Manipulation of encapsulated artificial phospholipid membranes using sub-micellar lysolipid concentrations.

Droplet Interface Bilayers (DIBs) constitute a commonly used model of artificial membranes for synthetic biology research applications. However, their practical use is often limited by their requirement to be surrounded by oil. Here we demonstrate in-situ bilayer manipulation of submillimeter, hydrogel-encapsulated droplet interface bilayers (eDIBs). Monolithic, Cyclic Olefin Copolymer/Nylon 3D-printed microfluidic devices facilitated the eDIB formation through high-order emulsification. By exposing the eDIB capsules to varying lysophosphatidylcholine (LPC) concentrations, we investigated the interaction of lysolipids with three-dimensional DIB networks. Micellar LPC concentrations triggered the bursting of encapsulated droplet networks, while at lower concentrations the droplet network endured structural changes, precisely affecting the membrane dimensions. This chemically-mediated manipulation of enclosed, 3D-orchestrated membrane mimics, facilitates the exploration of readily accessible compartmentalized artificial cellular machinery. Collectively, the droplet-based construct can pose as a chemically responsive soft material for studying membrane mechanics, and drug delivery, by controlling the cargo release from artificial cell chassis.

Formation of Polarized, Functional Artificial Cells from Compartmentalized Droplet Networks and Nanomaterials, Using One-Step, Dual-Material 3D-Printed Microfluidics.

The bottom-up construction of synthetic cells with user-defined chemical organization holds considerable promise in the creation of bioinspired materials. Complex emulsions, droplet networks, and nested vesicles all represent platforms for the engineering of segregated chemistries with controlled communication, analogous to biological cells. Microfluidic manufacture of such droplet-based materials typically results in radial or axisymmetric structures. In contrast, biological cells frequently display chemical polarity or gradients, which enable the determination of directionality, and inform higher-order interactions. Here, a dual-material, 3D-printing methodology to produce microfluidic architectures that enable the construction of functional, asymmetric, hierarchical, emulsion-based artificial cellular chassis is developed. These materials incorporate droplet networks, lipid membranes, and nanoparticle components. Microfluidic 3D-channel arrangements enable symmetry-breaking and the spatial patterning of droplet hierarchies. This approach can produce internal gradients and hemispherically patterned, multilayered shells alongside chemical compartmentalization. Such organization enables incorporation of organic and inorganic components, including lipid bilayers, within the same entity. In this way, functional polarization, that imparts individual and collective directionality on the resulting artificial cells, is demonstrated. This approach enables exploitation of polarity and asymmetry, in conjunction with compartmentalized and networked chemistry, in single and higher-order organized structures, thereby increasing the palette of functionality in artificial cellular materials.

Photopolymerization of polydiacetylene in hybrid liposomes: effect of polymerization on stability and response to pathogenic bacterial toxins.

Liposomes containing lipids and polydiacetylene (PDA) are hybrid systems encompassing both a fluid phospholipid membrane and a polymer scaffold (PDA). However, the biophysical role of PDA in such liposomes is not well understood. In this report, we studied the effects of photopolymerization of PDA on the stability of lipid-PDA liposomes, and their sensitivity to selected purified toxins and bacterial supernatants, using a fluorescence assay. Of the three different types of liposomes with variable lipid chain lengths that were chosen, the degree of polymerization had a significant impact on the long-term stability, and response, to external microbial exotoxins secreted by pathogenic bacteria, namely, Staphylococcus aureus and Pseudomonas aeruginosa. The degree of polymerization of TCDA played an important role in lipid-chain-length-dependent stabilization of lipid-PDA liposomes, as well as in their response to bacterial toxins of S. aureus and P. aeruginosa.

Genes that influence swarming motility and biofilm formation in Variovorax paradoxus EPS.

BACKGROUND: Variovorax paradoxus is an aerobic soil bacterium associated with important biodegradative processes in nature. We use V. paradoxus EPS to study multicellular behaviors on surfaces. METHODOLOGY: We recovered flanking sequence from 123 clones in a Tn5 mutant library, with insertions in 29 different genes, selected based on observed surface behavior phenotypes. We identified three genes, Varpa_4665, Varpa_4680, and Varpa_5900, for further examination. These genes were cloned into pBBR1MCS2 and used to complement the insertion mutants. We also analyzed expression of Varpa_4680 and Varpa_5900 under different growth conditions by qPCR. RESULTS: The 29 genes we identified had diverse predicted functions, many in exopolysaccharide synthesis. Varpa_4680, the most commonly recovered insertion site, encodes a putative N-acetyl-L-fucosamine transferase similar to WbuB. Expression of this gene in trans complemented the mutant fully. Several unique insertions were identified in Varpa_5900, which is one of three predicted pilY1 homologs in the EPS genome. No insertions in the two other putative pilY1 homologs present in the genome were identified. Expression of Varpa_5900 altered the structure of the wild type swarm, as did disruption of the chromosomal gene. The swarming phenotype was complemented by expression of Varpa_5900 from a plasmid, but biofilm formation was not restored. Both Varpa_4680 and Varpa_5900 transcripts were downregulated in biofilms and upregulated during swarming when compared to log phase culture. We identified a putative two component system (Varpa_4664-4665) encoding a response regulator (shkR) and a sensor histidine kinase (shkS), respectively. Biofilm formation increased and swarming was strongly delayed in the Varpa_4665 (shkS) mutant. Complementation of shkS restored the biofilm phenotype but swarming was still delayed. Expression of shkR in trans suppressed biofilm formation in either genetic background, and partially restored swarming in the mutant. CONCLUSIONS: The data presented here point to complex regulation of these surface behaviors.