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

合成生物学使用标准化的生物部件，由工程DNA序列形成，可以组合形成执行有用功能的设备和系统。给定功能所需的部分的组合称为合成遗传回路，当回路被放置在宿主细菌内部时，回路DNA由宿主的细胞机制表达。合成生物学的潜在应用包括农业、医疗保健、环境修复、生物传感和可持续制造。然而，合成电路DNA的表达使用了宿主细胞可用于细胞生长和繁殖的有限资源的一部分。因此，含有回路的细胞通常具有比它们的无回路对应物更低的生长速率和生物体适应性。我们将这种由于资源竞争导致的细胞活动的普遍减慢称为细胞上的全局负担。回路的表达或维持也可以抑制宿主基因组中特定基因的天然功能，这可能对生物体的适应性有害，但也可能反馈和损害回路功能本身。因此，来自电路的寄生性质的一般资源竞争，以及电路与其主机之间的上下文相关的相互作用可以降低电路的性能质量和主机的适应性。这给工程合成回路带来了重大问题，因为除了降低产量外，降低的生物体适应性还对含有细胞的回路产生了选择性压力，这意味着它们可能会被任何突变失去回路的细胞所击败，从而减轻它们所承受的负担。在这个项目中，我们的目标是精确地量化合成电路产生的全局负担和背景效应，并消除由于这些效应而导致的电路性能和进化鲁棒性的任何损失。为了实现这一目标，我们将使用延时显微镜对在高通量微流体室中生长的活细胞进行成像，其中细胞将在均匀的环境中生长，具有单细胞分辨率。通过诱导受控的电路损耗，我们将能够精确地测量包含和不包含电路的遗传相同的细胞之间的生长速率差异。此外，通过对电路和宿主基因组突变的长期观察，我们的目标是深入了解细胞如何进化以与合成电路共存。我们的目标是使用定制的自动化生长室来实现这一目标，该生长室控制细菌培养物的光密度。我们相信，这种方法将使我们能够在实验期间观察到比传统长期进化实验更多的单位时间世代。通过对这些进化实验中的样本进行定期DNA测序，我们将能够量化电路功能丧失的速率和方式。最后，我们的目标是将合成电路引入细菌菌株的集合中，其中每个菌株跟踪细菌基因组中不同基因的活性。通过诱导这些菌株的受控回路丢失，并观察基因组中每个基因在有和没有回路的情况下的活性，我们希望推断合成回路的操作是否以及如何抑制生物体其他基因的表达。该项目与合成生物学EPSRC研究领域最密切相关，该项目的一个关键成果可能是为细菌细胞中的负荷测量提供定量，可重复的基准和新方法。该项目还与传感器和仪器研究领域保持一致，因为重点是开发新的精确方法来测量合成基因电路造成的负担和环境影响。

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.