In vivo integral feedback control for robust synthetic biology

Lead Research Organisation: Imperial College London
Department Name: Bioengineering

Abstract

Biotechnology companies use single cells (bacteria, yeast, or mammalian) as 'cell factories' to produce molecules of use in many different sectors, such as pharmaceuticals, enzymes, biofuels, cosmetics or fragrances. In some cases this means that compounds that were previously produced from non-renewable sources (petroleum) can be produced from renewable sources. In other cases cell factories produce useful compounds that would be impossible, too difficult, or too expensive to produce in other ways (e.g. using chemistry). To date, innovation for biotechnological processes has focused on maximising output, but now the challenge is to use cell factories more efficiently by reducing the required input of energy and nutrients. Moreover, as we learn more about how to design and control living cells, we can begin to envision new exciting potential uses for these 'living machines', especially in the healthcare sector.

In order to do this, we need to be able to engineer living cells that behave controllably in the face of changing conditions. This is what this project aims to achieve. In electronic, mechanical and chemical engineering, robust control is typically accomplished through the use of 'Integral Feedback Control', which is an effective strategy to guarantee robustness to step-like perturbations and uncertainties. This requires an integrator. In a nutshell, the integrator accumulates information about the system's past behaviour and uses it to adjust and improve its activity as more information becomes available. Integral Feedback Control allows, for example, cruise control systems to maintain a car at constant speed irrespective of the slope of the road or the combined weight of the passengers; or the speed of an escalator to remain constant regardless of the number of people using it. In this project, we will design, model, construct and test a biological integrator to implement 'in-vivo robust control'.

A fully (re-)programmable and controllable cell is one of the core long-term objectives of the blossoming field of synthetic biology. However, no biological integrator currently exists. To fill this gap, we will engineer the first in vivo 'plug-and-play' bio-integrator device that can be customised for different applications. To demonstrate the functionality of our bio-integrator device, we will use it to create engineered cells that can robustly maintain the concentration of a chosen small molecule around a specified value. To accomplish this, the cell will be equipped with both the ability to sense the extracellular concentration of the molecule and to synthesise and secrete the molecule itself. A rigorous control design will allow for the secretion rate to change dynamically so as to counteract step-like perturbations in the extracellular concentration of the molecule. This will establish the necessary theoretical and experimental basis for future extension of this research into in vivo environments.

For example, a biological integrator device would make it possible to engineer microbes that reside symbiotically with or within other organisms, and that are able to sense and self-adjust to changing and uncertain external conditions. We anticipate that this in turn could lead to the emergence of a revolutionary new form of medicine that we are calling 'active in vivo medicine', i.e. cells that are implanted in patients and monitor the concentration of disease-related biomolecules (e.g. insulin), modulating their production of these molecules in response to patient need.

In order to investigate how active in vivo medicine might be implemented in real-world conditions, we have integrated into this project a programme of work on 'Responsible Research and Innovation' designed to incorporate the perspectives of a wide range of interested parties into any future development of active in vivo medicine, including: biomedical researchers, clinicians, patient groups, regulators, pharmaceutical firms, and bioethicists.

Planned Impact

The availability of automatic control mechanisms that can ensure robust and optimal operation of controlled systems is one of the key factors behind the tremendous advances in engineering fields like transportation, industrial production, and energy. However, we are currently unable to systematically design biological control systems and this is impeding the development of synthetic biology applications for bioprocess engineering, novel therapeutics, and advanced biomedicine. This project will bring synthetic biology one step closer to real-world applications, by demonstrating the feasibility of using Biological Integral Feedback Control in living cells. We envisage that our Biological Integrator Device (BID) could be employed in a variety of commercial applications and thus have designed it with modularity in mind so that inputs and outputs can be modified for different applications. We therefore anticipate that the results of this project will have impact on a wide range of beneficiaries over extended timescales.

In the short term (1 to 5 years) the key impact will be on academic research communities, not only in synthetic biology but also in medical and pharmacology research, control engineering, bio-design automation, and social studies of science. For synthetic biology, delivering academic impact on these associated fields is part of the critical pathway towards economic and societal impact because it is an emerging field that relies on - and contributes to - knowledge and skills from a wide range of disciplines.

In the medium term (5 to 10 years), this project will impact a whole range of commercial sectors that are important for the UK and global economic performance and in particular the industrial biotechnology sector. Firms in this sector rely on the development of bacteria capable of producing biofuels, bulk commodity chemicals, and high-value fine and speciality chemicals for food, cosmetic, and pharmaceutical products. Most biotechnological processes are designed to maximise output without taking robustness of the operating conditions into account. A better strategy would be to employ Integral Feedback Control in order to enable cells to dynamically and robustly control their output around an optimal level for the task at hand (see Case for Support and letters of support from firms for examples).

