This method measures the levels of the global regulator ppGpp within different growth conditions and stress situations. PpGpp can be found in gram positive and negative bacteria, as well as in chloroplast. PpGpp has a rapid onset in seconds, which lead to a fast second relation once stress is produced.
Simultaneously, it has a short half-life, up to 30 seconds. Therefore, there is a need to monitor many culture at time intervals that may vary from 10 seconds to hours, including stressed transitions. We also radiolabel bacterial cultures in microtiter dishes facilitates high throughput samplings that allow multiple technical and biological replicates.
Make sure to follow the proper safety measures when working with radioactive materials. To begin this procedure, prepare all media as outlined in the text protocol. Grow overnight cultures in MOPS media with three millimolar sodium phosphate.
Add 550 microliters of MOPS media, contining 0.2 micromolar sodium phosphate carrier and 30 microliters of each bacterial inoculum to the wells of a 24 well plate. This will result in an initial inoculum with an OD600 of 0.1. Add fresh media to one well as a sterility control.
Place the plate in a shaking incubator at 37 degrees celsius, shaking at 900 RPM for 30 minutes. Growth can be monitored using a plate reader with a replicate plate in parallel in media lacking p32 using a plate reader while incubating under the same conditions. Next, add 100 microcuries of p32 orthphosphate to each well of the plate.
Grow the cultures in a shaking incubator for one to two doublings to allow external label to largely equilibrate with intercellular nucleotide pools. For proper visualization, none of the active material was used in this video. In a real experiment, proper shielding is required as well as constant monitoring for contamination with a survey meter.
First, induce stress using a method appropriate for the sort of stress studied. Next, add 20 microliters of each labeled cell sample to a separate 0.2 milliliter PCR tube, containing 20 microliters of ice cold six molar formic acid. Immediately place the samples on dry ice.
Enhance cellular extraction efficiency by three cycles of freezing and thawing. Just before spotting PEI cellulose thin layer chromatograms, centrifuge the samples for one minute at maximum speed to pellet cell debris. First, set out a 20 by 20 centimeter PEI cellulose TLC plate.
Use scissors to remove the top five centimeters. And use a soft pencil to mark an origin line one centimeter from the edge. Apply a five microliter droplet of each sample to the PEI surface.
Before the spot can dry, transfer the plate to a tank containing a layer of 1.5 molar monopotassium phosphate that is shallow enough to not touch the origin sample spot. Cover the tank with an airtight seal, and allow liquid ascent to the top of the 15 centimeter trimmed sheet. After this, remove the fully developed chromatogram and air dry it at room temperature.
Cut and discard the top portion of the chromatogram containing the free P32 into radioactive waste. Use a phosphor screen to expose the audioradiographic films overnight. The next day, develop the film and use a phosphorimager to capture and quantitate the phosphor's green signal.
Then, use ImageJ software to quantitate the radioactive spots. After provoking stress or starvation for time sufficient to allow ppGpp accumulation, use an appropriate method to reverse the stress. Take samples every 20 to 30 seconds for up to two minutes and process the samples as previously described.
Once the TLC is developed, and the levels of ppGpp are obtained during the time course, plot the residual ppGpp content on a semilogarithmic plot versus time. In this study, cells grown in MOPS containing all amino acids except for ILV are labeled with P32. Once labeled, L valine is added to produce isoleucine starvation.
After five minutes, a two and two and a half old increase in the levels of tetra and pentaphosphate of guanosine occurred. A negative control of a cell free labeled sample is used to detect possible compounds that are not orthophosphate in the P32 source. Reversal of isoleucine starvation can be achieved by chloramphenicol, an inhibitor of protein synthesis, which reduces the consumption of charged isoleucine tRNA, and in turn, restores high ratios of charged to uncharged tRNA.
Activation of the strong rela-mediated ppGpp synthetase is abolished, which allows a measure of ppGpp degradation unperturbed by residual synthesis. In this case, ppGpp decays with a half-life of approximately 64 seconds. It is important to let the cells grow for a couple of generations to achieve a uniform labeling before inducing any stress or starvation.
Because ppGpp is widely spread among bacteria, this method can be applied to other organisms. Modification of the TLC development method could allow the proper separation of all the regulatory nucleotides, such as ppApp. P52 is a better emitter isotope, so it is important to use proper shielding and to properly discard any residue.
