Bu çalışma DOE Great Lakes Biyoenerji Araştırma Merkezi (DE-FC02-07ER64494 Bilim DOE BER ofis) bağışları ile desteklenmiştir. Biz Dr Xudong Zhang ve protokol tamamlanmasına yardımcı teknik tartışmalar ve değerli yorumlarınız için kendi grubunun üyelerine minnettarız.

1. Numune Hazırlama ve Asit Ekstraksiyon <ol><li> Freeze-kuru toprak örnekleri alan koleksiyon sonra. </li><li> Bir bilyalı değirmen, toprak değirmeni veya bir havanda kullanarak toprak örnekleri eziyet ve homojenize. </li><li> 25 ml&#39;lik hidroliz balona toprak örnekleri (&gt; 0.3 mg N içeren) tartılır. </li><li> Her hidroliz balona 10 ml 6M HCl ekleyin, sıkı N 2 şişe gaz ve kap doldurun. </li><li> Otomatik zamanlayıcı anahtarı kullanarak 8 saat boyunca 105 ° küvözde C&#39;de hidroliz. </li></ol> 2. Örnek Arıtma <ol><li> Inkübatör gelen şişeler çıkarın; oda sıcaklığına kadar soğutulur. </li><li> Dönen karıştırın, her balona 100 mcL iç standart myo-inositol (suda 1 mg ml -1) ekleyin. </li><li> ST / NS 24 / istikrar için plastik bardaklar ayarlanan 40 eklem, 200 ml armut şeklinde matara drene plastik huni ayarlayın. </li><li> Katlama Whatman # 2 Kalitatif Circles (11 cm çapında) mahalle ve huni içine setine. </li&gt; <li> Swirl her hidroliz şişesi, ve (daha fazla su ~ 3 mL Her balona durulayın olabilir) filtre hunisi içine şerbeti dökün. </li><li> ~ 45 ° C&#39;de, vakum uygulanarak bir döner buharlaştırıcı kullanılarak Filtrat kuruyana. </li><li> 3 ~ 5 mL su (ve istenirse ultrasonik banyoda kullanan) ile her bir armut şişesi kurutulmuş tortu tekrar süspansiyon, ve bir 40 mL Teflon tüpüne dökülür; suyun ikinci bir kısım ile birlikte şişeye durulanır. </li><li> 6.8 çökelti metal iyonları ve diğer organik molekülleri 1M KOH çözeltisi kullanılarak - 6.6 pH ayarlayın. </li><li> 10 dakika boyunca 2000 rcf santrifüj ile tortulaşır çıkarın. </li><li> Bir 40 mL cam tüp içine dökülür süpernatan, -20 azından tüp parafilm olan açıklığı, dondurularak ° C kapsayan, daha sonra parafilm delikler karıştırmak. </li><li> Tüm sıvı kaldırmak için dondurulmuş süpernatant dondurun-kurulayın. </li><li> , 3 ml kuru metanol ile Kalıntı çözünmesi (ve istenirse ultrasonik banyoda kullanmak) iyice vorteks; kap tüpü ve sonra getirilmesi için 10 dakika süreyle 2000 rcf santrifüjlenirtuzları dışarı. </li><li> Bir 3 mL konik bir reaksiyon şişesi için süpernatan aktarılması; 45 ° C (veya arzu edildiği takdirde, kuru azot gazı akımı altında yumuşak) azından RapidVap makine tarafından kuruyana kadar buharlaştınlmıştır. </li><li> Her flakon için, 100 uL kurtarma standart N-metil-(suda 1 mg ml -1) ve 1 mL H 2 O eklemek -20 flakon parafilm ağızlara, donma ° C kapsayacak, parafilm delebilir ve sonra kuru dondurma . </li><li> Standartları hale: 100 uL muramic asit (metanol içerisinde 0.5 mg mL -1), 100 uL glukozamin (su içinde 1 mg mL -1), 100 uL myo-inositol (su içinde 1 mg mL -1), 100 uL N-ekleme metilglukamin (su içinde 