In the longer term (10 to 50 years), we believe that this research could open up a revolutionary new field of biomedicine, which we have called 'active in vivo medicine'. Live engineered cells or microbes would be inserted into patients in order to produce robust homeostatic regulation, inside their bodies, of the concentration of specific molecules of high clinical importance (e.g. insulin or uric acid). Beneficiaries would be patients, medical practitioners and health services, as well as the biomedical firms involved in the production of engineered cells and associated medical devices.

This project will also serve as an exemplar of how to implement 'Responsible Research and Innovation' (RRI) into the life cycle of a synthetic biology project, by incorporating a workpackage (WP) based on the social science concept of Anticipatory Governance. This WP will integrate the perspectives of a wide range of academic and non-academic stakeholders in order to explicitly investigate the real world prospects of the envisaged applications for Biological Integral Feedback Control (especially active in vivo medicine). This will clarify which applications are considered to be more or less desirable and/or realistic, and will also generate additional ideas about the directions in which this research could be developed. Moreover, this WP will provide evidence-based guidance on how to enhance the governance of emerging biosciences and biotechnologies, which will benefit public bodies involved in the funding, regulation and promotion of research and innovation (e.g. Research Councils, HSE, DEFRA, TSB).

Publications

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Wright O (2013) Building-in biosafety for synthetic biology. in Microbiology (Reading, England)

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

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Wei Pan (2014) Inference of Switched Biochemical Reaction Networks Using Sparse Bayesian Learning in Proceedings of the European Conference on Machine Learning and Principles and Practice of Knowledge Discovery in Databases (ECML PKDD 2014)

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Pan W (2016) A Sparse Bayesian Approach to the Identification of Nonlinear State-Space Systems in IEEE Transactions on Automatic Control

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OyarzĂșn DA (2015) Noise propagation in synthetic gene circuits for metabolic control. in ACS synthetic biology

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Hancock EJ (2015) Simplified mechanistic models of gene regulation for analysis and design. in Journal of the Royal Society, Interface

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Borkowski O (2016) Overloaded and stressed: whole-cell considerations for bacterial synthetic biology. in Current opinion in microbiology

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Arpino JAJ (2013) Tuning the dials of Synthetic Biology. in Microbiology (Reading, England)

 
Description As was our original intention, we have a much better understanding of the practical realities and the challenges associated with building a biological integral feedback controller. We have established physical reasons as to why integral control is difficult to implement in the cellular context. During this project we have worked to design potential solutions to these challenges.

We have made significant progress building a synthetic biology system in E. coli that serves as a proof-of-concept device and a testbed for a biomolecular feedback integral control system engineered in living cells. This feedback system is in the form of an external molecular concentration regulator, and we hope that the initial prototype will be extendable to various specific cases.
Exploitation Route The 'controller' portion of the system we have built, which is designed to implement near-integral control, is modular in the sense that the chemical input signal can be swapped by changing the extracellular sensor domain of the membrane-bound receptor protein. This has been demonstrated in previously published work using the same receptor protein we have used. Furthermore, the output of the controller is the activity of a transcription factor. Thus it can be used to control arbitrary genes by placing them downstream of the appropriate regulated promoter.
Thus, this controller has the potential to be used by other researchers to control different biological processes.
Sectors Agriculture

Food and Drink

Healthcare

Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

Other

 
Description This is one of the first synthetic biology project that has taken a forward engineering driven approach to build, in a modular way, an integral feedback control system inside a living cell (E. coli). The various modules of this system (sensor, control, and actuator modules) have been implemented and can be changed to allow other biomolecular feedback loops to be created. The project has revealed the importance of input-output balancing, a core concept that is now widely accepted and used in the community (see for example: Nielsen, A. A. K., Der, B. S., Shin, J., Vaidyanathan, P., Paralanov, V., Strychalski, E. A., et al. (2016). Genetic circuit design automation. Science (New York, N.Y.), 352(6281), aac7341-aac7341. http://doi.org/10.1126/science.aac7341). Furthermore, the concepts developed during this research have laid down the foundations for the field of host-aware synthetic biology enabled via biomolecular feedback control systems built in living cells (see Boo, A., Ellis, T., & Stan, G.-B. (2019). Host-aware synthetic biology. Current Opinion in Systems Biology, 14, 66-72. http://doi.org/10.1016/j.coisb.2019.03.001 and Aoki, S. K., Lillacci, G., Gupta, A., Baumschlager, A., Schweingruber, D., & Khammash, M. (2019). A universal biomolecular integral feedback controller for robust perfect adaptation. Nature, 97, 4649. http://doi.org/10.1038/s41586-019-1321-1 ) The findings of this work have served as the basis for the organisation of an international workshop, whose presentations and research outputs are accessible online at http://www.bg.ic.ac.uk/research/g.stan/group/CCBT_Workshop/CCBT_Workshop.html Our work has lead to collaborations with the MIT Centre for Synthetic Biology and to further interest from Synlogic (http://www.synlogictx.com).
First Year Of Impact 2015
Sector Agriculture, Food and Drink,Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Cultural

