An equation for biomimicking macromolecular crowding using Escherichia coli MG1655 strain

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 OD600. 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 OD600 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.