e. without need for much additional re-tuning) in other strains or even other organisms. The mathematical characterisation of the interplay between and fundamental limits of negative feedback and buffering opens avenues for the design and implementation of new homeostatic regulation mechanisms in living cells that allow for better robust performance based on the combination of these two fundamental mechanisms. Finally the theoretical design of genetic oscillators with independently tuneable amplitude and frequency opens novel research avenues for the implementation of effective multi-input biosensors and periodic signal generators in biology.

These results have also led to the award to Prof Guy-Bart Stan of a Royal Academy of Engineering Chair in Emerging Technologies in Engineering Biology (2019-2029): https://www.raeng.org.uk/news/news-releases/2019/april/royal-academy-of-engineering-awards-£20-million-in
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other

 
Description A broad audience summary of the findings and future prospects of this work was presented at the Meeting of the New Champions at the World Economic Forum in Dalian, China, in September 2015: http://www.imperialtechforesight.com/future-visions/87/visionary/guy-bart-stan.html, http://www.imperialtechforesight.com/future-visions/87/event/wef-amnc-2015.html, The corresponding video of the talk of Dr Guy-Bart Stan at the World Economic Forum is available at https://www.youtube.com/watch?v=hs55KCM8Uyo Our recent and exciting finding on the first burden-based biomolecular control system implemented in living cells (E. coli bacteria) have been presented at the Mathematical Biosciences Institute at Ohio State University, USA, in October 2017. The talk is available online at: https://mbi.osu.edu/video/player/?id=4377 and summarises our recent efforts to engineer living E. coli cells that exhibit improved robustness and performance during synthetic network operations. Furthermore some of the findings on designing, tuning, and implementing de novo biomolecular feedbacks in living cells of formed the basis for the International Workshop "Prospects for Controllable Cell-Based Therapies" that I co-organised at City University London, on 22 and 23 February 2016. This workshop brought together diverse stakeholders to discuss the prospects for an emerging category of medical applications of synthetic biology that we are calling 'controllable cell-based therapies' (CCBTs) including: * Scientists conducting cutting-edge research to enable CCBTs * Firms seeking to commercialise CCBTs * Staff from agencies involved in the regulation of CCBTs * Independent experts on regulatory frameworks and translation for CCBTs * Staff from patient advocacy groups with expertise in medical innovation and regulation * Social scientists with expertise in medical innovation and regulation A summary of the talks and of the workshop can be found at http://www.bg.ic.ac.uk/research/g.stan/group/CCBT_Workshop/CCBT_Workshop.html
First Year Of Impact 2017
Sector Education,Pharmaceuticals and Medical Biotechnology
Impact Types Cultural,Societal,Economic

 
Description A novel, fast and efficient resource recycling system for improving the performance of engineered bacteria
Amount £445,502 (GBP)
Funding ID EP/P009352/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 02/2017 
End 07/2020
 
Description EPSRC Centre for Doctoral Training in BioDesign Engineering
Amount £7,001,622 (GBP)
Funding ID EP/S022856/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 03/2019 
End 09/2027
 
Description Genetically Encoded Nucleic Acid Control Architectures
Amount £642,353 (GBP)
Funding ID EP/P02596X/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 07/2017 
End 02/2021
 
Title BSID 
Description Matlab toolbox for nonlinear systems identification from time-series data 
Type Of Technology Software 
Year Produced 2015 
Impact Prediction and control of behaviour and abnormalities in any complex dynamical network, and in particular in those encountered in biology require the development of multivariate predictive models that integrate large dataset from different sources. Although, a large amount of data are being collected on a daily basis, very few methods allow the automatic creation from these data of nonlinear Ordinary Differential Equation models for understanding and (re-)design/control, and an inordinate amount of time is still being spent on the manual aggregation of information and expert development of models that explains these data. In this context, the problem of reconstruction or identification of biological systems from experimental time series data is of fundamental importance. Yet, the development of general reconstruction techniques remains challenging, especially for nonlinear system identification. We are currently developing new methods to identify both parametric structure and parameter values in nonlinear Ordinary Differential Equation models from heterogeneous datasets. Applications of such nonlinear systems identification methods cover fundamental questions in systems biology, synthetic biology (debugging and design of cellular systems) and modelling of complex dynamical networks. 
URL https://github.com/panweihit/BSID
 
Description Invited Plenary Speaker at the Design, Optimization and Control in Systems and Synthetic Biology International Workshop, Paris, France, November 12-13, 2015. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Broad audience international workshop at Ecole Normale Supérieure, Paris, France
Year(s) Of Engagement Activity 2015
 
Description Invited Talk at the Raymond and Beverly Sackler USA-UK Meeting: Scientific Forum on Trends in Synthetic Biology and Gain of Function Research, and Regulatory Implications, hicheley Hall, Chicheley, U.K., November 15-17, 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This prestigious event is co-organised by the Royal Society and the US National Academy of Sciences, Chicheley Hall, Chicheley, U.K.
Year(s) Of Engagement Activity 2015
 
Description Invited Talk at the World Economic Forum in Dalian, China, 9-11 September 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Invited Talk at the World Economic Forum in Dalian, China, 9-11 September 2015 as part of the prestigious "Meeting of the New Champions". The Imperial relationship with the World Economic Forum is led from the President's office and is personally with Prof Alice Gast, President of Imperial College London. Further information can be found on these websites: http://www.imperialtechforesight.com/future-visions/87/event/wef-amnc-2015.html and http://www.imperialtechforesight.com/future-visions/87/visionary/guy-bart-stan.html
Year(s) Of Engagement Activity 2015
URL https://youtu.be/hs55KCM8Uyo
 
