Haimov, Y. | Search bioRxiv

bioRxiv2019

Sticky Situations: Bacterial Attachment Deciphered by Interferometry of Silicon Microstructures

The peculiarities of surface-bound bacterial cells are often overshadowed by the study of planktonic cells in clinical microbiology. Thus, we employ phase-shift reflectometric interference spectroscopic measurements to observe the interactions between bacterial cells and abiotic, microstructured material surfaces in a label-free, real-time manner. Both material characteristics (i.e., substrate surface charge and wettability) and characteristics of the bacterial cells (i.e., motility, cell charge, biofilm formation, and physiology) drive bacteria to adhere to a particular surface. We conclude that the attachment of bacterial cells to a surface is determined by the culmination of numerous factors. When specific characteristics of the bacteria are met with factors of the surface, enhanced cell attachment and biofilm formation occur. Such knowledge can be exploited to predict antibiotic efficacy, biofilm development, enhance biosensor development, as well as prevent biofouling.

microbiology↗

bioRxiv2017

Demonstration of de novo chemotaxis in E. coli using a real-time, quantitative, and digital-like approach

Chemotaxis is the movement of an organism in response to an external chemical stimulus. This system enables bacteria to sense their immediate environment and adapt to changes in its chemical composition. Bacterial chemotaxis is mediated by chemoreceptors, membrane proteins that bind an effector and transduce the signal to the downstream proteins. From a synthetic biology perspective, the natural chemotactic repertoire is of little use since bacterial chemoreceptors have evolved to sense specific ligands that either benefit or harm the cell. Here we demonstrate that using a combined computational design approach together with a quantitative, real-time, and digital detection approach, we can rapidly design, manufacture, and characterize a synthetic chemoreceptor in E. coli for histamine (a ligand for which there are no known chemoreceptors). First, we employed a computational protocol that uses the Rosetta bioinformatics software together with high threshold filters to design mutational variants to the native Tar ligand binding domain that target histamine. Second, we tested different ligand-chemoreceptors pairs with a novel chemotaxis assay, based on optical reflectance interferometry of porous silicon (PSi) optical transducers, enabling label-free quantification of chemotaxis by monitoring real-time changes in the optical readout (expressed as the effective optical thickness, EOT). We found that different ligands can be characterized by an individual set of fingerprints in our assay. Namely, a binary, digital-like response in EOT change (i.e. positive or negative) that differentiates between attractants and repellants, the amplitude of change of EOT response, and the rate by which steady state in EOT change is reached. Using this assay, we were able to positively identify and characterize a single mutational chemoreceptor variant for histamine that mediated chemotaxis comparably to the natural Tar-aspartate system. Our results demonstrate the possibility of not only expanding the natural chemotaxis repertoire, but also provide a new quantitative assay by which to characterize the efficacy of the chemotactic response.

synthetic biology↗