In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

In our protocol, we use film re-hydration from small glass beads to form reaction compartments made of peptides. In experiments, we benefit from the protocol's simplicity and robustness. The main advantage of this technique is the ability to incorporate even sensitive samples, such as the used crude cell extract.

Contact with organic solvents will significantly affect the sample. To begin this procedure, use a centrifugal vacuum concentrator to concentrate the ELP solution to 1.1 millimolar. Mix 200 microliters of this concentrated solution with 1, 250 microliters of a two to one chloroform and methanol mixture.

Vortex the solution to mix thoroughly. Next, add 1.5 grams of spherical glass beads to a ten milliliter round bottom flask. Add the ELP and chloroform methanol solution to the round bottom flask, and gently shake to mix.

Connect the flask to a rotary evaporator. Adjust the speed to 150 RPM and regulate the pressure to 20, 000 pascals for approximately four minutes, until the liquid is evaporated at room temperature. It's important to be careful when using the rotary evaporator due to possible boiling retardation.

After this, loosely wrap aluminum foil around the opening of the round bottom flask to prevent the loss of glass beads and place the flask into a desiccator for at least one hour, to ensure that the remaining chloroform and methanol are evaporated. For a single experiment, mix 100 milligrams of the peptide covered glass beads with 60 microliters of a swelling solution. Incubate this sample at 25 degrees Celsius for five minutes.

Use a table top centrifuge to centrifuge the samples quickly and sediment the glass beads. Then use a pipette to collect the supernatant, which contains the vesicles. First, mix 100 microliters of the pre-purified plasmid DNA with 100 microliters of either Roti-Phenol, chloroform, or isoamyl alcohol in a micro centrifuge tube to enable better phase separation.

Gently invert the tube up to six times and centrifuge at 16, 000 times g and room temperature for five minutes. Then, add 200 microliters of chloroform to the upper phase, and invert the tube up to six times. Centrifuge the sample at 16, 000 times g and room temperature for five minutes.

After this, pipette the supernatant to a separate tube and add 10 microliters of three molar sodium acetate for ethanol precipitation. Add one milliliter of cold ethanol at 80 degrees Celsius and store the sample at 80 degrees Celsius for one hour. Next, centrifuge the sample at 16, 000 times g and four degrees Celsius for 15 minutes.

Decant the supernatant and add one milliliter of cold 70%ethanol at 20 degrees Celsius. Centrifuge at 16, 000 times g and four degrees Celsius for five minutes. Then, remove the liquid by pipetting, being careful to not disturb the DNA pellet.

Store the sample at room temperature for approximately 15 minutes to evaporate the remaining ethanol. After this, add ultra pure water to the sample to adjust the sample concentration to approximately 300 nanomolar, which is measured by absorption at 260 nanometers. To prepare the transcription-translation reaction, follow the prepared crude cell extraction and the reaction buffer on ice.

For a 60 microliter reaction mix, add the plasma DNA to 37.5 microliters of the reaction buffer, add 28.7 microliters of crude cell extract and fill with ultra pure water to a final volume of 58.8 microliters. Right before the reaction starts, add 1.2 microliters of the T7RNA polymerase solution, and pipette up and down to mix. Incubate the sample and a swelling solution at 29 degrees Celsius for the duration of the experiment, which is typically four to eight hours.

Transmission electron microscopy images of vesicles show that various swelling solutions, such as TX/TL or even only PBS can be used to form vesicles. For both solutions, size determination is a straight forward step. Dynamic light scattering shows that vesicles prepared without the glass beads method result in a diameter of 134 nanometers, with a polydispersity of 25%When glass beads are used, the diameter results in 168 nanometers, with a polydispersity of 21%The fluorescence intensity of two fluorescent proteins, which are expressed inside the ELP vesicles, are then measured using a fluorescence plate reader.

It is important to note that after vesicle formation, the contents of the vesicles and the outer solution are the same, thus, the antibiotic Kanamycin is added to the exterior solution to suppress protein expression. As a control, Kanamycin is also added to the swelling solution, in which case protein expression inside of the vesicle is suppressed. This indicates that Kanamycin does not diffuse through the membrane, and suppresses internal expression constantly, all the time.

The FRET assay is then performed to demonstrate ELP incorporation into the membrane. Upon expression of the membrane ELPs, additional peptides incorporate into the membrane, which increases the average distance between the FRET pairs, and which results in an increase of donor signal. The presented technique can be used to produce simple reaction containers or to create artificial cells with peptide-based membranes.

The content inside can be chosen as needed. By using these peptide vesicles, we can now look forward on more complex synthesis reactions within them.