Japan_IPAP: High-Throughput Prototyping of Heterogeneity in genetic networks using Artificial Cells with femtolitre volume (HT-PHAC)

Synthetic biology engineers living systems to perform useful functions. For example, we engineer small bacteria's genomes to produce expensive vitamins or to degrade plastic waste. However, cells do not behave the same even when their genetic information is the same. For example, when we engineer cells to produce a specific molecule, some cells produce it efficiently while other cells do not. This is a problem because the overall yield of production is reduced because of inefficient cells. This increase in the production cost is one of the major obstacles that need to be overcome to commercialise many synthetic biology applications.
To solve this problem, we need to know what is happening inside each cell. However, it is not an easy task because a cell is a complex object. Even a simple bacterial cell has more than one million molecules inside its cytoplasm. In this proposal, we will develop a simple cell mimic - an artificial cell system made from scratch using synthetic elements - to observe what is happening inside a cell. This will help us to understand why cells show different responses despite sharing the same genetic information. A microfluidic device will be used to produce artificial cells at a scale large enough to analyse different populations. Then we will observe individual cells and their responses. The result will be analysed with mathematical modelling to understand why certain cells behave differently from other cells. This knowledge will allow us to engineer cells that exhibit homogeneous and consistent behaviour. In a long term, this work will help commercialise a lot of synthetic biology applications by reducing their production costs.

Cellular heterogeneity arises when cells show different phenotypes despite sharing the same genome. With recent developments in single-cell technologies, cellular heterogeneity has been studied widely in various subjects of biology including microbiology, cancer biology and neuroscience. While it has been mainly overlooked in synthetic biology, heterogeneity has significant implications as heterogeneous subpopulations with reduced production yield hamper the productivity of the entire population. However, it is difficult to understand how cellular heterogeneity arises due to the complexity of cells.
In this project, we will build an artificial cell system and study it with single-cell techniques to understand the heterogeneous behaviour of E. coli cells in the context of synthetic biology. The cell-free transcription-translation (TXTL) system will be encapsulated inside of a 1-micrometre compartment produced by a high-throughput microfluidic device. The TXTL system allows the implementation of genetic circuits that can be analysed at single-cell resolution using a fluorescence-activated cell sorting device. The subpopulations will be further analysed at single-molecule resolution in real-time with high-resolution fluorescence microscopy by immobilising the cells onto a glass surface. We will maximise the benefit of using an artificial cell system by enhancing the imaging using external fluorescence reporters added inside the cells. Through mathematical modelling, we will gain insight into how different factors affect cellular heterogeneity when different types of circuits are implemented. This will allow us to control the population homogeneity to optimise yield for synthetic biology. Therefore, our work will play an important role in the commercialisation of synthetic biology applications.