Description Invited inaugural talk at the Cambridge University Synthetic Biology Society, 3 March 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact I was invited to give the first inaugural lecture for the synthetic biology seminar series organised by the newly founded Cambridge University Synthetic Biology Society.
The audience consisted of around 40 undergrad students, 5 postgrad students and several staff members of Microsoft Research Cambridge.
Year(s) Of Engagement Activity 2016
URL http://www.meetup.com/Cambridge-Synthetic-Biology-Meetup/events/228878586/
 
Description Invited plenary speaker at the Imperial Alumni Event, Beijing, China, 12 September 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact The Imperial Alumni Event was organised and lead by Imperial College's President Office and is personally with Prof Alice Gast, President of Imperial College London. Further information can be found on http://www.synbicite.com/news-events/2015/sep/23/synbicite-academics-meet-imperial-alumni-beijing/
Year(s) Of Engagement Activity 2015
URL http://www3.imperial.ac.uk/newsandeventspggrp/imperialcollege/newssummary/news_18-9-2015-12-0-16
 
Description Invited talk at BBN Technologies, Boston, USA, 3 September 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Invited talk by Dr Jacob Beal, BBN Technologies. The goal of the talk was to explore the application of the Control Engineering methods in Synthetic Biology for BBN Technologies applications and projects and investigate possible collaborations. Dr Stan and Dr Beal are both active members of the SBOL work group (http://sbolstandard.org) aiming at defining a world-wide standard for the exchange of synthetic biology designs in both machine and human readable format. Dr Stan was the first invited European member to the SBOL work group (http://sbolstandard.org/development/developers/).
Year(s) Of Engagement Activity 2015
 
Description Invited talk at the Edinburgh Centre for Synthetic and Systems Biology, SynthSys, 21 January 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact A very large audience from the Edinburgh SynthSys Centre, which sparked questions and discussion afterwards, with various meetings planned during the day with PIs and undergrad and postgrad students
Year(s) Of Engagement Activity 2016
URL http://www.synthsys.ed.ac.uk/event/synthsys-seminar-guy-bart-stan-imperial-college-london
 
Description Invited talk at the MIT Synthetic Biology Center, 13 August 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Invited wide audience talk at the MIT Centre for Synthetic Biology.
My talk was entitled "Systems and Control Engineering of Cells for Robust and Efficient Synthetic Biology: Feedback Considerations to Engineer Cells with Better Performance", and was invited and hosted by Prof. Tim Lu.
Year(s) Of Engagement Activity 2015
URL http://cisb.mit.edu/event/sbc-seminar-series-systems-and-control-engineering-of-cells-for-robust-and...
 
Description Organisation of the International Workshop "Control Engineering and Synthetic Biology: What Next", 17-18 July 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 100 participants attended this international workshop that gathered world-leading academics, industry representatives, and funders. The full programme and summary of the event can be found on the website associated with the event: http://sysos.eng.ox.ac.uk/wiki/index.php/SynBioControl2017
Year(s) Of Engagement Activity 2017
URL http://sysos.eng.ox.ac.uk/wiki/index.php/SynBioControl2017
 
Description Organisation of the international workshop: On the Prospects for Controllable Cell-Based Therapies, City University London, 22-23 February, 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This workshop brought together diverse stakeholders to discuss the prospects for an emerging category of medical applications of synthetic biology that we are calling 'controllable cell-based therapies' (CCBTs) including:

* Scientists conducting cutting-edge research to enable CCBTs
* Firms seeking to commercialise CCBTs
* Staff from agencies involved in the regulation of CCBTs
* Independent experts on regulatory frameworks and translation for CCBTs
* Staff from patient advocacy groups with expertise in medical innovation and regulation
* Social scientists with expertise in medical innovation and regulation

Core aspects and questions discussed during the workshop included:
* What is the current scientific, economic and regulatory landscape for CCBTs?
* How do CCBTs compare to alternative approaches to health and medicine?
* How can we ensure that these new therapies reach the clinic and provide actual benefits to patients without generating unreasonable risks?
* What implications might the use of bacterial (as opposed to human) cells have in terms of safety, regulatory frameworks and other challenges for translation to the clinic?

A summarising report on the basis of the discussions and interviews conducted as part of the workshop will be made publicly available soon on the website of the workshop (http://www.bg.ic.ac.uk/research/g.stan/group/CCBT_Workshop/CCBT_Workshop.html)
Year(s) Of Engagement Activity 2016
URL http://www.bg.ic.ac.uk/research/g.stan/group/CCBT_Workshop/CCBT_Workshop.html
 
Description Organiser of the Launch of the expanded Imperial College Centre for Synthetic Biology, 7 December 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Organisation and launch of the expanded Imperial College Centre for Synthetic Biology (http://www.imperial.ac.uk/synthetic-biology/centre/), of which I am the Co-Director
Year(s) Of Engagement Activity 2018
URL https://www.imperial.ac.uk/news/189595/expanded-centre-synthetic-biology-launches-imperial/
 
Description Scientific Committee for Work Package 3 ("Community Building") of the European Research Area Network on Synthetic Biology (ERASynBio Twinning programme), EU FP7, 2012-2013 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Member of the Scientific Committee for Work Package 3 ("Community Building") of the European Research Area Network on Synthetic Biology (ERASynBio Twinning programme), EU FP7, 2012-2013 .

Around 15-20 twinning projects involving a minimum of 3 research institutions in different countries have been awarded through this activity
Year(s) Of Engagement Activity 2012