The overall goal of this procedure is to prepare, purify, and use liposomes comprised in part or whole of fatty acids. This method can help to answer key questions in synthetic biology and origin of life studies such as defining minimal cells and constructing artificial protocells. The main advantage of this protocol is that it introduces an easy and inexpensive way to produce high yield monodispersed fatty acid vesicles and handle them in a range of experimental conditions.
Generally, individuals new to this method will struggle because fatty acid vesicles are more delicate than phospholipid vesicles, especially in the presence of To prepare thin films of dried lipids, use gas-tight syringes to transfer a specific amount of lipid in chloroform to a glass vial. Evaporate the chloroform under a stream of nitrogen or argon in the fume hood. Subject the resulting thin film to house vacuum for at least two hours to remove any residual chloroform.
The lipid could be left under vacuum overnight at this point. To rehydrate the vesicles, pipette 250 microliters of the hydration buffer into an empty tube, then add 1.875 microliters of 10 molar sodium hydroxide for a final 75 millimolar base. Transfer the solution to the thin film of dry lipid to form vesicles with the total fatty acid concentration of 0.15 molar, then use a high-speed vortexer to vortex the resulting mixture for four to five seconds.
Agitate the lipid buffer mixture on a low-speed rotating shaker to rehydrate overnight. To generate vesicles of uniform size, use tweezers to apply one filter support to each inner surface of the syringe ports of the liposome extruder. Wet each filter support with 250 millimolar Tris-HCL pH 8.
Using tweezers, apply one track etch to 100 nanometer polycarbonate membrane to one of the extruder o-rings and filter supports. Taking care to not tear or puncture the membrane, gently push the membrane into the o-ring so as to make good contact between the two surfaces. Next, assemble the extruder, taking care not to displace the membrane and filter supports.
Fill an extruder syringe with approximately 0.5 milliliters of 250 millimolar Tris-HCL pH 8. Insert this syringe into one side of the extruder. Insert an empty syringe into the other side of the extruder.
Push the plunger and the syringe-containing buffer slowly by hand and verify that resistance is felt indicating the track etched membrane is in place and intact. It is helpful to practice this step without a membrane in place in order to gauge the expected level of resistance. Remove and empty the two syringes.
It is not necessary to clean the two syringes at this stage since they contain buffer of identical composition to the liposome preparation. Next, load the liposome preparation into one of the two syringes. Reassemble the extruder with the syringe containing the vesicle sample on the left side and an empty syringe on the right side.
Then push the plunger of the syringe containing the vesicle sample very slowly by hand. Carefully observe the syringe on the right side of the extruder. Clear buffer will initially enter the right side of the extruder due to the inner dead volume of the extruder, followed by a small plume of extruded liposomes.
Immediately stop pushing at this point. Remove the syringe on the right side of the extruder and discard the solution. Replace the syringe on the right side of the extruder and extrude until the left syringe is empty.
Reverse the orientation of the extruder and repeat the syringe replacement. Continue for the desired number of cycles. An odd number is always used to ensure liposomes are not collected from the syringe originally containing unextruded liposomes.
Finally, gently dispense the contents of the right syringe into an Eppendorf tube. Place the tube on a low-speed rotating shaker for around 30 minutes. To prepare the vesicle purification mobile phase, prepare five milliliters of 250 millimolar Tris-HCL pH 8 hydration buffer in a 15 milliliter Falcon tube.
Then add 37.5 microliters of 10 molar sodium hydroxide to the hydration buffer. Pipette 235 microliters of pure oleic acid into the Falcon tube, resulting in a vesicle solution with 0.15 molar total lipids. Next, use a high-speed vortexer to vortex the mixture for four to five seconds.
Then tumble on a low-speed rotating shaker for at least two hours. The lipid preparation can be left overnight on the rotating shaker at this point. Filter the mobile phase through a 0.22 micron syringe filter unit to remove any potential aggregates.
To purify the vesicles, first prepare the Sepharose 4B column as detailed in the text protocol and clamp it on the retort stand. Then connect the tip of the column with the stop cock connector and connect the tubing to the fraction collector. Add another portion of buffer to flush the tubing and close the stop cock when the liquid level in the column approaches the top of resin.
Next, apply the extruded vesicles to the top of the resin using a 200 microliter pipetter taking care to apply the vesicle preparation as evenly as possible to the resin without touching the resin bed or column wall. Open the stop cock to begin the flow and start collecting fractions into a 96-well plate. Apply the vesicle purification mobile phase to the top of the resin bed in 0.5 to one milliliter portions as the buffer depletes, taking care not to allow the resin bed to dry out.
Then collect the eluent in five-drop fractions, collecting at least 36 wells. To characterize the purification fractions, read the 96-well plate on a plate reader. Plot the resulting fluorescence data as fluorescence versus fraction number.
Vesicles elute first, followed by the unencapsulated fraction. When using liganded magnesium, premix magnesium chloride and potassium citrate at the specified ratio in 250 millimolar Tris-HCL pH 8 buffer. Here, a ratio of one to four is used for stable oleic acid vesicles.
Remember always to premix magnesium and ligand solution. Never expose fatty acid vesicles to the unchelated magnesium alone. Add the premixed magnesium citrate solution to the vesicle sample.
Briefly vortex the mixture. Leave the vesicle sample on a tumbler for at least 30 minutes before repurification as described in the text protocol. Be sure to add the same concentration of magnesium and citrate as in the vesicle sample to the repurification mobile phase.
Mix the magnesium solution with purified vesicles as described in the text protocol to initiate the self-cleavage reaction. For kinetic studies, take 100 microliters of the mixture at each time point and directly repurify this aliquot through a Sepharose 4B size exclusion column with 250 millimolar Tris-HCL pH 8 as the mobile phase. Collect the vesicle fraction.
Prepare the RNA loading sample as described in the text protocol. Place the commercially-casted 15%TBE urea gel into the gel box and fill the gel box with 1X TBE gel-running buffer. After heating the samples on an 80 degree Celsius heat block for one minute, load five microliters of each sample per well.
Run the gel with a constant 200 volts for approximately one hour. Finally, scan the gel and quantify with an analysis program. To demonstrate that RNA can function inside protocells, hammerhead ribozyme self-cleavage with the fluorescently-labeled substrate strand was employed as a model RNA catalytic reaction.
This reaction requires free magnesium to facilitate catalysis. Therefore, oleic acid/glycerol monooleate vesicles were used since they are stable in the presence of five millimolar magnesium. The hammerhead ribozyme self-cleavage reaction can be monitored by gel electrophoresis.
After quantifying the page image, a linear fit of the natural logarithm of the ratio of amount of substrate remaining at a given time point to the initial amount of substrate versus time is generated. This plot can be used for ribozyme activity calculation. Once mastered, fatty acid liposomes with encapsulated dye or RNA can be prepared and purified within 24 hours.
While attempting this procedure, it is important to remember to form fatty acid liposomes in the right pH range and keep the total lipid concentration above the lipid's critical aggregation concentration to avoid the dissolution of vesicles. After watching this video, you should have a good understanding of how to prepare fatty acid liposomes and how to use them to host biochemical reactions.
