The overall goal of the following experiment is to determine suitable conditions to study FTSZ, polymerization and activity using simple assays and equipment. This is achieved by performing FDSC polymer sedimentation assays under various conditions to find suitable conditions at which FDSZ polymerizes. As a second step, we perform light scattering assays to study FDSZ polymerization in real time.
Next, we perform GTP hydrolysis assays to determine the polymerization associated g gtpa activity of FT SC.Results are obtained that show which conditions are best suited for the study of FTSC of a specific bacterial source based on sedimentation, light scattering and GTP hydrolysis. The technique shown here are easy to perform in any laboratory and will provide a quick idea of the polymerization capabilities of specific FT a z. We find that the choice of the right buffer is especially important when working with FTAZ in the presence of other proteins demonstrating the technique will be EVA crawl, a graduate student from my laboratory.
To begin add five microliters of 100 millimolar magnesium chloride, and 12 micromolar of the bacterial proteins, FDSC and or SEP F into one of the chosen polymerization buffers that have various pH values and compositions as described in the accompanying text protocol. Next, place the tubes into an incubator with a shaking function and incubate the samples for two minutes at 30 degrees Celsius and 300 RPM. Once the tubes have been thoroughly mixed, begin polymerization by adding one microliter of either GTP or GDP mixture from a 100 millimolar stock solution so that the final concentration is two millimolar.
Then place the samples back into the shaking incubator for an additional 20 minutes. Next, spin down the tubes at room temperature for 15 minutes at 20, 000 RPM using a TLA 55 rotor or an equivalent 24, 600 Gs if using something else. Once the centrifuge has finished, transfer the supernatants into clean tubes and save the pellets for later.
Take 20 microliters of the transferred supernatants and mix them in a separate tube with 20 microliters of two times concentrated sample buffer. Secure the caps and boil the samples for 10 minutes at 98 degrees Celsius. Next, add 50 microliters of two times concentrated sample buffer to each pellet fraction that was previously set aside and suspend the polymers by boiling the samples for 10 minutes at 98 degrees Celsius.
Once the samples have finished boiling, add an additional 50 microliters of demineralized water to the tubes containing the pellets. Next load 10 microliters of both the supernatants and pellets from each sample side by side on a 10%SDS page gel and run the gel at 150 volts. Once the gel is complete, remove the gel and stain it with kumasi brilliant blue G two 50.
Then image the gel and quantify the protein using cytometry as described in the accompanying text protocol. First turn on a fluorescent spectrometer and allow the lamp to warm up for several minutes to avoid thermal fluctuations. Also, turn on a circulating water bath and set the water temperature to 30 degrees Celsius to maintain the vete chamber at a constant temperature.
As the system warms up, define the operating parameters of the spectrometer. Set the detector high voltage to 300 volts and the emission and excitation wavelengths to 350 nanometers with a slit width of four nanometers. Then program the data acquisition protocol by choosing a time-based acquisition with a duration of 3, 600 seconds.
Carefully clean a 200 microliter fluorescence vete with a one centimeter path length by fornicating it in a cleaning solution in a water bath for five minutes. Next, prepare 294 microliters of a master mix for each of the nine polymerization buffers described in the accompanying text protocol, each with 10 millimolar magnesium chloride, and 12 micromolar FDSE as the final concentrations calculated for 300 microliters. Then briefly vortex the mixtures transfer 196 microliters of the first polymerization buffer to the vet.
Place it in the spectrometer and incubate for two minutes to bring the temperature of the sample up to 30 degrees Celsius. After two minutes, start the data acquisition and wait for 90 seconds to verify that the signal is stable. Then add four microliters of 100 millimolar GTP or GDP.
Mix the sample and continue data acquisition for a full hour. Repeat these steps for each buffer listed. Prepare 1.5 milliliters of two millimolar GTP in a chosen polymerization buffer.
Also prepare a phosphate standard dilution in the zero to 40 micromolar range in the same buffer. Next, prepare malachite green working reagent as described in the bioassays kit. Then make up four master mixes with a total volume each of 360 microliters master mix one contains FDSZ derived from B subtilis and master mix two contains FDSE derived from e coli.
