Summary

Пример подготовки<em> Микобактерий туберкулеза</em> Экстракты для ядерно-магнитного резонанса исследований Metabolomic

Published: September 03, 2012
doi:

Summary

Metabolomic профиля<em> Микобактерий туберкулеза</em> Определяется после роста в бульоне культуры. Условия могут быть изменены, чтобы проверить влияние пищевых добавок, окислители, и противотуберкулезных препаратов на метаболический профиль этого микроорганизма. Порядок подготовки экстракт применяют как для 1D<sup> 1</sup> H и 2D<sup> 1</sup> H-<sup> 13</sup> C-ЯМР-анализа.

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

1. Текст протокола Этот протокол выдвигает на первый план адаптации методологии ЯМР M. туберкулез (класс III агента). Таким образом, уровень биобезопасности 3 (BSL3) практики необходимо следовать при проведении M. туберкулез исследований в ежегодной сертифицированно?…

Discussion

Значительное число исследований были проанализированы транскриптомных и протеомных профилей M. туберкулезом в различных в пробирке и в естественных условиях. 11-16 конечном счете, изменения в экспрессии генов и активность фермента приводит к изменениям в концентра?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Авторы хотели бы поблагодарить всех членов лаборатории д-ра Барлетта и д-р держав за полезные замечания при разработке протокола. Мы благодарим Wendy Остина за полезные обсуждения и редактирование рукописи. Работа, описанная в этой рукописи была профинансирована за счет грантов семян пилот каждого следователя, перечисленные выше из Университета Небраски-Линкольна окислительно-восстановительного центра биологии (родитель грант № NCRR 2P20RR 017675, Д. Беккер, PI). Кроме того, мы благодарим доктора Офелия Чакона для предоставления средств из своего гранта R21 (1R21AI087561-01A1) для исследования материалов и частичная поддержка зарплата г-н Halouska, чтобы стандартизировать методы ЯМР включены в эту публикацию.

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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Cite This Article
Zinniel, D. K., Fenton, R. J., Halouska, S., Powers, R., Barletta, R. G. Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies. J. Vis. Exp. (67), e3673, doi:10.3791/3673 (2012).

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