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Direct Observation of Bacterial Growth in Giant Unilamellar Vesicles: A Novel Tool for Bacterial Cultures

Abstract Bacterial cultivation techniques are classic, basic, and common processes used to characterize the physiological activity of bacteria in their environment. Owing to recent advances in bacterial cultivation techniques, the physiological activity of bacteria can be elucidated at the single‐cell culture level. Here, we report a novel method to monitor the real‐time activity of bacterial growth at the single‐cell level inside giant unilamellar vesicles (GUVs). This method consists of two steps: 1) encapsulation of single bacteria in 1–33 pL scale GUVs and 2) immobilization of the GUVs on a planar lipid bilayer membrane on a glass surface. We directly observed single E. coli cells actively growing to a great number of cells inside GUVs. GUVs also protected the bacteria from external antibiotic compounds during prolonged cultivation for more than 24 h. This approach can be applied widely in the fields of biochemistry, biotechnology, microbiology, and synthetic biology.

Glucose, sucrose, and sodium chloride were purchased from Wako Pure Chemicals (Tokyo, Japan). Yeast extract and bactotryptone were purchased from BD Biosciences (Franklin Lakes, NJ, USA), and deionized water was obtained from a Millipore Milli-Q system (Burlington, MA, USA).

Preculture of E. coli
E. coli was inoculated in 1× LB medium (1 g yeast extract, 2 g bactotryptone, and 2 g sodium chloride in 200 mL of deionized water) from a LB plate and incubated at 37°C overnight (12-14 hours). After incubation, 20 μL of culture solution was collected, transferred to fresh 1× LB medium to a total volume of 2 mL, and cultured for 2 hours.

Synthesis of Giant Unilamellar Vesicles (GUVs) Containing E. coli
We prepared lipid stock solutions of POPC (10 mM) and biotin-PEG-DSPE (0.1 mM) in chloroform/methanol (2:1 v/v). First, 20 μL of POPC solution and 4 μL of biotin-PEG-DSPE solution were poured into a glass tube. These two different lipid compositions were used at a POPC:biotin-PEG-DSPE molar ratio of 100:0.2. To prepare the oil phase with lipids, the organic solvent was evaporated by airflow to form a lipid film at the bottom of the glass tube, which was then placed in a desiccator and the lipid film was dried for 1 hour. Mineral oil (200 μL) was then added to the glass tube, followed by sonication in an ASU-3D ultrasonic bath (AS ONE, Osaka, Japan) for at least 1 hour, with final concentrations of 1 mM POPC and 0.002 mM biotin-PEG-

DSPE.
GUVs were prepared by adapting a previously published protocol. [1] To create an oilwater interface in a 1.5 mL-sized plastic tube, we introduced 50 μL of 1× LB medium with 200 mM glucose (external solution of GUVs) into the 1.5 mL-sized plastic tube and added 150 μL of the mineral oil containing lipids. The plastic tube was then incubated at room temperature (25°C) for 10-15 min. A glass capillary was placed in a holder and this holder set was inserted into the plastic tube. We added 2 μL of E. coli solution consisting of 1× LB medium with 200 mM sucrose (internal solution of GUVs) into the glass capillary. The plastic tube was then centrifuged for 10 minutes at 1600 × g at room temperature (25°C) in a ATT101 desktop centrifuge (Hi-Tech Co., Tokyo, Japan). The oil phase was aspirated from the plastic tube using a pipette, and the GUVs containing E. coli were collected from the plastic tube.

Preparation of Small Unilamellar Vesicles (SUVs)
For the preparation of the supported lipid bilayer membrane, small unilamellar vesicles (SUVs) were prepared. Using the same lipid composition for GUV preparation, 20 μL of the POPC solution and 4 μL of biotin-PEG-DSPE solution were poured into a glass tube. The organic solvent was then evaporated by airflow to form a lipid film at the bottom of the glass tube, which was then placed in a desiccator and the lipid film was dried for 1 hour. A 200-μL aliquot of 1× LB medium with 200 mM glucose (external solution of GUVs) was then added to the glass tube, which was sonicated in an ultrasonic bath for at least 1 hour, with final concentrations of 1 mM POPC and 0.002 mM biotin-PEG-DSPE. After sonication, SUVs were prepared by the extrusion method [2] using a Mini-extruder (Avanti Polar Lipids, Alabaster, AL, USA) and polycarbonate membranes with 100-nm pores (Whatman, Cambridge, UK).

Construction of the GUV Observation System (Bacterial Culture System)
GUVs were observed using a handmade chamber system. A chamber with a doublefaced seal (10 × 10 × 1 mm) was drilled to create a 5-mm hole and pasted on a cover glass (40

Fluorescence Imaging of GUVs Containing E. coli
Confocal laser-scanning imaging of GUVs containing E. coli (Figure 2a and d) was performed using an IX-73 inverted microscope (Olympus, Tokyo, Japan) equipped with a CSU10 spinning disk confocal scanning unit (Yokogawa, Tokyo, Japan), and LUCPLFLN 40×/0.6 NA and 60×/0.7 NA objective lenses (Olympus). Images were captured using a Zyla 4.2 plus sCMOS camera (Andor, Belfast, UK), and images shown in Figure 2b and c were captured with an inverted microscope (Eclipse Ti-E, Nikon, Tokyo, Japan) equipped with an A1R laser-scanning confocal microscopy system (Nikon), a GaAsP detector (Nikon), and an CFI Apo 60×/1.4 NA oil ls oilimmersion objective lens (Nikon). Sectioned images were deconvoluted by the imaging software NIS-Elements version 4.51 (Nikon). All confocal images of Z-projection were processed with ImageJ. For the imaging of GUVs containing E. coli, rhodamine-DHPE was used to stain the GUV membrane, and rhodamine-DHPE was mixed with POPC and biotin-PEG-DSPE in preparation of the oil phase at a final molar ratio of 0.001 against POPC. E. coli cells were stained using SYTO9 as follows: 50 μL of E. coli solution (1× LB medium with 200 mM sucrose) with 5 μM SYTO9 was incubated at room temperature (25°C) for 20 minutes before encapsulation. After incubation, encapsulation into GUVs was carried out as described above.
To detect cell viability, E. coli-containing solution was mixed with 5 μM of SYTO9 and propidium iodide (PI) (Dojindo, Kumamoto, Japan) and incubated at room temperature (25°C) for 20 minutes. Dead E. coli cells were prepared by treatment with 100% methanol for 1 minute [3] .
We checked the cell viability of E. coli before and after encapsulation in GUVs.

Bacterial Culture Inside GUVs Against Antibiotic Compounds
To evaluate the protection of E. coli within GUVs against external antibiotics, 1 μL of ampicillin (1 μg/mL, Wako Pure Chemicals, Osaka, Japan) was added to E. coli-containing GUVs in the hole of chamber, and the chamber was sealed with a cover glass. The E. coli-containing GUVs were incubated at 37°C on a microscopic heating stage system by using a TP-110R-100 thermoplate (TOKAI HIT, Shizuoka, Japan). Bacterial growth was captured with a Zyla 4.2 plus sCMOS camera (Andor, Belfast, UK).

Image Processing and Quantification of E. coli Growth
To quantify E. coli growth, the original phase-contrast microscopic images were used and analyzed with ImageJ ( Figure S8a). We determined the edges of E. coli cells inside a GUV ( Figure S8b). The image was binarized and the region excluding E. coli (A ex ) was measured