Functionalizing cell-mimetic giant vesicles with encapsulated bacterial biosensors

Abstract

The design of vesicle microsystems as artificial cells (bottom-up synthetic biology) has traditionally relied on the incorporation of molecular components to impart functionality. These cell mimics have reduced capabilities compared with their engineered biological counterparts (top-down synthetic biology), as they lack the powerful metabolic and regulatory pathways associated with living systems. There is increasing scope for using whole intact cellular components as functional modules within artificial cells, as a route to increase the capabilities of artificial cells. In this feasibility study, we design and embed genetically engineered microbes (Escherichia coli) in a vesicle-based cell mimic and use them as biosensing modules for real-time monitoring of lactate in the external environment. Using this conceptual framework, the functionality of other microbial devices can be conferred into vesicle microsystems in the future, bridging the gap between bottom-up and top-down synthetic biology.

Keywords: artificial cells; biosensing; cellular bionics; giant lipid vesicles; microfluidics; synthetic biology.

Figure 1.

Lactate biosensor used in this study. (a) Diagram of the lactate biosensor in E. coli. (b) Characterization of lactate biosensor in bulk (non-encapsulated form) using a flow cytometer. The whole-cell E. coli biosensor and control cells were spiked with different concentrations of l-lactate.

Figure 2.

Hybrid lactate biosensor construction via the phase transfer of water-in-oil droplets. (a) Bacteria were encapsulated in lipid-coated water-in-oil droplets loaded with a mixture of sucrose and cell medium, and were expelled above a water–oil column stabilized with a lipid monolayer. The bacteria-loaded droplets sank through the column due to their higher density and picked up another lipid monolayer, forming lipid vesicles. (b) Schematic of the vesicle system that was generated for the hybrid lactate biosensor. Lactate permeates the vesicle via the α-HL pores and initiates a fluorescence response (GFP) from the encapsulated E. coli.

Figure 3.

Microfluidic encapsulation of E. coli biosensor in droplets. (a) Microfluidic device design consisting of a flow-focusing junction and a large reservoir. Channel depth is 20 µm. Micrograph of the reservoir containing water-in-oil droplets. Scale bar, 400 µm. (b) Fluorescence images of bacteria encapsulated in water-in-oil droplets with 50 mM concentration after 300 min of incubation. Scale bars, 40 µm.

Figure 4.

Full operational system showing the hybrid lactate biosensor. (a) Bright field images showing encapsulated microbes in vesicles over 240 min. Scale bar, 20 µm. (b) Fluorescence images of the hybrid lactate biosensor at 50 mM concentration of lactate at t = 0 and t = 270 min. Brightness and contrasts enhanced equally across the fluorescence images. Scale bar, 20 µm. (c) Fluorescence images of hybrid biosensors at 50 mM lactate concentration at t = 240 min. Scale bar, 20 µm. (d) Graph showing fluorescence intensity over time of the produced GFP at different lactate concentrations as lactate penetrates the vesicle system via the α-HL pores. The full two-step reaction cascade takes place. (e) Fluorescence signal at 15 mM lactate in the presence and absence of α-HL pores. (f) Characterization data of the hybrid biosensor at different concentrations of lactate. Error bars represent the standard deviation of five technical replicates.