Master mixes three and four are used as controls and contain EDTA instead of magnesium chloride. Next pipette 20 microliter aliquots of the master mixes and phosphate standards. In A-Q-P-C-R 96 well plate so that there are 16 wells for each master mix and the phosphate standards arranged as shown here.
Pipette 180 microliters of two millimolar GTP stock to the last lane of the plate. Then place the plate in a PCR machine. Run a program with a single cycle set to 30 degrees Celsius for 40 minutes.
In a second 96 well plate pipette, 20 microliter aliquots of the malachite green working reagent into wells, A one through H 10. Then at 60 microliters of the polymerization buffer from the experiment claims A one through H eight, next at 20 microliters of the two millimolar GTP solution to the wells in each lane of plate. One at the predetermined intervals as shown here to examine the reaction at various time points.
Pipe at the mixtures up and down to mix them after 30 minutes, starting with lane one in plate one, transfer 20 microliters from lane one of plate one into lane one of plate two and pipette up and down to mix. This is the starting point of malachite green color development for the 30 minute reaction after 30 and a half minutes, transfer 20 microliters from lane two of plate one into lane two of plate two and mix. This is the starting point of malachite green color development for the 20 minute reaction.
Repeat these steps with the remaining lanes three through seven at 32nd intervals after 33 and a half minutes at 20 microliters of GTP to lane eight on plate one and mix. Then transfer 20 microliters to lane eight on the second plate and mix. This represents the zero minute reaction and is the starting time point for malachite green color development.
After 34 minutes, add 80 microliters from lane nine of plate one containing the phosphate standard to lane nine of plate two and mix after 34 and a half minutes, transfer 80 microliters from the second phosphate standard in lane 10 of the first plate to lane 10 on plate two and mix. Then remove all the air bubbles from the samples and incubate the plate at room temperature for another 25 and a half minutes. Following incubation.
Place the plate in a 96 well plate reader starting at 60 minutes from the start of the experiment. Measure the plate at 630 nanometers every 30 seconds for five minutes so that 10 time points are taken. The first plate measurement will correspond with lane one, the second with lane two, and so that each lane has had exactly the same time for the malachite green color to develop.
Next, calculate the free phosphate in every sample using the phosphate standard calibration curve and plot the data as phosphate release over time. Then calculate the F-D-S-Z-G-T-P hydrolysis activity from the linear range of the curve. Shown here are the percent of SEP F and FDSZ in the pellets of the samples.
FDSZ was recovered in the pellet above background levels only when both SEP F and GTP were also present in the sample. The exact stoichiometry of SEPPE and FDSZ in these tubules is not known, but these results suggest that there is more seppe present than FDSZ. Light scattering of FDSZ from B subtilis and e coli are shown here, assembled in the presence of two millimolar GTP in the 50 millimolar potassium chloride buffer.
F-D-S-E-B-S gives a 20 to 40 fold higher light scattering signal than FDSC EC depending on buffer pH. Interestingly, increasing the potassium chloride concentration in the buffer did not significantly influence the light scattering signal of F-D-S-Z-E-C, but the signal of F-T-S-Z-B-S decreased about 80 fold at pH 7.5 30 fold at pH 6.8 and 45 fold at pH 6.5 in 300 millimolar potassium chloride compared to buffers with 50 millimolar potassium chloride. The GTP hydrolysis activity of FDSZ was measured under different buffer conditions using a colorimetric assay for free phosphate.
The GTPS activity of FT SZ was found to increase with increasing potassium chloride concentration at each of the different pH conditions. At 50 millimolar potassium chloride, the gtpa activity was reduced due to bundling of F-D-S-Z-B-S filaments at 50 millimolar. Potassium chloride FDSZ EEC had a three to sixfold higher GTPS activity than F-D-S-Z-B-S due to quicker disassembly of the FDSC EEC polymers.
Finally, the difference in GTP hydrolysis activity between F-D-S-Z-B-S and F-D-S-Z-E-C was reduced at 300 millimolar potassium chloride, possibly because of reduced bundling of F-D-S-Z-B-S filaments. Following this procedure, other metals like electromicroscopy can be performed in order to answer additional questions relating to the structure of fts polymers in the sample.