1 mg mL -1), ve 1 ml H2O, parafilm herbir şişe, -20 ° C&#39;de dondurulmuş, daha sonra dondurularak kuru, parafilm perfore. </li></ol> 3. Türevlendirme <ol><li> 32 mg mL -1 hidroksilamin hidroklorür ve 40 mg mL -1 4-dimetilamino-py içeren türevlendirme reaktifi hazırlamakin ridine piridin-metanol (4:1 v / v). </li><li> Kapağı sıkıca ve iyice girdap, reactivials her birine türevlendirme reaktifi 300 uL ekleyin. </li><li> 35 dakika boyunca 75-80 ° C su banyosunda (de herhangi bir su şişeleri girmesine izin vermemek için flakon mühürlü) içinde şişeleri koyun. </li><li> Su banyosu, ve oda sıcaklığında serin şişeleri çıkarın. </li><li> 3 mL reactivials, sıkı bir şekilde kap, ve iyice vorteks her birine 1 mL asetik anhidrit ekleme, daha sonra 75-80 dakika boyunca 25 ° C&#39;de su banyosu içinde yeniden ısıtma. </li><li> Su banyosu, ve oda sıcaklığında serin şişeleri çıkarın. </li></ol> 4. Ayırma ve Ölçüm <ol><li> Her flakon, kapağı sıkıca ve iyice girdabına 1.5 mL diklorometan ekleyin. </li><li> Çözümler iki sözcük ayrı kadar rahatsız oturup bekleyin Her flakon için, kapağı sıkıca ve iyice girdabına 1 ml 1M HCl ekleyin, aspire ve 1000 uL pipet kullanarak üst (sulu) faz atın. </li><li> Aynı fashion, organik faz 3 kez ayıklamak 1 ml H 2 O ile (son yıkama adımla, sulu üst aşaması tüm kaldırıldı sağlamak için özel dikkat). </li><li> 45 de RapidVap kullanarak nihai çözelti kurumaya ° C (ya da kuru istenirse azot gazı kullanarak). </li><li> 300 uL, etil asetat-hekzan (1:1 v / v) ve küçük hacimli ve sıkı bir şekilde uç kapaklı 2 mL amber vida kapaklı küçük şişelerden transfer çözünmüştür. </li><li> ; Sabit faz% 5-fenil,% 95 metil-polisiloksan (DB-5 veya eşdeğeri) hidrojen ile 30 m uzunluğunda, 0.25 mm 0.25 um film kalınlığı: ölçümü için, erimiş silika apolar kapiler kolon kullanılarak GC-FID tarafından analiz veya helyum taşıyıcı gaz olarak, 0.5 mL min -1 sabit debide. Kromatografisi prim &quot;etkisiz&quot; fazları ve hidrojen taşıyıcı gaz daha iyi, ama daha yaygın çeşitleri ve helyum ile kabul edilebilir. Dedektör 300 ayarlar ve bunlar ° C; 400 mL dak -1 hava ve 30 mL dak -1 nitrojen ve hem dehidrojen makyaj gazlar. Enjeksiyon ve fırın parametreler şunlardır: ° C, 250 GC girişi ile 1 uL bölünmüş enjeksiyon (30:1); ilk fırın sıcaklığı 120 ° C; 1 dakika tutun; 10 fırın sıcaklığı artırmak ° C min. -1 ila 250 ° C; 2.5 dk tutun;. 270 rampa ° C ile 20 ° C min -1; 2 dakika tutun; 290 rampa ° C ile 20 ° C min -1, 5 dakika tutun. Taşıyıcı gaz akış hızını ayarlama böylece 250 ° C&#39;de inositol, glukozamin ve muramic asit türevleri elüt edilmesi, ve 270 de N-metilglukamin elutes ° C. </li></ol> 5.. Türev Doğrulama <ol><li> Moleküler iyon tanımlamak ve türevinin moleküler ağırlıkların belirlenmesi için yumuşak bir iyonizasyon LC-ESI-TOF-MS kullanabilir. </li><li> Türevin hedeflenen iyonları araştırmak için duyarlılık geliştirme için GC-EI-MS-SIM veya GC-EI-MSMS