Julien Reboud*, Hang Zhao, Kimia Mohammadi, David Paterson, Ellie Puleine <sup>1</sup>, Huabing Yin, Jonathan M. Cooper
Division of Biomedical Engineering, University of Glasgow, Glasgow, UK
* corresponding author -- Jon.Cooper@glasgow.ac.uk
Artificial Protocells for Water Remediation.
Background. The provision of clean drinking water for all has been recognised as a key societal challenge by numerous stakeholders, and wastewater treatment in particular has been highlighted as a crucial technological need, that would benefit from the emergence of radically new techniques. Bacteria have been identified as valuable tools to sense, uptake and dispose of a large range of pollutants (including heavy metals ). In particular, synthetic biology, which aims at the assembly of biological parts into new processes such as metabolic or transport pathways, has extended the potential for the large scale use of specific organisms in water remediation . However, the practical implementation of these solutions has been limited, in part by the difficulties linked to maintaining functioning consortia of organisms, in a fine balance to prevent extensive growth, while maintaining high activity and function.
Aims and Objectives. Here we introduce the potential of artificial protocells in water remediation strategies. These synthetic cell chassis  generate a controlled reservoir that can be used to accumulate toxic or high value substances from the water environment, inside cell-like vesicles . Protocells are double emulsions (as mayonnaise) of water-based micrometre sized drops, surrounded by a cell-like hydrophobic membranes, in a water environment. Our group and others have developed microfluidic platforms to produce protocells of a highly controlled size and composition, using polymers to increase their robustness . Although these systems have been used efficiently as models of cells to study biochemical processes, they remain in isolation from their environment.
Here we demonstrate an engineered microfluidic platform to control the composition and thickness of the membrane of these systems, while producing them at a high throughput (kHz), as would be necessary for practical applications. Ultra-thin membranes (<10nm) are required to insert membrane proteins that will provide communication and energy source to the protocells.
Materials and Methods. We first manufactured a microfluidic system out of chemically modified glass capillaries to fabricate the ultra-thin membrane protocells, comprising an outer phase (10 wt% polyvynilalcool --PVA- solution), inner phase (5 wt% PVA solution in water) and middle phase (3-5 mg/ml 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) + 0.1 mol% Rhodamine- 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE)). Round glass capillaries of 1 mm outer diameter purchased from World precision instruments, Inc., were tapered using a Sutter Instruments model P-97 micropipette puller into the tip of approximately 0.5 mm and inserted in a square capillary of 1.05 mm inner dimension purchased from Atlantic International Technologies. Capillary surfaces were modified with n-octadecyltrimethoxyl silane (Sigma-Aldrich), and 2-[methoxy(polyethylene-oxy)propyl] trimethoxyl silane (Gelest, Inc.), for making them hydrophobic and hydrophilic, respectively .
We then produced the protocells using a middle phase (lipids) composed of 2% Span 80 (Sigma-Aldrich) in mineral oil (Sigma-Aldrich). Typical flow rate ranges for the inner, outer and middle phases, were 500, 1000 and 5000 μl/ h, respectively using syringe pumps (Harvard Apparatus). Flow motion was observed using upright microscope (Axio Lab.A1, Zeiss) equipped with Andor sCMOScamera (100 fps).
We observed the membranes using a combination of confocal fluorescence microscopy, by incorporating dyes within the inner phase (FITC, green) and the middle phase (Rhodamine, red), and atomic force microscopy (AFM) to study the thickness of the membranes.
Results and Discussion. We created artificial protocells at high throughput with a highly controlled size (180 Âµm +/- 10 Âµm) in a monodisperse fashion. Using AFM, we confirmed that the membrane of the chassis was ultrathin (between 4 and 7 nm), a size conducive to the introduction of membrane proteins within this highly controlled system.
Conclusions. We have demonstrated a microfluidic system to produce artificial protocells with an ultrathin shell that is amenable to protein insertion. The proteins of interest, a metal transporter, will be inserted into the middle phase and self-assembled into the membrane of the protocells. In future we will use cell-free gene expression machinery, which we have already utilized successfully in thicker systems, to obtain a self-sustained protocells, that could be deployed in water treatment applications.
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