Societal

Economic

 
Title DH10B E. coli knockout strains 
Description Variants of the E. coli cloning strain DH10B with one or more genes knocked out. These genes will be subsequently reused within a repurposed synthetic network. DH10Bf: OmpF knockout DH10Bfc: OmpF + OmpC knockout DH10Bfcz: OmpF + OmpC + EnvZ knockout DH10Bfcrz: OmpF + OmpC + OmpR + EnvZ knockout DH10BZ1fcz: OmpF + OmpC + EnvZ knockout with constitutively expressed transcriptional repressors LacI and TetR integrated into the genome. DH10BZ1fcrz: OmpF + OmpC +OmpR + EnvZ knockout with constitutively expressed transcriptional repressors LacI and TetR integrated into the genome. 
Type Of Material Cell line 
Provided To Others? No  
Impact The EnvZ, OmpR, OmpC, OmpF E. coli osmoregulation system is a commonly modified system in synthetic biology. Therefore these knockout strains will surely be of use to other researchers. 
 
Title Engineered synthetic gene network 
Description A closed-loop synthetic gene network serving two purposes: (1) an testbed to prototype the in vivo implementation of integral feedback control, (2) an early, generic version of a automated extra-cellular concentration regulator. An early version of this synthetic gene network has been constructed and tested, and we are currently refining the network for better performance. 
Type Of Material Cell line 
Provided To Others? No  
Impact (1) The demonstration that steady-state dose-response data (which is relatively easy to measure) from separated network modules can infer the performance of a full system, where network modules have been connected. (2) Establishment of a basic, working synthetic biology system that can be adapted in the future to the concentration regulation of various molecules of interest. (3) A functional testbed to prototype and refine the various control strategies regarding regulation, and their real-world implementation. 
 
Description Collaboration with King's College London 
Organisation King's College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration with Dr Claire Marris and Dr Catherine Jefferson of the Department of Social Science, Health and Medicine at King's College London. We are working to identify the current and potential regulatory framework surrounding "active in vivo medicine"---i.e., the use of engineered living cells for therapeutic purposes within the body. The intention of this collaboration is to clarify the regulatory roadmap for future implementations of synthetic biology in human health. This is of interest to both social scientists and policy makers, as well as physical and biological scientists who may potentially choose to direct their research toward such real-world application.
Collaborator Contribution Dr Claire Marris and Dr Catherine Jefferson lead the investigation of societal, economic, ethical and regulatory aspects of this collaborative project. We have monthly meetings between engineers, experimental biologists and social scientists to truly embed Responsible Research and Innovation in our research activities.
Impact International workshop in preparation around the theme of engineered cells for active in vivo medicine. Direct collaboration and participation in the frontiers paper: Synthetic biology and biosecurity: challenging the "myths", http://journal.frontiersin.org/Journal/10.3389/fpubh.2014.00115/abstract
Start Year 2013
 
Description Collaboration with University of Oxford & University of Sydney 
Organisation University of Oxford
Department Department of Engineering Science
Country United Kingdom 
Sector Academic/University 
PI Contribution We have collaborated in a theoretical project studying the effects of buffering and feedback in cellular regulation. We are currently involved in drafting a journal article.
Collaborator Contribution All parties have equal partnership in this collaboration.
Impact Currently drafting an academic paper.
Start Year 2014
 
Description Collaboration with University of Oxford & University of Sydney 
Organisation University of Sydney
Country Australia 
Sector Academic/University 
PI Contribution We have collaborated in a theoretical project studying the effects of buffering and feedback in cellular regulation. We are currently involved in drafting a journal article.
Collaborator Contribution All parties have equal partnership in this collaboration.
Impact Currently drafting an academic paper.
Start Year 2014
 
