This protocol provides a means for culturing bacterial cells at the single-cell level inside giant vesicles. It's easy to develop the giant vesicles, and only a very small volume of sample solution is required to prepare the vesicles. Demonstrating the procedure will be Yuri Ota, a PhD candidate from our laboratory.
Begin by adding 20 microliters of freshly prepared POPC solution and four microliters of freshly prepared biotin-PEG-DSPE solution to a glass vial. Evaporate the organic solvent by air flow until a lipid film is formed. Place the film in a desiccator for one hour to completely evaporate the organic solvent.
Then add 200 microliters of mineral oil to the vial. Cover the opening of the vial with plastic paraffin film, and sonicate the vial contents in an ultrasonic bath at 120 watts for at least one hour. For a small vesicle preparation, create a lipid film, as just demonstrated, and add 200 microliters of 200 millimeter glucose in LB medium to the film.
Then sonicate the film at 120 watts for at least an hour, followed by extrusion of the resulting giant vesicles into small vesicles using a mini extruder and a polycarbonate membrane with a 100-nanometer pore size. For bacterial cell pre-culture, inoculate E.coli from an LB plate into milliliters of LB medium for an overnight culture at 37 degrees Celsius. The next morning, transfer 20 microliters of the culture's supernatant to 1.98 milliliters of fresh LB medium for an additional two hours of culture.
At the end of the incubation, check the optical density at 600 nanometers, or OD 600 value, of the pre-culture solution on a spectrophotometer. A pre-culture solution with an OD 600 of one to 1.5 should be used. Then mix the pre-culture solution with freshly prepared sucrose solution and fresh LB medium, according to table one.
For giant vesicle formation, add 50 microliters of a freshly prepared outer aqueous solution of giant vesicles to a 1.5 milliliter lidded plastic tube. Gently layer 150 microliters of the lipid-containing oil solution over the outer aqueous solution of giant vesicles. Incubate the resulting solution for 10 to 15 minutes at room temperature, before checking to ensure that the interface of the oil and aqueous solutions is flat.
For water and oil bacterial cell containing droplet formation, add two microliters of a freshly prepared inner aqueous solution of giant vesicles to 50 microliters of the sonicated lipid-containing oil solution in a 0.6 milliliter lidded plastic tube. Then, tap the tube to emulsify the two components. Next, use a pipette to add 50 microliters of water and oil droplet solution to the interface of the oil and aqueous solution, and sediment the bacterial cell-containing giant vesicles by centrifugation.
Then aspirate the top oil layer and collect the giant vesicles. To prepare a handmade chamber, drill a seven-millimeter hole with a hollow punch on a 10 by 10 by one millimeter double-faced seal, then paste the double-faced seal onto a 30 by 40 millimeter cover glass. To prepare a supported bi-layer membrane, add 30 microliters of the small vesicle solution to the hole of the handmade chamber.
Incubate the vesicles at room temperature for 30 minutes. At the end of the incubation, gently wash the hole two times with 20 microliters of LB medium, supplemented with 200 millimiller glucose. To immobilize giant vesicles on the supporter bi-layer membrane, introduce 10 microliters of neutravidin in outer aqueous giant vesicle solution to the hole for a 15 minute incubation at room temperature.
At the end of the incubation, gently wash the hole two times with 20 microliters of LB medium, supplemented with 200 millimiller glucose, and add the entire volume of giant vesicle solution to the hole in the chamber. Then seal the hole with an 18 by 18 millimeter cover glass. To monitor bacterial growth within the giant vesicles, select the 40X objective on an inverted microscope, and place the chamber on the microscope heating stage system.
Then incubate the bacterial cell-containing giant vesicles in the chamber under static conditions for six hours at 37 degrees Celsius, capturing and recording images of the bacterial cell growth every 30 minutes, with the scientific complementary metal oxide semiconductor camera. Giant vesicles containing bacterial cells typically range in size from 10 to 30 micrometers. For both sizes of giant vesicles, the E.coli cells undergo elongation and division processes, with some E.coli cells growing to a very large number of cells over the six hour observation period.
The relative frequency of giant vesicles containing a single cell level is approximately 10%of the obtained giant vesicles, and the giant vesicles encapsulated at the single cell level are approximately 50%of the giant vesicles containing bacterial cells. The stability of the oil-water interface is important for obtaining giant vesicles containing bacterial cells. Take care to aspirate the oil carefully before the giant vesicle correction.
After watching this video, you should have a good understanding of how to culture bacterial cells at the single cell level within individual giant vesicles. Our bacterial culture method is a new tool in microbiology for culturing unknown environmental bacteria to obtain or analyze their metabolic products.
