This work was supported by a Leading Initiative for Excellent Young Researchers (LEADER, No. 16812285) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, a Grant-in-Aid for Young Scientist Research (No. 18K18157, 16K21034) from Japan Society for the Promotion of Science (JSPS) to M.M., and Grant-in-Aid from MEXT to K.K. (No. 17H06417, 17H06413).

1. Preparation of GVs Containing Bacterial Cells by the Droplet Transfer Method
<ol>
	<li>Prepare lipid stock solutions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, 10 mM, 1 mL) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000] (biotin-PEG-DSPE, 0.1 mM, 1 mL) in chloroform/methanol solution (2/1, v/v) and store the stock at -20 &#176;C.</li>
	<li>Preparation of a lipid-containing oil solution
	<ol>
		<li>Pour 20 &#956;L of the POPC solution and 4 &#956;L of the biotin-PEG-DSPE solution into a glass tube (Figure 1b (i)).</li>
		<li>Evaporate the organic solvent by air flow to form a lipid film and place the film in a desiccator for 1 h to completely evaporate the organic solvent (Figure 1b (ii)). 
		NOTE: It is necessary to evaporate the organic solvent in a fume hood.</li>
		<li>Add 200 &#956;L of mineral oil (0.84 g/mL, Table of Materials) to the glass vial (Figure 1b (iii)).</li>
		<li>Wrap the opening part of the glass vial with film and sonicate it in an ultrasonic bath (120 W) for at least 1 h (Figure 1b (iii)). The final concentrations of POPC and biotin-PEG-DSPE are 1 mM and 0.002 mM, respectively.</li>
	</ol>
	</li>
	<li>Pre-culture of bacterial cells
	<ol>
		<li>Inoculate E. coli into 1x LB medium (1 g yeast extract, 2 g bacto tryptone, and 2 g sodium chloride in 200 mL of deionized water) from an LB plate and incubate at 37 &#176;C for 12&#8211;14 h (overnight).</li>
		<li>After incubation, collect 20 &#956;L of the culture solution and transfer to 1.98 mL of fresh 1x LB medium, and culture the cells again for 2 h.</li>
		<li>Check the optical density at 600 nm (OD600) value of the pre-culture solution (prepared in step 1.3.2). A pre-culture solution of OD600 = 1.0&#8211;1.5 should be used.</li>
	</ol>
	</li>
	<li>Preparation of the outer and inner aqueous solutions of GVs
	<ol>
		<li>Dissolve glucose in 1x LB medium to prepare an outer aqueous solution of GVs. Prepare 20 mL of a stock glucose solution (500 mM).</li>
		<li>Dilute the stock glucose solution with 1x LB medium to 200 mM (Table 1).</li>
		<li>Dissolve sucrose in 1x LB medium to prepare an inner aqueous solution of GVs. Prepare 20 mL of a stock sucrose solution (500 mM).</li>
		<li>Mix the pre-culture solution (OD600 = 1.0&#8211;1.5), sucrose solution (500 mM), and 1x LB medium (Table 1). The final OD600 value of the culture solution should be 0.01&#8211;0.015 and the final sucrose concentration should be 200 mM. 
		NOTE: Take care to avoid osmotic pressure. It is necessary to balance the concentration between the inner and outer aqueous solution.</li>
	</ol>
	</li>
	<li>Preparation of water-in-oil (W/O) droplets containing bacterial cells
	<ol>
		<li>Add 2 &#956;L of the inner aqueous solution of GVs (prepared in step 1.4.4) to 50 &#956;L of the oil solution containing lipids (mineral oil with POPC and biotin-PEG-DSPE) in a 0.6 mL lidded plastic tube (Figure 1b (iv)).</li>
		<li>Emulsify the two components in the plastic tube by tapping the tube by hand (Figure 1b (v)).</li>
	</ol>
	</li>
	<li>Formation of GVs containing bacterial cells
	<ol>
		<li>Add 50 &#956;L of the outer aqueous solution of GVs (prepared in step 1.4.2) in a 1.5 mL lidded plastic tube (Figure 1b (vi)) and gently layer 150 &#956;L of the oil solution containing lipids (mineral oil with POPC and biotin-PEG-DSPE) on the surface of the outer aqueous solution (Figure 1b (vii)). Incubate this sample at room temperature (RT, 25 &#176;C) for 10&#8211;15 min. Check to ensure that the interface of the oil and aqueous solutions is flat.</li>
		<li>Add 50 &#956;L of the W/O droplet solution (prepared in step 1.5.2) on the interface of the oil and aqueous solution using a pipette (Figure 1b (viii)).</li>
		<li>Centrifuge the 1.5 mL lidded plastic tube (from step 1.6.2) for 10 min at 1,600 x g at RT in a desktop centrifuge (Figure 1b (ix)). After centrifugation, aspirate the oil (top layer) from the 1.5-mL lidded plastic tube using a pipette, and collect the GVs containing bacterial cells (Figure 1b (x)).</li>
	</ol>
	</li>
</ol>
2. Preparation of a GV Observation System (Bacterial Cell Culture System)
<ol>
	<li>Preparation of small vesicles (SVs) for constructing a supported bilayer membrane
	<ol>
		<li>Pour 20 &#956;L of the POPC solution and 4 &#956;L of the biotin-PEG-DSPE solution into a glass tube (using the same lipid composition as used for GV preparation in step 1.1).</li>
		<li>Evaporate the organic solvent by air flow to form a lipid film and place this sample in a desiccator for 1 h to completely evaporate the organic solvent.</li>
		<li>Add 200 &#956;L of 200 mM glucose in 1x LB medium (the outer aqueous solution of GVs) to the glass vial.</li>
		<li>Wrap the opening part of the glass vial with film and sonicate it in an ultrasonic bath (120 W) for at least 1 h.</li>
		<li>Prepare SVs by the extrusion method21 using a mini-extruder and polycarbonate membrane with 100 nm pore size.</li>
	</ol>
	</li>
	<li>Preparation of a handmade chamber
	<ol>
		<li>Drill a 7 mm hole with a hollow punch on a double-faced seal (10 mm x 10 mm x 1 mm).</li>
		<li>Paste the double-faced seal with the hole on a cover glass (30 mm x 40 mm, thickness 0.25&#8211;0.35 mm).</li>
	</ol>
	</li>
	<li>Preparation of a supported bilayer membrane on the cover glass in the hole of the chamber
	<ol>
		<li>Add 30 &#956;L of the SV solution to the hole of the chamber (prepared in section 2.2) and incubate at RT for 30 min.</li>
		<li>Gently wash the hole twice with 20 &#956;L of 1x LB medium containing 200 mM glucose (the outer aqueous solution of GVs) by pipetting.</li>
	</ol>
	</li>
	<li>Immobilization of GVs on the supported bilayer membrane on the cover glass in the hole of the chamber
	<ol>
		<li>Introduce 10 &#956;L of neutravidin with the outer aqueous solution of GVs (1 mg/mL) into the hole and incubate at RT for 15 min.</li>
		<li>Gently wash the hole twice with 20 &#956;L of 1x LB medium containing 200 mM glucose (the outer aqueous solution of GVs) by pipetting.</li>
		<li>Add all solution containing GVs (prepared in step 1.6.3) into the hole of the chamber and seal with a cover glass (18 mm x 18 mm, thickness 0.13&#8211;0.17 mm) (Figure 2b).</li>
	</ol>
	</li>
	<li>Microscopic observation of bacterial cell growth inside GVs
	<ol>
		<li>Set a microscopic heating stage system with an inverted microscope equipped with a 40x/0.6 numerical aperture (NA) objective lens with a long working distance (Figure 2b).</li>
		<li>Place the chamber on the microscopic heating stage system (Figure 2b). Incubate the GVs containing bacterial cells in the chamber at a static condition for 6 h at 37 &#176;C.</li>
		<li>Capture and record microscope images of bacterial cell growth inside GVs every 30 min by using a scientific complementary metal oxide semiconductor (sCMOS) camera.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Here, we describe a method for culturing bacterial cells at the single-cell level inside GVs. This simple method involves forming GVs containing bacterial cells at the single-cell level by using the droplet transfer method. Compared with other approaches for obtaining GVs containing bacterial cells, this method has two advantages: (i) it is easy to develop, and (ii) a small volume (2 &#956;L) of the sample solution is required to prepare the GVs. The droplet transfer method20 for preparing GVs con...

