Engineered burden-based feedback for robust and optimised synthetic biology

Synthetic biology is an exciting new subject that is accelerating the research and development of new biotechnologies by rigorously applying engineering design principles to the way we work with biological systems. The most prominent application of synthetic biology is the rational modification and redesign of living organisms like microbes for new efficient use in sectors such as energy production, biomedicine, drug production and food technology. Crucial to developing and applying synthetic biology is the rigorous quantification, modelling, analysis and control of the synthetic biology designs. By using this engineering framework we aim to be able to reliably predict and robustly control how engineered biological systems will operate.

Although synthetic biology has had numerous successes in research, it is still difficult to predict how engineered cells behave when new synthetic genetic information is added to these host cells. One of the major reasons for this is that new synthetic genes add an as-yet unquantified burden to cells, particularly to commonly used microbes like E. coli. This burden effect is due to the new genes requiring resources to be maintained and function. This means that the introduced genes take resources away from those needed by their host cell in order to grow and survive. Usually the result of this is the unpredictable failure of the synthetic biology design to behave as expected or the creation of designs that only function in a narrow set of ideal conditions.

The work proposed in this project seeks to address and make use of the understudied effect of synthetic biology we know as burden. To achieve this goal, we will use novel genetic tools to quantify the effect of burden for several typical synthetic biology devices and do this work in the well-characterised microbe E. coli so that our results are useful to the many researchers who work with this model organism. To see how the cell naturally reacts to burden we will use the high-resolution tool of RNA sequencing to quantify the gene expression changes that a cell triggers when it is burdened. Quantified burden combined with the quantified gene expression changes in response to burden will together give us crucial data that can be used to build a mathematical model of how a cell reacts to new synthetic genes being added and used. This model will allow future applications of synthetic biology to predict how synthetic systems will interact with their host cells and therefore open the door for rigorous optimisation of the robustness/performance compromise inherent to any control engineering design. It will also allow for a new generation of synthetic biology devices that automatically account for the burden effect. To demonstrate this final point, this project will use our quantified understanding of burden to engineer novel synthetic plasmid vectors that are designed to auto-regulate their copy number via feedback mechanisms that take into account burden, thereby serving as general purpose burden-based controllers. We will show how these plasmid systems work by building and testing a self-regulating biological nightlight that emits bioluminescence in the dark without any significant loss in growth. The new plasmid systems we generate will be extremely valuable for synthetic biology as they will allow synthetic devices and systems to respond directly to cell health thereby endowing them with the robust and predictable behaviours needed for future applications in health, energy and biosensing.

Synthetic biology is an EPSRC priority research area where scientists apply engineering principles to modify and reconstruct biology, in order to make biological engineering a scalable, predictable and economically successful industry for the 21st century. The USA has so far led the way in synthetic biology, but the UK's strong record in fundamental science puts us in an enviable position to deliver the foundational tools needed to move synthetic biology from ad-hoc custom designs to an advanced engineering discipline. The key to delivering this lucrative future is the predictable and repeatable engineering of biology from parts, using a rational approach incorporating modelling, design, analysis, control and simulation. This method is still non-trivial, partly due to a lack of well-characterised parts and predictive models, but also due to a major gap in our understanding of how engineered biology interacts with the natural cellular systems in which it is hosted. The burden synthetic biology imparts on its host cell is a crucial part of this understudied gap. Without sufficient understanding and control over burden, many designs in synthetic biology fail unexpectedly. Clearly, to accelerate the realisation and predictability of synthetic biology designs for research and industry, we need to bridge this gap. This project is a rational plan to address this, by understanding, quantifying, modelling and controlling the burden of synthetic biology and so advance our ability to engineer biology for human needs.

The deliverables leading to major impact on knowledge are four-fold: (i) a major advance in our understanding of the burden interactions between synthetic biology systems and their host cell, (ii) a new set of well-characterised biological parts regulated by burden effects and useful for future designs, (iii) a mathematical model of the cellular response to burden that will inform and predict future constructs, and (iv) a set of new burden-regulated plasmids capable of feedback-regulated self-optimisation. Each of these deliverables will immediately have economic and societal impact on current and future synthetic biology research projects, improving predictability of designs and therefore accelerating turn-over times from ideas to realisation. The fundamental research that we will perform will also provide new insights for microbiology and have useful synergy to systems biology research aiming at a greater understanding of the processes underlying natural cell behaviour.

The private sector is actively involved in synthetic biology and its presence in industry is growing at a rate where it is not unreasonable to believe that it will eventually replace large parts of the petrochemical industry. This project will impact here by enabling more complex yet robust designs using our proposed self-optimising plasmids and burden-responsive parts. We especially expect to make an impact on the next challenge in biosynthesis of fuels and high-value compounds by incorporating regulation to optimise production in a range of conditions.

This project will also impact on educational training. Synthetic biology is an increasingly popular subject with students, specifically because of its increasing repeatability, ease-of-use and due to its anticipated impact on the future. The members of this project play an active role in the training and education of the next generation through synthetic biology teaching at Imperial and intend to incorporate the findings, models and parts from this project into courses and suitable research competitions such as iGEM (http://igem.org/Main_Page) or CAGEN (http://openwetware.org/wiki/CAGEN). The UK has had remarkable success in teaching parts-based synthetic biology, producing many world-class undergraduate projects, so investment in further research here in the UK is critical to retaining the best students in the country and building a successful UK-based biotechnology industry.