Genetic Control of Radical Cross-linking in a Semisynthetic Hydrogel

Abstract

Enhancing materials with the qualities of living systems, including sensing, computation, and adaptation, is an important challenge in designing next-generation technologies. Living materials seek to address this challenge by incorporating live cells as actuating components that control material function. For abiotic materials, this requires new methods that couple genetic and metabolic processes to material properties. Toward this goal, we demonstrate that extracellular electron transfer (EET) from Shewanella oneidensis can be leveraged to control radical crosslinking of a methacrylate-functionalized hyaluronic acid hydrogel. Crosslinking rates and hydrogel mechanics, specifically storage modulus, were dependent on a variety of chemical and biological factors, including S. oneidensis genotype. Bacteria remained viable and metabolically active in the crosslinked network for a least one week, while cell tracking revealed that EET genes also encode control over hydrogel microstructure. Moreover, construction of an inducible gene circuit allowed transcriptional control of storage modulus and crosslinking rate via the tailored expression of a key electron transfer protein, MtrC. Finally, we quantitatively modeled dependence of hydrogel stiffness on steady-state gene expression, and generalized this result by demonstrating the strong relationship between relative gene expression and material properties. This general mechanism for radical crosslinking provides a foundation for programming the form and function of synthetic materials through genetic control over extracellular electron transfer.

In Situ Optical Quantification of Extracellular Electron Transfer using Plasmonic Metal Oxide Nanocrystals

“…Given the aerobic preparation of the cell culture and sample, they also suggest that dissolved oxygen is first consumed through aerobic respiration, followed by activation of anaerobic EET pathways ( i . e ., reduction of PAAPEO-ITO), as we have previously observed for other electron acceptors such as copper and fumarate (45, 46). We then measured EET from a S. oneidensis MR-1 population deprived of a carbon source ( e .…”

“…This quantification revealed two consistent electron transfer regimes across samples – a variable regime during the first ∼1 h, where electron transfer was nonlinear (Figure 3a, inset; Figure S8a), followed by a steady-state electron transfer regime. In previous studies, aerobic S. oneidensis at OD 600 = 0.2 took between 10 min to 1 h to fully consume dissolved oxygen and turn on anaerobic EET pathways, depending on the electron acceptor and reaction conditions (45, 46). Therefore, we attribute this initial variable domain to a combination of oxygen removal via aerobic respiration and cell stress as they adjust to a new electron acceptor and undergo a metabolic shift.…”

“…Toward this goal, we used an S. oneidensis strain that tailors expression of a critical EET gene, mtrC , in response to inducing molecule concentration. Genetically engineered S. oneidensis Δ mtrC Δ omcA Δ mtrF + mtrC proportionally expresses mtrC in the presence of isopropyl ß-D-1-thiogalactopyranoside (IPTG) as an inducer (12, 46). Cultures of S. oneidensis Δ mtrC Δ omcA Δ mtrF + mtrC were pregrown anaerobically in varying concentrations of IPTG (ranging from 0 to 1000 µM), leading to differing expression levels of mtrC and resultant EET flux.…”

Recent advancements in enzyme-mediated crosslinkable hydrogels: In vivo-mimicking strategies

“…12(a-ii-iii) ]. 183 Crosslinking may be adjusted by knocking out the EET-associated genes of S. oneidensis . MtrC, a key electron transfer protein, can also be regulated under transcriptional control.…”

“…Copyright 2020 American Chemical Society. 183 (b) Schematic illustration of neuronal membrane-specific polymerization of aniline by genetically targeted chemical assembly (i). Schematic illustration of aniline polymerization mediated by Apex2 (ii).…”