kullanın. </li><li> Türevlerinin hazırlanması için birden çok izotopik olarak etiketlenmiş bir reaktif kullanmak ve sonra kütle kayma kullanımıMS olanlar izotopik olarak etiketlenmiş türevlerinin molekül iyonu ve iyonu parçacıkları incelemek. </li></ol> 6. Temsilcisi Sonuçlar Yöntemin protokolü esas olarak dört adımları ihtiva etmektedir: sindirimi asit, Örnek saflaştırma, türevlendirme ve GC tayini (Şekil 1). Glukozamin ve standart stoktan ve bir toprak örneğinden muramic asit analizi için bir örnek, Şekil 2&#39;de gösterilmiştir. Glukozamin ve muramic asit, mannosamine ve galaktosamin (glukozamin iki izomerler) yanı sıra, aynı zamanda yöntemi kullanılarak eş zamanlı olarak tespit edilebilir. İç standart myo-inositol ile ilgili standartların yanıt faktörlere dayanarak, toprak örneklerinde bu biyobelirteçleri ölçmek olabilir. Kurtarma standart nitelik türevlendirme sürecini izlemek üzere kullanılmıştır. Aldononitrile asetat türevlendirilmiş glukozamin ve muramic asit oluşumu için şemalar Şekil 3 &#39;de gösterilmiştir </strong&gt;. Türevlerinin önerilen yapılar duyarlılığı geliştirme, ya da yumuşak iyonizasyon LC-ESI-TOF-MS 8 için GC-EIMS-SIM ile belirlenmiştir; elektron iyonizasyon üzerinde oluşmuş iyon parçalarının önerilen yapıları arasında iyon spektrumu karşılaştırarak incelenmiştir örnekleri, çeşitli izotop birleşmeleri 11 ile hazırlanır. Şekil 4, baskın iyon kütle kayması göstermektedir non-etiketli ajanlar, D-asetik anhidrit ve U -13 C-glukosamin ile hazırlanan üç senaryo, in aldononitrile asetat türevlendirilmiş glukozamin m / z 187. Diğer bilgi ve açıklamalar bizim son yayınları 8, 11 sevk edilebilir. Bu strateji, türev kimya okumak için bir model olarak hizmet verebilir. <img alt="Şekil 1" src="/files/ftp_upload/3767/3767fig1.jpg" /> Glukozamin analizi ve toprak muramic asit protokolü Şekil 1. Ölçüm akış şemasıörnekleri. Asit sindirim, Arıtma, türetme ve GC belirlenmesi: protokolü, 4 adımları içerir. <img alt="Şekil 2" src="/files/ftp_upload/3767/3767fig2.jpg" /> Şekil 2. Standartlara ve toprak için inositol, glukozamin, muramic asit, mannosamine, galaktosamin ve metilglucamin ve aldononitrile asetatlar GC-FID kromatogramları. <img alt="Şekil 3" src="/files/ftp_upload/3767/3767fig3.jpg" /> Şekil 3. Aldononitrile asetat türevlendirilmiş glukozamin ve muramic asit oluşumu için Şema. 1 numara nitril reaksiyonu temsil eder. 2 numaralı asetilasyonu temsil eder. <a href="http://www.jove.com/files/ftp_upload/3767/3767fig4_lg.jpg"><img alt="Şekil 4" src="/files/ftp_upload/3767/3767fig4.jpg" /></a> Şekil 4. Baskın iyon structur ile ilişkili aldononitrile asetat türevlendirilmiş glukozamin Kütle spektrumlarıEI modu altında es (A) non-etiketli maddeleri ile hazırlanmıştır, (B), D-asetik anhidrit, (C) U -13 C-glukosamin. Yıldız ağır izotop atomu veya izotop grubu temsil eder.

<ol>
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Biz ifşa hiçbir şey yok.

