The overall goal of this procedure is to build cellular mimics from scratch through the encapsulation of transcription translation machinery in vesicles. This is accomplished by first preparing a thin lipid film. Next, after forming the vesicles, the components are assembled for in vitro transcription translation.
The final step is to place the transcription translation machinery inside of the vesicles. Ultimately, the activity of the cellular mimic can be monitored by microscopy. The main advantage of this technique over other existing encapsulation methods, such as water and oil emulsions, micro retic devices, or a water and oil to vesicle conversion process is that this method is easy, inexpensive, and yields robust vesicles with permeability properties that are well suited for the construction of cellular mimics that exploit genetically encoded protein function.
This Method cannot help to explore key areas in cell-free and bottom up synthetic biology, such as defining the minimal basis of a life and developing non-living lifelike technologies. To make a thin lipid film begin by dissolving POPC in chloroform at a concentration of 40 milligrams per milliliter, then aliquot 12 micromoles of POPC into a five milliliter round bottom flask. Next, use a circular clip to securely attach the flask to the distillation tube of a rotary evaporator.
Then using setting six, start a strong, steady rotation of the flask. Next, start circulating the water through the condenser coil. The water does not need to be heated because chloroform has a low boiling point of 61.2 degrees Celsius.
At one atmosphere, ensure that the system is open to the atmosphere by checking that the stop cock is open to apply the vacuum to the system simultaneously. Turn on the vacuum pump while slowly closing the stop cock. Let the rotary evaporation continue for 0.5 to two hours after the chloroform is no longer visible by eye.
To stop the process, slowly release the vacuum pressure and stop the rotation. Remove the flask and opaque thin lipid film is visible on the walls of the flask to resuspend the lipid. Begin by adding one milliliter of 18.2 mega ohm deionized water to the thin lipid film in the round bottom flask vigorously vortex the solution at approximately 3, 200 RPM until the lipid film detaches from the glass.
This should take about one minute. Next, transfer the lipid dispersion to a two milliliter micro centrifuge tube. Set up a ring stand to hold a homogenizer equipped with a five millimeter tip and place a micro centrifuge.
Stand below the homogenizer to securely hold the lipid dispersion. Then place the dispersing element directly into the lipid dispersion, ensuring that the tip does not touch the bottom of the tube. Homogenize the lipid dispersion for one minute.
At power level four, assemble the parts of the mini extruder, which consists of an outer casing and a retainer nut that houses two Teflon internal membrane supports two O-rings and one Teflon bearing Place the two O-rings into the grooves of the internal membrane. Supports now pre-wet two filters and one 400 nanometer cutoff membrane and place the wet filters against the Teflon inside of the O-rings. The 400 nanometer cutoff membrane is positioned between the two filters.
Place the membrane supports into the extruders outer casing with the membrane surrounded by two filters between the O-rings. Place the Teflon bearing inside the casing and secure it with a retainer nut by hand. Next, rinse two syringes and fill one with 18.2 mega ohm water.
Insert the syringe needles into the small holes in the Teflon on either end of the extruder assembly. The needles should slide in easily. Do not force the needles.
Then fasten the assembly into the extruder housing. Now pass the water through the extruder by slowly pushing the water out of one syringe and into the other. This represents one passage.
Make three passages to check for leaks, and then remove the syringes. Fill a new syringe with the vesicle solution. Attach it to the extruder and slowly pass the solution through the membrane.
Repeat this for a total of 11 passages. With the sample, gradually the sample becomes less turbid and easier to push across the membrane. A sudden decrease in resistance, however, usually indicates a rupturing of the membrane When completed, make 40 microliter aliquots of the extruded vesicle solution.
Flash freeze each aliquot in dry ice or liquid nitrogen. Once frozen, use a centrifugal evaporator to lyophilize each aliquot overnight At 30 degrees Celsius, the lyophilized empty vesicles can be stored at minus 20 degrees Celsius. Begin the encapsulation by mixing the components of the transcription translation reaction and adding 20 units of RNAs inhibitor.
Incubate the reactants on ice, then add the DNA template for a control, use 250 nanograms of a plasmid encoding m venous, or a similar fluorescent protein behind a T seven transcriptional promoter and a strong e coli ribosome binding site. Bring the final volume of the reactions to 25 microliters. With RNAs free water now hydrate 40 microliters of lyophilized vesicles in 10 microliters of the reaction mixture.
Briefly vortex the mixture until the vesicles are resuspended. This should take less than 30 seconds. Incubate the reaction on ice for 30 minutes to allow the vesicles to swell.
Next, dilute the vesicle mixture 20 fold to a final volume of 30 microliters by combining 1.5 microliters of vesicles with 27.0 microliters of tris chloride solution and 1.5 microliters of proteinase K stock solution. Now incubate the diluted vesicle mixture for at least two and a half hours. At 37 degrees Celsius.
Prepare a sample chamber by placing a 20 by five millimeter silicone spacer onto a standard microscope. Slide pipette 10 microliters of vesicles into the sample chamber and place a siliconized glass cover slip over the chamber. Then observe the vesicles with a 63 x oil dispersion or similar objective by brightfield and fluorescence microscopy.
Using the appropriate filter set for the exploited fluorescent protein fluorescence microscopy reveals that fluorescence is only observed inside of the vesicles. Extra vesicular material is enzymatically degraded for the expression of en venous intravesicular. Fluorescence begins to be observed after 1.5 hours at 37 degrees Celsius.
M Venus reaches a maximum fluorescence by six hours. Here it is seen at 2.5 hours. This protein production depends upon protein synthesis and folding and also on chromophore formation.
An analogous in vitro transcription translation reaction of a construct and coating m Venus without vesicles is used as a control. Such reactions have higher fluorescence intensities due to encapsulation efficiency and a dilution effect. Therefore, they are monitored by spectroscopy, not microscopy.
This procedure can be used to answer question pertaining reregulation and metabolic load effects on artificial cell activity. After watching this video, you should have a good understanding of how to construct cellular mimics by directly encapsulating transcription translation machinery into synthetic vesicles.