Description Collaboration with University of Toronto 
Organisation University of Toronto
Country Canada 
Sector Academic/University 
PI Contribution Collaboration with Professor David McMillen at the University of Toronto. We are currently investigating theoretical aspects of robust feedback control. We expect the collaboration to yield one or more journal publications.
Collaborator Contribution Experimental and theoretical intellectual contributions
Impact Multi-disciplinary collaboration: control engineering, systems engineering, microbiology.
Start Year 2014
 
Description Invited Plenary Speaker, BioBricks Foundation SB6.0: The 6th International Meeting on Synthetic Biology. Imperial College London, UK, July 9-11, 2013. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation poster presentation
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Title of the invited plenary talk: Control Engineering Synthetic Biology, given at the largest synthetic biology conference in the world. BioBricks Foundation SB6.0: The 6th International Meeting on Synthetic Biology. Imperial College London, UK, July 9-11, 2013.

Increased awarness of the novel method proposed by our research group
Year(s) Of Engagement Activity 2013
 
Description Invited talk at Information, probability and inference in systems biology, Edinburgh, UK, July 15-17, 2013. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation poster presentation
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Stimulated discussions and novel research questions.

Increased awarness of the novel methods proposed by our research group
Year(s) Of Engagement Activity 2013
 
Description Invited talk at the Centre for Organelle Research, University of Stavanger, Stavanger, Norway, 02 December 2015. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Talk given by Dr Jordan Ang at the Centre for Organelle Research, University of Stavanger, Stavanger, Norway, 02 December 2015.
Year(s) Of Engagement Activity 2015
 
Description Invited talk at the Department of Mathematics Biomaths Seminar, Imperial College London, London, UK, 25 November 2014 
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 given by Jordan Ang at the Department of Mathematics Biomaths Seminar, Imperial College London, London, UK, 25 November 2014
Year(s) Of Engagement Activity 2014
 
Description Invited talk at the Workshop on Control Engineering and Synthetic Biology, University of Oxford, Oxford, UK, 04 August 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited talk by Dr Jordan Ang at the international Workshop on Control Engineering and Synthetic Biology, University of Oxford, Oxford, UK, 04 August 2014
Year(s) Of Engagement Activity 2014
URL http://sysos.eng.ox.ac.uk/wiki/index.php/Workshop_on_Control_Engineering_and_Synthetic_Biology
 
Description Organisation of the international Workshop on "Control Engineering and Synthetic Biology", University of Oxford, Oxford, 10-12 September 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Over 100 fellow researchers (professors, postdocs, graduate students) attended the workshop that Dr Stan co-organised at the University of Oxford. Furthermore, Dr Jordan Ang gave an talk describing the EPSRC funded project EP/K020617/1.

Many requests for additional discussions.
Year(s) Of Engagement Activity 2014
URL http://sysos.eng.ox.ac.uk/wiki/index.php/Workshop_on_Control_Engineering_and_Synthetic_Biology
 
Description Taking a Forward-Engineering Approach to the Design of Synthetic Biology Systems, GARNet Synthetic Biology Workshop, 21-22 May, 2013. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact GARNet Synthetic Biology Workshop, 21-22 May, 2013.

Invited by Ruth Bastow and Charis Cook.

Invited Speaker by the organisers
Year(s) Of Engagement Activity 2013
 
Description Taking a Systems Control Approach in Biology : exogenous and endogenous control of biological systems, Department of Mathematics and Statistics, University of Reading, March 20th, 2013 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Department of Mathematics and Statistics, University of Reading, March 20th, 2013. Invited by Marcus Tindall.

Invited Speaker for Departmental Seminar
Year(s) Of Engagement Activity 2013
 
Description Taking a Systems Control Approach in Biology, Dept. d'Enginyeria de Sistemes i Automatica, Universitat Politecnica de Valencia, Spain, April 11th, 2013. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Dept. d'Enginyeria de Sistemes i Automatica, Universitat Politecnica de Valencia, Spain, April 11th, 2013.

Invited by Prof Jesús Andrés Picó Marco.

Invited Plenary Speaker by the organisers
Year(s) Of Engagement Activity 2013
 
Description Workshop on Decision Making in Nature. Imperial College London, UK, May 2-4, 2013. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation poster presentation
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Stimulated discussions and novel research ideas

increased awarness of the novel methods proposed by our research group
Year(s) Of Engagement Activity 2013