The culture of bacterial cells at the single-cell level has received increasing attention. Culturing bacterial cells at the single-cell level inside a confined space can elucidate bacterial functions such as phenotypic variability1,2,3,4, cell behavior5,6,7,8,9, and antibiotic resistance10,11. Because of recent advances in culture techniques, the culture of single bacteria can be achieved inside a confined space, such as in a well-chip4,7,8, gel droplet12,13, and water-in-oil (W/O) droplet5,11. To promote understanding or utilization of single bacterial cells, further technical developments of cultivation techniques are needed.
Vesicles that mimic the biological cell membrane are spherical compartments consisting of amphiphilic molecules and can hold various materials. Vesicles are classified according to size and include small vesicles (SVs, diameter &#60; 100 nm), large vesicles (LVs, &#60;1 &#956;m), and giant vesicles (GVs, &#62;1 &#956;m). SVs or LVs are commonly used as drug carriers because of their affinity to the biological cell membrane14. GVs have also been used as a reactor system for the construction of protocells15 or artificial-cells16. Encapsulation of biological cells into GVs has been reported17,18, and thus GVs show potential as a cell culture system when combined with the reactor system.
Here, along with a video of experimental procedures, we describe how GVs can be used as novel cell-culture vessels19. GVs containing bacteria were made by the droplet transfer method20 and were then immobilized on a supported membrane on a cover glass. We used this system to observe bacterial growth at the single-cell level inside GVs in real-time.

