नमूने की तैयारी<em> माइकोबैक्टीरियम क्षयरोग</em> परमाणु चुंबकीय अनुनाद metabolomic अध्ययन के लिए अर्क
Summary
के metabolomic प्रोफाइल<em> माइकोबैक्टीरियम क्षयरोग</em> शोरबा संस्कृतियों में वृद्धि के बाद निर्धारित किया जाता है. स्थितियां इस microorganism की चयापचय प्रोफ़ाइल पर पोषक तत्वों की खुराक, oxidants, और विरोधी तपेदिक एजेंटों के प्रभाव का परीक्षण करने के लिए अलग किया जा सकता है. निकालने की तैयारी के लिए प्रक्रिया दोनों 1D के लिए लागू है<sup> 1</sup> एच और 2d<sup> 1</sup> एच<sup13></sup> सी एनएमआर विश्लेषण करती है.
Abstract
Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 °C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.
Protocol
Discussion
अध्ययन का एक महत्वपूर्ण संख्या एम. transcriptomic प्रोटिओमिक और प्रोफाइल का विश्लेषण किया है इन विट्रो में और vivo परिस्थितियों में की एक किस्म के तहत तपेदिक 11-16 अंततः, जीन अभिव्यक्ति और एंजाइ?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
लेखकों के लिए उपयोगी टिप्पणी के लिए डा. Barletta और डॉ. पॉवर्स की प्रयोगशालाओं के सभी सदस्यों को धन्यवाद करते हुए प्रोटोकॉल विकासशील करना चाहते हैं. हम पांडुलिपि के सहायक और विचार विमर्श proofreading के लिए वेंडी ऑस्टिन धन्यवाद. प्रत्येक नेब्रास्का – लिंकन Redox जीवविज्ञान केंद्र (मूल अनुदान # NCRR 2P20RR 017675, डी. बेकर, पीआई) के विश्वविद्यालय से ऊपर सूचीबद्ध अन्वेषक इस पांडुलिपि में वर्णित काम बीज पायलट अनुदान द्वारा वित्त पोषित किया गया था. इसके अलावा, हम उसे R21 अनुदान (1R21AI087561 01A1) से अनुसंधान आपूर्ति और श्री Halouska आंशिक वेतन के इस प्रकाशन में शामिल एनएमआर तकनीक मानकीकृत समर्थन के लिए धन उपलब्ध कराने के लिए डॉ. Ofelia Chacon धन्यवाद.
Materials
| Name of the Reagent/Equipment | Company | Catalogue Number | Comments |
| ADC Enrichment | BD BBL Middlebrook | 212352 | |
| BACS-120 Sample Changer | Bruker | ||
| Bruker Avance NMR | Bruker | 500 MHz | |
| Bovine Serum Albumin | Fisher Scientific | BP1600-100 | Fraction V |
| Centrifuge | Beckman Coulter | Allegra X-15R | Benchtop |
| Centrifuge Tubes | Corning | 430291 | 50 ml sterile polypropylene |
| Cryogenic Vials | Corning | 430488 | 2.0 ml sterile polypropylene |
| Cycloheximide | A.G. Scientific | C-1189 | Toxic |
| D(+) – Glucose | ACROS | 41095-0010 | |
| Deuterium Oxide | Sigma Aldrich | 617385 | |
| Erlenmeyer Flask | VWR | 89095-266 | Sterile, flat base, polycarbonate, 0.22 μm PTFE membrane vented cap |
| Flash Freeze Flask | VWR | 82018-226 | 750 ml |
| Freeze Dryer | VWR | 82019-038 | 4.5 L Benchtop |
| Glycerol | GibcoBRL | 15514-029 | |
| Incubator | New Brunswick | Innova 40 | Benchtop shaker |
| Lysing Matrix B | MP Biomedicals | 6911-100 | |
| Lysis Machine | MP Biomedicals | FastPrep-24 | |
| Microcentrifuge | Eppendorf | 5415D | Benchtop |
| Microcentrifuge | Beckman Coulter | Microfuge 22R | Benchtop |
| Middlebrook 7H9 Broth | Difco | 271310 | |
| NMR tubes | Norell | ST500-7 | 5mM |
| OADC Enrichment | BD BBL Middlebrook | 212351 | |
| Oleic Acid | Sigma | O1008 | |
| Potassium Phosphate Dibasic | VWR | BDH0266 | |
| Potassium Phosphate Monobasic | VWR | BDH0268 | |
| Rotor – Microfuge 22R | Beckman Coulter | F241.5P | Sealed and polypropylene |
| Rotor – Allegra X-15R | Beckman Coulter | SX4750 | With bio-certified covers |
| Sodium Chloride | Fisher Scientific | S271-3 | |
| Sodium-3-trimethylsilylpropionate-2,2,3,3-D4 | Cambridge Isotope | DLM-48 | |
| Spectrophotometer | Beckman Coulter | DU-530 | |
| Spectrophotometer Cuvettes | LifeLINE | LS-2410 | 1.5 ml polystyrene, 2 clear sides |
| Syringe | Becton Dickinson | 309585 | Sterile, 3 ml Luer-Lok |
| Syringe Filter | Nalgene | 190-2520 | 0.2 μm sterile cellulose acetate |
| Tween 80 | Fisher Scientific | BP338-500 |
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