The aim of this procedure is to analyze mycobacterium tuberculosis, cell-free extracts by NMR metabolomics first culture, and harvest the cells at an appropriate stage of growth. Then lysa cells and obtain cell-free extracts for analysis. Re suspend the lyophilized extracts in buffer for NMR analysis.
Finally, collect NMR data and analyze spectra with software to identify metabolites. Ultimately, NMR analysis of mycobacterium tuberculosis is used to show preservations in metabolite levels upon various treatments. NMR metabolomics can be applied to key aspects of the physiology of mycobacterium tuberculosis, such as important metabolic pathways.
This understanding is critical to elucidate the mechanisms of act of drug action and resistance and to identify novel targets for drug design. Generally, individuals new to this method would struggle because of inability to handle all samples exactly the same, which will introduce unnecessary variations and results from my laboratory. Steven Luka PhD graduate student will demonstrate the procedures to handle the samples for NMR processing From my laboratory, the research laboratory manager and Robert Fenton, research technician three, will now demonstrate the procedures to handle mycobacterium tuberculosis cultures When conducting M tuberculosis.
Research followed biosafety level three practices as defined by institutional guidelines. Although the labels in this video say microbacterium tuberculosis for biosafety considerations, this demonstration used the non-pathogenic mycobacterium magmatic. Start by thawing a 50%glycerol stock of M tuberculosis on ice.
Inoculate 0.15 milliliters into 110 milliliters of M-A-D-C-T-W broth in the 250 milliliter ER and my flask grow the cultures at 37 degrees Celsius with shaking at 100 RPM for approximately six days. If no antibiotics, alternative additions or other treatments are needed. Harvest the cultures for cultures requiring treatments.
Reserve a 0.5 milliliter Eloqua on ice for culture, titration, and quality control. Then perform the desired treatment and place the cultures back in the shaker after the treatment time period. Remove one milliliter and determine the OD 600.
Also, reserve a 0.5 milliliter sample for culture, titration, and quality control. Maintain the cells on ice throughout the entire remaining protocol. Harvest the cells in four 50 milliliter tubes by centrifugation after washing with 15 milliliter ice cold double distilled water.
Reese suspend the pellets in 10 milliliter aliquots. Pull two aliquots and pellet by centrifugation, then resuspend the cell pellet in a final volume of one milliliter of double distilled water. If a single cell suspension free of clumping is desired, pass the cells through a 27 gauge needle.
Three times proceed to transfer the one milliliter cell suspension to a two milliliter screw cap containing lysing matrix B.Position the fast prep 24 lysis homogenizer inside the biosafety cabinet. Place the samples into the sample holder securing the retention spoke plate. Then process for 60 seconds at a speed setting of six meters per second.
Next, centrifuge the samples to pellet cell debris and intact cells. Then pass the supinate through a 0.2 micron syringe filter into a sterile tube to control for the absence of viable cells. Plate 0.1 milliliter of sample on MADC.Agar.
Freeze the samples in an ethanol dry ice bath and store at minus 80 degrees Celsius until they're ready to be lyophilized and processed in an NMR facility. After two months, check the plates to verify the absence of colony forming units. If no CF user are found, the samples may be removed from the BSL three laboratory for further analysis in a standard NMR facility.
After izing the samples to dryness resuspend in 0.7 milliliters of NMR buffer and transfer to a micro centrifuge tube centrifuge for three minutes at 13, 000 times. G, remove 0.6 milliliters and transfer to a five millimeter NMR tube. From media analysis, a customized rack facilitates loading multiple NMR tubes into spinners at the correct depth.
Then transfer the samples to the NMR spectrometer to insert into A-B-A-C-S one 20 sample changer. Alternating between untreated and drug treated cultures. The first sample needs to be preloaded into the magnet at the start of an automated run.
To calibrate the instrument, optimize the 90 degree pulse length with a single sample and tune the instrument for the remaining samples. Use icon NMR and grad shim. Automate locking shimming and NMR data collection for every sample.
For A 1D hydrogen one NMR spectrum, select the pulse sequence with water suppression and excitation sculpting. Set a total of 32, 000 data points, 128 scans and 16 dummy scans for a two DH one C 13 hs QC spectrum. Select the pulse sequence.
Select a total of 2048 data points with sweep width along the direct H one dimension and 64 data points with sweep width along the indirect C 13 dimension. Also specify spectrum collection with 128 scans and 16 dummy scans to obtain good signal to noise. A spectrum of m tuberculosis generated approximately 400 peaks, including 50 known metabolites.
For example, data processing as detailed in the accompanying manuscript produced this spectrum of metabolites that are directly or indirectly associated with the D alanine pathway. The magnitude of peak intensities is proportional to metabolite concentrations present in the cell extract, so relative perturbations in metabolites and metabolic pathways can be quantified between samples and treatments. LMR metabolomics paves the way for researches in the field of bacteriology to explore physiology and pathogenesis in visual and microbacteria another pathogenic or non-pathogenic microorganisms.
After watching this video, you should have a good understanding of how to prepare multiple tuberculosis cell extracts for NMR metabolomics, and importantly maintain consistency throughout the entire protocol to obtain reliable results.