<table><tbody><tr><td>Bactotryptone</td><td>BD Biosciences</td><td>211705</td><td></td></tr><tr><td>Chloroform</td><td>Wako Pure Chemicals</td><td>032-21921</td><td></td></tr><tr><td>Cover glass (18 &amp;times; 18 mm)</td><td>Matsunami Glass Ind.</td><td>C018181</td><td>thickness 0.13&amp;ndash;0.17 mm</td></tr><tr><td>Cover glass (30 &amp;times; 40 mm)</td><td>Matsunami Glass Ind.</td><td>custom-order</td><td>thickness 0.25&amp;ndash;0.35 mm</td></tr><tr><td>Desktop centrifuge</td><td>Hi-Tech Co.</td><td>ATT101</td><td>swing rotor type</td></tr><tr><td>Double-faced seal (10 &amp;times; 10 &amp;times; 1 mm)</td><td>Nitoms</td><td>T4613</td><td></td></tr><tr><td>Glass vial</td><td>AS ONE</td><td>6-306-01</td><td>Durham fermentation tube</td></tr><tr><td>Glucose</td><td>Wako Pure Chemicals</td><td>049-31165</td><td></td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>IX-73</td><td></td></tr><tr><td>Methanol</td><td>Wako Pure Chemicals</td><td>133-16771</td><td></td></tr><tr><td>Microscopic heating stage system</td><td>TOKAI HIT</td><td>TP-110R-100</td><td></td></tr><tr><td>Mineral oil</td><td>Nacalai Tesque</td><td>23334-85</td><td></td></tr><tr><td>Mini-extruder</td><td>Avanti Polar Lipids</td><td>610000</td><td></td></tr><tr><td>Neutravidin</td><td>Thermo Fisher Scientific</td><td>31000</td><td></td></tr><tr><td>Objective lens</td><td>Olympus</td><td>LUCPLFLN 40&amp;times;/0.6 NA</td><td></td></tr><tr><td>Polycarbonate membranes</td><td>Avanti Polar Lipids</td><td>610005</td><td>pore size 100 nm</td></tr><tr><td>sCMOS camera</td><td>Andor</td><td>Zyla 4.2 plus</td><td></td></tr><tr><td>Sodium chloride</td><td>Wako Pure Chemicals</td><td>191-01665</td><td></td></tr><tr><td>Sucrose</td><td>Wako Pure Chemicals</td><td>196-00015</td><td></td></tr><tr><td>Ultrasonic bath</td><td>AS ONE</td><td>ASU-3D</td><td></td></tr><tr><td>Yeast extract</td><td>BD Biosciences</td><td>212750</td><td></td></tr><tr><td>0.6 mL lidded plastic tube</td><td>Watson</td><td>130-806C</td><td></td></tr><tr><td>1.5 mL lidded plastic tube</td><td>Sumitomo Bakelite Co.</td><td>MS4265-M</td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline</td><td>Avanti Polar Lipids</td><td>850457P</td><td>POPC</td></tr><tr><td>1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000]</td><td>Avanti Polar Lipids</td><td>880129P</td><td>Biotin-PEG-DSPE</td></tr></tbody></table>

bacterial cell culture at the single-cell level inside giant vesicles

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for understanding the function of bacterial cells in the natural environment. Because of technological advances, various bacterial cell functions can be revealed at the single-cell level inside a confined space. GVs are spherical micro-sized compartments composed of amphiphilic lipid molecules and can hold various materials, including cells. In this study, a single bacterial cell was encapsulated into 10&#8211;30 &#956;m GVs by the droplet transfer method and the GVs containing bacterial cells were immobilized on a supported membrane on a glass substrate. Our method is useful for observing the real-time growth of single bacteria inside GVs. We cultured Escherichia coli (E. coli) cells as a model inside GVs, but this method can be adapted to other cell types. Our method can be used in the science and industrial fields of microbiology, biology, biotechnology, and synthetic biology.

