Method for the Isolation of Francisella tularensis Outer Membranes

Isolation of Francis Cellis outer membrane vesicles and their associated outer membrane proteins starts with osmotic lysis of bacterial cells. Logarithmic phase bacteria are sequentially treated with hypertonic sucrose, EDTA and lysozyme solutions before being slowly diluted into deionized water. The resulting bacteria lysates are subjected to low speed centrifugation, which removes cellular debris and unbroken cells, followed by high speed centrifugation to pellet membrane vesicles.

Next, total membrane vesicles are layered onto sucrose density gradients in order to separate inner membrane or IM and outer membrane or om vesicles. Om vesicles can be separated from IM vesicles based on differences in buoyant densities believed to be predicated largely on the presence of lipopolysaccharide or LPS in the om immuno blotting of sequential fractions from sucrose density gradients show spatial separation of inner membrane proteins from outer membrane proteins. Hi, I'm Dr.Michael Norgaard, and this is Dr.Jason Huntley from the Department of Microbiology at UT Southwestern Medical Center at Dallas.

Today, Dr.Huntley will show you a procedure for extracting, enriching and isolating France cellis outer membranes and their associated membrane proteins. We use this procedure in our laboratory to study the outer membrane localized and potentially surface exposed molecules for this intracellular bacterial pathogen. We have a busy day, so let's get started.

To begin this procedure, prepare and grow two 500 milliliter cultures of FCIs bacteria as described in the accompanying written protocol. Retrieve the bacterial cultures from the shaking incubator and remove a one milliliter Eloqua from each culture for OD measurement F to lorenza cells in early logarithmic phase of growth, correlating with an OD 600 between 0.2 and 0.4. Give optimal membrane extraction and isolation results when the cultures reach the desired OD 600.

Use four 250 milliliter centrifuge bottles to centrifuge the cultures at 7, 500 Gs for 30 minutes at 15 degrees Celsius in order to collect the cells. After centrifugation, carefully remove the media supernatant from each centrifuge bottle and firmly tap the bottles on absorbent material to remove excess growth medium. Within 10 minutes of completing the centrifugation, suspend each bacterial pellet in 8.75 milliliters of 0.75 molar sucrose in five millimolar triss pH 7.5.

After suspension of each pellet, transfer the solution to a sterile 250 milliliter flask. Holding a small stir bar. Repeat for all four bacterial pellets with a final bacterial pellet suspension totaling 35 milliliters Thoroughly mix the cell suspension on a star plate, but not too fast.

In order to avoid frothing or bubbles while mixing slowly, add 70 milliliters of 10 millimolar EDTA interests to the solution over the course of 10 minutes. Hold the tip of the pipette below the cell suspension level to avoid elevated local concentrations of EDTA. After adding the EDTA, incubate the solution for 30 minutes at room temperature.

Then while still mixing slowly, add 11 milliliters of a two milligram per milliliter lysozyme solution to a final concentration of 200 micrograms per milliliter. Continue to mix the cell suspension during the lysozyme solution addition. As before, hold the tip below the cell suspension level.

Incubate the cells with the lysozyme for 30 minutes at room temperature. During this incubation, prepare a sterile one liter flask with a small stir bar and 530 milliliters of room temperature. Cell grow molecular grade deionized water.

When the cells have finished incubating osmotically lysed the cell suspension using a 25 milliliter pipette, resting on the bottom of the flask adjacent to the stir bar, slowly dilute the cells into the water in the one liter flask over the course of 10 to 15 minutes with gentle mixing. This step is challenging and is critical for overall success of the experiment. Stirring should be fast enough to thoroughly disperse the cell suspension, but not too fast leading to frothing or bubble formation as needed.

Slowly increase the stir bar speed to ensure proper mixing. Carefully monitor the dilution process and adjust the flow rate or rotate the flask as necessary to disperse local accumulations of cells, cell clumps, and cell wisps. Once all the cells have been added, incubate the mix for 30 minutes at room temperature at the end of the incubation transfer 40 milliliter aliquots of the osmotic lysis solution into 50 milliliter conical tubes and centrifuge at 7, 500 Gs for 30 minutes at 10 degrees Celsius.

To remove intact cells and debris following centrifugation carefully remove 27 to 30 milliliters of supernatant from each conical tube and pool the supernat in a sterile one liter flask. Transfer 25 milliliters of pooled supernat into each of 16 ultracentrifuge tubes and centrifuge at 200, 000 Gs for two hours at four degrees Celsius. 10 minutes before centrifugation is complete.

