Living material assembly of bacteriogenic protocells

Abstract

Advancing the spontaneous bottom-up construction of arti cial cells with high organisational complexity and diverse functionality remains an unresolved issue at the interface between living and non-living matter. To address this challenge, a living material assembly process based on the capture and on-site processing of spatially segregated bacterial colonies within individual coacervate micro-droplets is developed for the endogenous construction of membrane-bounded, molecularly crowded, compositionally, structurally and morphologically complex synthetic cells. The bacteriogenic protocells inherit diverse biological components, exhibit multi-functional cytomimetic properties and can be endogenously remodelled to include a spatially partitioned DNA/histone nucleus-like condensate, membranized water vacuoles and a self-supporting 3D network of F-actin proto-cytoskeletal laments. The ensemble is biochemically energized by self-sustainable ATP production derived from implanted live E. coli cells to produce a cellular bionic system with amoeba-like external morphology and integrated lifelike properties. Our results demonstrate a novel bacteriogenic strategy for the bottom-up construction of functional protoliving micro-devices and provide opportunities for the fabrication of new synthetic cell modules and augmented living/synthetic cell constructs with potential applications in engineered synthetic biology and biotechnology.

Membranized Coacervate Microdroplets: from Versatile Protocell Models to Cytomimetic Materials

“…Other studies have assembled arrays of negatively charged hydrophilic liposomes, ca. 180 nm in size, on the surface of positively charged coacervate droplets to produce model protocells with multicompartmentalized organization (Figure c). − Finally, in very recent work, we used living bacteria as building blocks for the membranization of PDDA/ATP coacervate microdroplets . In this system, populations of negatively charged Pseudomonas aeruginosa bacteria with diameters of 1–2 μm are trapped specifically at the coacervate/water interface to produce a living cell/coacervate hybrid architecture (Figure e,f).…”

“…(f) 2D CLSM (bottom) and 3D reconstruction (top) images of bacteria-coated PDDA/ATP coacervate microdroplets ( P. aeruginosa , green fluorescence; coacervate, red fluorescence). Reproduced with permission from ref ( 39 ). Copyright 2022 Springer Nature.…”

“… 36 − 38 Finally, in very recent work, we used living bacteria as building blocks for the membranization of PDDA/ATP coacervate microdroplets. 39 In this system, populations of negatively charged Pseudomonas aeruginosa bacteria with diameters of 1–2 μm are trapped specifically at the coacervate/water interface to produce a living cell/coacervate hybrid architecture ( Figure 3 e,f). Assembly of the membrane into a closely packed array of viable bacterial cells stabilizes the coacervate droplets and facilitates further processing into complex multifunctional bacteriogenic protocells.…”

Open questions on liquid–liquid phase separation

“…Recently, it was reported that complex coacervates show no signs of Ostwald ripening, possibly because of the complexed nature of their contents 6 . Peptides, proteins, lipids, and even small molecules can also adsorb at the surface of some coacervates and effectively stabilize them 13 , 14 . And spreading of coacervates at an air-water interface could lead to droplet splitting, thereby providing a way to proliferation 8 .…”

Complex Coacervate Materials as Artificial Cells

“…Indeed, coacervate droplets (both with and without outer membranes) have been studied by various groups with recent successes in elucidating RNA localization as well as duplex thermodynamics and mechanisms, along with incorporation of bacterial systems for dynamic/growing artificial cells. 14,15 In this Account, we will highlight new developments of synthetic and semisynthetic macromolecular coacervate materials in mimicking cellular systems. We start with briefly discussing some fundamental aspects of coacervate formation, including an overview of the synthetic macromolecules used for coacervate artificial cells to date.…”