Quantifying global burden and contextual effects of synthetic genetic circuits in their bacterial chassis

Synthetic biology uses standardised biological parts, formed from engineered DNA sequences, which can be combined to form devices and systems which perform useful functions. The combination of parts necessary for a given function is called a synthetic genetic circuit, and when a circuit is placed inside a host bacterium the circuit DNA is expressed by the cellular machinery of the host. Potential uses for synthetic biology include applications in agriculture, healthcare, environmental remediation, biosensing and sustainable manufacturing.
However, the expression of synthetic circuit DNA uses a portion of the limited resources available to the host cell for cell growth and reproduction. Therefore, circuit containing cells typically have a lower growth rate and organism fitness than their circuit free counterparts. We refer to this general slow-down of cellular activity due to resource competition as a global burden on the cell. The expression or maintenance of the circuit can also inhibit the natural function of specific genes in the host genome, which can be detrimental to organism fitness, but can also feedback and impair the circuit function itself.
Therefore, the general resource competition from the parasitic nature of the circuit, and context dependent interactions between the circuit and its host can reduce both the performance quality of the circuit and the fitness of the host. These pose major problems for engineering synthetic circuits, since in addition to reducing yields, the reduced organism fitness creates a selective pressure against the circuit containing cells, meaning they are likely to be outcompeted by any cells which mutate to lose the circuit and hence reduce the burden they experience. In general, this means useful functions performed by circuits are very quickly lost from a population of cells.
In this project, we aim to precisely quantify the global burden and the contextual effects arising from a synthetic circuit, and to characterise any loss in circuit performance and evolutionary robustness due to these effects. To achieve this, we will use time-lapse microscopy to image live cells growing in a high-throughput microfluidic chamber where cells will grow in a uniform environment, with single cell resolution. By inducing a controlled circuit loss, we will be able to precisely measure the difference in growth rates between genetically identical cells containing and free from the circuit.
Furthermore, through long term observation of mutations in the circuit and host genome, we aim to gain insight into how cells evolve to co-exist with a synthetic circuit. We aim to achieve this using a custom built, automated growth chamber which controls the optical density of the bacterial culture. We believe this methodology will allow us to observe many more generations per unit time over the length of the experiment than traditional long-term evolution experiments. Through regular DNA sequencing of samples from these evolution experiments, we will be able to quantify the rate and manner by which circuit function is lost.
Lastly, we aim to introduce a synthetic circuit into collections of bacterial strains, where each strain tracks the activity of a different gene in the bacterial genome. By inducing a controlled circuit loss from these strains, and observing the activity of each gene in the genome both with and without the circuit, we hope to deduce whether and how the operation of the synthetic circuit inhibits the expression of others genes of the organism.
This project most closely aligns with the Synthetic Biology EPSRC research area, and a key outcome of this project could be to provide quantitative, metrological benchmarks and new methods for the measurement of burden in bacterial cells. The project also aligns with the Sensors and Instrumentation research area, due to the focus on developing new and precise methods for measuring burden and contextual effects caused by synthetic genetic circuits.

The primary outputs from the CDT will be cohorts of highly qualified, interdisciplinary postgraduates who are experts in a wide range of sensing activities. They will benefit from a world leading training experience that recognises sensor research as an academic discipline in its own right. The students will be taught in all aspects of Sensor Technologies, ranging from the physical and chemical principles of sensing, to sensor design, data capture and processing, all the way to applications and opportunities for commercialisation, with a strong focus in entrepreneurship, technology translation and responsible leadership. Students will learn in extensive team and cohort engaging activities, and have access to cutting-edge expertise and infrastructure. 90 academics from 15 different departments participate in the programme and more than 40 industrial partners are actively involved in delivering research and business leadership training, offering perspectives for impact and translation and opportunities for internships and secondments. End users associated with the CDT will benefit from the availability of outstanding, highly qualified and motivated PhD students, access to shared infrastructure, and a huge range of academic and industrial contacts.

Immediate beneficiaries of our CDT will be our core industrial consortium partners (MedImmune, Alphasense, Fluidic Analytics, ioLight, NokiaBell, Cambridge Display Technologies, Teraview, Zimmer and Peacock, Panaxium, Silicon Microgravity, etc., see various LoS) who incorporate our cross-leverage funding model into their corporate research strategies. Small companies and start-ups particularly benefit from the flexibility of the partnerships we can offer. We will engage through weekly industry seminars and monthly Sensor Cafés, where SME employees can interact directly with the CDT students and PIs, provide training in topical areas, and, in turn, gain themselves access to CDT infrastructure and training. Ideas can be rapidly tested through industrially focused miniprojects and promising leads developed into funded PhD programmes, for which leveraged funding is available through the CDT.

Government departments and large research initiatives are formally connected to the CDT, including the Department for the Environment, Food and Rural Affairs (DEFRA); the Cambridge Centre for Smart Infrastructure and Construction (CSIC); the Centre for Global Equality (CGE); the National Physics Laboratory (NPL); the British Antarctic Survey (BAS), who all push our CDT to generate impacts that are in the public interest and relevant for a healthy and sustainable future society. With their input, we will tackle projects on assisted living technologies for the ageing population, diagnostics of environmental toxins in the developing world, and sensor technologies that help replace the use of animals in research. Developing countries will benefit through our emphasis on open technologies / open innovation and our exploration of responsible, ethical, and transparent business models. In the UK, our CDT will engage directly with the public sector and national policy makers and regulators (DEFRA, and the National Health Service - NHS) and, with their input, students are trained on impact and technology translation, ethics, and regulatory frameworks.