Prepare modified membrane resus suspension buffer. Combine eight milliliters of membrane suspension buffer with one tablet of EDTA free protease inhibitor cocktail and 375 units of benzo nase in a sterile tube and mixed gently following centrifugation of the lysed cells. Proceed to recover the membrane pellet.

When ultracentrifugation of the lysed cells is complete, carefully pour off the supernat, then invert the tubes absorbent material and firmly tap them. To remove excess supernatant, mark the location of each membrane pellet with a marker to aid in visualization. Gently resuspend eight membrane pellets with 600 microliters each of modified membrane resus suspension buffer.

It is difficult to resuspend the membrane pellets. So continue gently passing the modified membrane Resus suspension Buffer over the pellet until it peels away from the wall of the tube. This is another step which is challenging and critical for overall success of the experiment.

If necessary, use the micro pipette tip to scrape the membrane pellet off the tube wall. When pipetting avoid frothing or bubble formation, excess shear forces will lead to mixed my cell formation, which precludes membrane separation. Transfer each membrane resus suspension to the next set of eight tubes.

Gently resus suspend the remaining pellets and pool all suspensions in a sterile tube. After removing the membrane pellets from all 16 tubes, wash every four tubes with 600 microliters of modified membrane Resus suspension buffer. Focus the wash around the marked area of each tube and combine the wash with the resuspended membranes.

Repeat the wash for the remaining tubes. Incubate the membrane resus suspension with gentle rocking for 30 minutes at room temperature to degrade DNA and aid in reduction of flocculent material. Next, remove a small Eloqua of the membrane suspension and perform protein quantitation.

To determine total membrane yield, prepare one to one and one to three dilution of membrane suspension in 2.5%SDS heat, the undiluted one-to-one and one to three membrane suspensions at 90 degrees Celsius for 10 minutes. SDS and heat are required to release O MPS from the extracted membranes. Use the BioRad DC protein assay to quantitate the membrane resuspension protein concentration.

Total protein yield is generally between one and 1.6 milligrams per milliliter. Once the concentration of protein in the membrane preparation is determined, the samples can be separated on a sucrose gradient. Start by preparing a series of sucrose solutions in EDTA.

To prepare the linear sucrose gradients, use a bulb pipetter to layer 1.8 milliliters of each sucrose solution into 14 by 95 millimeter UltraClear ultracentrifuge tubes in the following order, 55%50%45%40%35%and 30%Next layer 1.5 milligrams of membrane resus suspension on top of each gradient. Do not load more than 1.7 milliliters of membrane resus suspension per gradient individually weigh each tube and carefully adjust the final weight by adding or removing membrane resuspension so that all tubes weigh the same. Transfer the sucrose gradients into an SW 40 TI swinging bucket rotor and centrifuge at 256, 000 GS for a minimum of 17 hours at four degrees Celsius.

After 17 hours of centrifugation, stop the centrifuge run and carefully remove the sucrose gradients from the rotor. Next, collect the fractions. Carefully clean the bottom of the sucrose gradient tube with 70%ethanol.

Then puncture the bottom of the tube with the 21 gauge needle and collect sequential 500 microliter fractions from the gradient into sterile micro fuge tubes. The gradient should be allowed to drip by gravity flow so as not to disturb the sucrose gradient. However, if the dripping ceases from the bottom of the gradient tube, a small amount of pressure may be applied to the top of the tube using one's finger.

Finally, determine the refractive index of each sucrose gradient fraction using a refractometer correlate the refractive index with a specific density in grams per milliliter. Assess protein localization of representative sucrose gradient fractions by SDS page separation and immuno blotting. Here is an immuno blot of fractions from the sucrose gradient used to separate the membranes extracted from lysed Freis live vaccine strain freis outer membrane proteins or O mps, localized between densities of 1.17 and 1.2 grams per milliliter.

By comparison, inner membrane proteins or ips localized between densities of 1.13 and 1.14 grams per milliliter. Similar results were obtained for the FHU four strain. I've just demonstrated how to extract and isolate Francis Cellis out membrane vesicles through osmotic lysis and sucrose density gradient ultracentrifugation.

When performing this procedure, it's important to remember to use bacterial cells in the early logarithmic phases of growth to slowly but consistently dilute those bacterial cells into deionized water during the osmotic glyco step, and to take care when Resus suspending the ultracentrifuge membrane pellets. This concludes our membrane separation procedure. Thanks for watching and good luck with your future experiments.