We present a simple method for generating GVs containing single bacterial cells using the droplet transfer method (Figure 1). Figure 1a shows a schematic image of the precipitation of GVs containing bacteria. W/O droplets containing bacteria are transferred across the oil-water (lipid monolayer) interface by centrifugation to form GVs. The difference in density between sucrose (inner aqueous solution) and glucose (outer aqueous s...

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Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

We demonstrate single-cell culture of bacteria inside giant vesicles (GVs). GVs containing bacterial cells were prepared by the droplet transfer method and were immobilized on a supported membrane on a glass substrate for direct observation of bacterial growth. This approach may also be adaptable to other cells.

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for ...

bacterial-cell-culture-at-the-single-cell-level-inside-giant-vesicles

Research

JoVE Journal

Immunology and Infection

6.9K Views.  National Institute of Advanced Industrial Science and Technology (AIST). Dit protocol biedt een middel voor het kweken van bacteriële cellen op eencellig niveau in gigantische vikjes. Het is gemakkelijk om de gigantische akeltjes te ontwikkelen, en slechts een zeer klein volume van monster oplossing is nodig om de vesikels voor te bereiden. Het aantonen van de procedure zal Yuri Ota zijn, een promovendus uit ons laboratorium.Begin met het toevoegen van 20 microliter vers bereide POPC-oplossing en vier microliter vers bereide biotine-PEG-DSPE-oplossing aan ...

Video: Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Biochemistry

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Bioengineering

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up cellular mimics that better represent the complexity of cellular life. To date there are a number of paths that could be taken to build compartmentalized cellular mimics, including the exploitation of water-in-oil emulsions, microfluidic devices, and vesicles. Each of the available options has specific advantages and disadvantages. For example, water-in-oil emulsions give high encapsulation efficiency but do not mimic well the permeability barrier of living cells. The primary advantage of the methods described herein is that they are all easy and cheap to implement. Transcription-translation machinery is encapsulated inside of phospholipid vesicles through a process that exploits common instrumentation, such as a centrifugal evaporator and an extruder. Reactions are monitored by fluorescence spectroscopy. The protocols can be adapted for recombinant protein expression, the construction of cellular mimics, the exploration of the minimum requirements for cellular life, or the assembly of genetic circuitry.

Herein we describe simple methods for the preparation of vesicles, the encapsulation of transcription and translation machinery, and the monitoring of protein production. The resulting cell-free systems can be used as a starting point from which to build increasingly complex cellular mimics.

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles

Giant unilamellar vesicles (GUVs) are frequently used as models of biological membranes and thus are a great tool to study membrane-related cellular processes in vitro. In recent years, encapsulation within GUVs has proven to be a helpful approach for reconstitution experiments in cell biology and related fields. It better mimics confinement conditions inside living cells, as opposed to conventional biochemical reconstitution. Methods for encapsulation inside GUVs are often not easy to implement, and success rates can differ significantly from lab to lab. One technique that has proven to be successful for encapsulating more complex protein systems is called continuous droplet interface crossing encapsulation (cDICE). Here, a cDICE-based method is presented for rapidly encapsulating cytoskeletal proteins in GUVs with high encapsulation efficiency. In this method, first, lipid-monolayer droplets are generated by emulsifying a protein solution of interest in a lipid/oil mixture. After being added into a rotating 3D-printed chamber, these lipid-monolayered droplets then pass through a second lipid monolayer at a water/oil interface inside the chamber to form GUVs that contain the protein system. This method simplifies the overall procedure of encapsulation within GUVs and speeds up the process, and thus allows us to confine and observe the dynamic evolution of network assembly inside lipid bilayer vesicles. This platform is handy for studying the mechanics of cytoskeleton-membrane interactions in confinement.

This article introduces a simple method for expeditious production of giant unilamellar vesicles with encapsulated cytoskeletal proteins. The method proves to be useful for bottom-up reconstitution of cytoskeletal structures in confinement and cytoskeleton-membrane interactions.

Giant unilamellar vesicles (GUVs) are frequently used as models of biological membranes and thus are a great tool to study membrane-related cellular ...

Cell-Free Expression of Membrane Proteins in Giant Unilamellar Vesicles

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

Author Spotlight: Membrane Protein Reconstitution in Synthetic Cells

This protocol describes the inverted emulsion method used to encapsulate a cell-free expression (CFE) system within a giant unilamellar vesicle (GUV) for the investigation of the synthesis and incorporation of a model membrane protein into the lipid bilayer.

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

Biology

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models. Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles -&#160;small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis. Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.

The present protocol providesdetailed descriptions for isolation, concentration,and characterization of extracellular vesicles from cyanobacterial cultures. Approaches for purifying vesicles from cultures at different scales, trade-offs among methodologies, and considerations for working with field samples are also discussed.

Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

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