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

Lead Research Organisation: University of Cambridge
Department Name: Engineering

Abstract

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.

Publications

10 25 50

Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/S023046/1 01/10/2019 31/03/2028
2262510 Studentship EP/S023046/1 01/10/2019 30/09/2023 Charles David Wedd