Advanced Characterization of Engineered Living Materials

Abstract

Engineered Living Materials (ELMs) are biological structures composed of cells embedded in a self-produced protein matrix [Molinari et al. 2021]. Our lab pioneered de novo ELMs made of elastin-like polypeptides (ELPs) displayed on the cell surface [Molinari et al. 2022]. Previous studies show that a fraction of ELPs detach from the outer membrane to be secreted into the media, contributing to the formation of macroscopic ELP-cell aggregates [Molinari et al. 2022]. Since the secretion of ELPs was not intentional, it is impossible to calculate the exact ELP-cell ratio within ELMs. However, this ratio is critical for the control of final ELM mechanical properties. To characterize the ELP-cell ratio within ELMs, I inserted a Tobacco Etch Virus protease (TEVp) cut site between the ELP and the surface anchor. This allows the ELP to be cleaved from the cell surface by adding TEVp to the media. In this way, I will quantify the protein content via immunoblotting and the cell number by measuring colony-forming units and the production of fluorescent proteins.