Autonomous translation modulation in a homeostatic artificial cell

通过解构细胞，无细胞基因表达已经被用来回答基本的生物学问题。最近，我们可以使用基于无细胞的平台来重建细胞。细菌中负责基因表达的反应网络紧密相连，消耗了一半以上的细胞能量。大量的成分投入到蛋白质合成和基因调控的校对机制中。在这个项目中，我们将设计和研究通过调节基因表达率在合成细胞内创造稳态的遗传电路。该电路将使用来自基因表达机制的组件，因此将与氨基酰化等核心过程耦合。因此，我们将为基因表达设定一个稳定的状态，并使合成细胞更加自主。合成细胞是基于微流体隔间内的表面结合基因，微流体隔间用重组的基因表达溶液连接到流动通道。该装置允许使用TIRF显微镜单分子分辨率长时间（bb0 - 12h）观察基因表达。将基因表达核心过程的组成部分整合到遗传电路中，将向我们展示我们对这些过程的理解的局限性和数量。我们将详细了解直接参与基因表达的组分（如带氨基酸的tRNAs分子）的调控动力学。这个项目的假设是，我们能够构建基因回路，在合成细胞中创造稳态，这是生物体的基本工作原理。

Cell-free gene expression have been used to answer fundamental biological question by deconstructing the cell. More recently we can employ cell-free based platforms towards the reconstruction of a cell. The reaction network responsible for gene expression in bacteria is tightly connected and consumes more than half of the cells energy. A great amount of components are invested in proof-reading mechanisms for protein synthesis and gene regulation. In this project, we will design and study genetic circuits that create homeostasis within a synthetic cell through the modulation of gene expression rates. The circuit will be using components from the gene expression machinery and therefore be coupled to core processes such as aminoacylation. We will thus set a steady-state for gene expression and make the synthetic cell more autonomous. The synthetic cells are based on surface-bound genes inside microfluidic compartments that are connected to a flow channel with reconstituted gene expression solution. The set-up allows for observing gene expression for prolonged time periods (>12h) with single molecule resolution using TIRF microscopy. The integration of components from core processes of gene expression into genetic circuit will show us the limitations and the amount of our understanding of these processes. We will gain detailed insights into the dynamics of the regulation of components that are directly involved in gene expression such as amino acid charged tRNAs molecules. The hypothesis in this project is that we are able to construct genetic circuits to create homeostasis in a synthetic cell, which is a fundamental working principle of living organisms.