Department of Biological Sciences and Biotechnology, Institute of Advanced Research, Koba Institutional Area, Gandhinagar 382426, India.
Department of Biological Sciences and Biotechnology, Institute of Advanced Research, Koba Institutional Area, Gandhinagar 382426, India; Present address: Department of Biosciences, School of Sciences, Indrashil University, Rajpur, Mehsana-382740, Gujarat, India.
Biophys Chem. 2019 Nov;254:106244. doi: 10.1016/j.bpc.2019.106244. Epub 2019 Aug 6.
Macromolecules present in the intracellular environment of a cell are densely packed, resulting in a highly crowded cytosolic environment. This crowded milieu influences several biochemical equilibria such as diffusibility and association constant of biomolecules which impose a serious impact on cellular functions as well as its processes. A number of in silico and in vitro studies have been reported till date about using synthetic crowding agents for resembling such a crowding environment within the cell. Lately, it has been realized that synthetic crowders are not suitable for mimicking the intrinsic environment of the cell. In this study, proteins were assumed to be the major biological molecule which contributes to the crowding environment. We have semi-theoretically determined the total protein concentration within an individual E. coli MG1655 cell which changes notably as the growth curve proceeds from 0.2 to 1.0 OD. The average range of total cellular protein concentration throughout the batch culture was found to be in the range of 15.2 to 178 fg/fL of cytoplasmic volume. The fundamental knowledge gained through the study was translated to applied research in the form of an equation. We propose an equation that could help to mimic the OD dependent crowding environment present within a single cell of E. coli in the desired volume of reaction solution. In a nutshell, the equation provides quantitative estimation of the volume of culture required to prepare the cell lysate for biomimicking the intracellular crowding environment in vitro. This finding provides a new insight into the cellular cytosolic environment that could be used as a platform to frame more cells like environment in cell-free protein synthesis (CFPS) system for synthetic biology applications.
细胞内环境中存在的大分子物质排列紧密，导致细胞胞质环境高度拥挤。这种拥挤的环境会影响到一些生化平衡，如生物分子的扩散性和结合常数，从而对细胞功能和过程产生严重影响。迄今为止，已经有许多关于使用合成拥挤剂来模拟细胞内这种拥挤环境的体内和体外研究报告。最近，人们已经意识到合成拥挤剂并不适合模拟细胞的固有环境。在这项研究中，我们假设蛋白质是导致拥挤环境的主要生物分子。我们半理论上确定了单个大肠杆菌 MG1655 细胞内的总蛋白浓度，该浓度随着生长曲线从 0.2 到 1.0 OD 的进展而显著变化。在整个批次培养过程中，总细胞蛋白浓度的平均范围在 15.2 到 178 fg/fL 细胞质体积之间。通过这项研究获得的基础知识被转化为应用研究，形成了一个方程。我们提出了一个方程，可以帮助模拟大肠杆菌单个细胞中存在的 OD 依赖性拥挤环境，所需的反应溶液体积。简而言之，该方程提供了定量估计所需培养体积的方法，以制备细胞裂解物，用于体外模拟细胞内拥挤环境。这一发现为细胞胞质环境提供了新的认识，可作为一个平台，用于在无细胞蛋白质合成 (CFPS) 系统中构建更多类似细胞的环境，以用于合成生物学应用。