Aldononitrile asetat türevlendirilmiş glukozamin ve muramic asit analizi için sunulmuş GC-bazlı yöntem toprak ekstrakte bu amino şekerler, ölçmek için göreceli olarak hızlı bir yöntem sağlar. Türevleri kimyasal açıdan kararlı olan ve bir analize tespit edilebilir. Yöntemi toprak örnekleri ile sınırlı değildir, ve non-toprak matris örnekler için basitleştirilmiş olabilir. Bu yöntemde kullanılan vakum pompası aside dirençli olması için yapılmıştır. Biz da...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Muramic asit </td><td> Sigma-Aldrich </td><td> M2503-25mg </td></tr><tr><td> D-(+)-glukosamin hidroklorür </td><td> Sigma-Aldrich </td><td> G1514-100G </td></tr><tr><td> N-metil-D-glukamin </td><td> Sigma-Aldrich </td><td> M2004-500G </td></tr><tr><td> Myo-inositol </td><td> Fisher Scientific </td><td> A307003G025 </td></tr><tr><td> Metanol (kuru) </td><td> Acros organik </td><td> AC326950010 </td></tr><tr><td> 4-dimetilamino-piridin </td><td> Acros organik </td><td> AC148270050 </td></tr><tr><td> Etil asetat </td><td> VWR International </td><td> BJGC100-4 </td></tr><tr><td> Hydroxlamine hidroklorür </td><td> Fisher Scientific </td><td> H330-100 </td></tr><tr><td> Piridin </td><td> Fisher Scientific </td><td> P368-500 </td></tr><tr><td> Asetik anhidrit </td> &lt;td&gt; Fisher Scientific </td><td> A10-100 </td></tr><tr><td> Diklorometan (Metilen klorür) </td><td> Fisher Scientific </td><td> D37&#39;ye-500 </td></tr><tr><td> Hekzan </td><td> Fisher Scientific </td><td> H303-4 </td></tr><tr><td> Hidroklorik asit (6M) </td><td> Fisher Scientific </td><td> S456-4 </td></tr><tr><td> Hidroksilamin hidroklorür -15 N </td><td> Simge hizmetleri </td><td> IN5280 </td></tr><tr><td> Asetik anhidrid -2 H (D 6 C 4 O 3) </td><td> Acros organik </td><td> AC174670050 </td></tr><tr><td> D-glukoz-U -13 C </td><td> Cambridge izotop laboratuvarı </td><td> CLM-1396-1 </td></tr><tr><td> Amonyum sufate -15 N </td><td> Cambridge izotop laboratuvarı </td><td> NLM-713-1 </td></tr><tr><td></td><td></td><td></td></tr><tr><td> Ekipman Adı </td><td> Şirket </td><td> Tip </td></tr><tr><td> Rapid-Vap </td&gt; <td> Labconco </td><td> 790002 </td></tr><tr><td> Vakum pompası </td><td> KNF Neuberger </td><td> D-79112 </td></tr><tr><td> Hidroliz şişesi </td><td> Fisher Scientific </td><td> 06 423A </td></tr><tr><td> Türevlendirme microvial </td><td> Fisher Scientific </td><td> 06-100E </td></tr><tr><td> GC </td><td> Hewlett-Packard </td><td> 6890 </td></tr><tr><td> MS </td><td> Hewlett-Packard </td><td> 5972 </td></tr><tr><td> LC-ESI-TOF-MS </td><td> Agilent </td><td> Agilent 1200 serisi HPLC sistemi Agilent LC / MSD-TOF birleştiğinde </td></tr></tbody></table>

gc-based detection of aldononitrile acetate derivatized glucosamine and muramic acid for microbial residue determination in soil

Karakterize eden mikroorganizmalar nicel yaklaşımlar ekosistemler içindeki mikrobik durumu ve işlevini daha geniş bir anlayış için çok önemlidir. Mikrobiyal analiz için güncel yaklaşımlar, geleneksel laboratuvar kültür-bağımlı teknikleri ve doğrudan çıkarma ve bazı biyo 1, 2 belirlenmesi esasına dayanan bu iki içerir. Toprak yaşayan mikrobiyal türlerin çeşitliliği arasında çok az kültüre, dolayısıyla kültür-bağımlı yöntemleri önemli önyargıları, biyomarker analizinde eksik bir sınırlama tanıtmak edilebilir. Glukozamin, mannosamine, galaktosamin ve muramic asit de toprak sistemleri 3 en önemli unsurlar olan bu glukozamin (en bol) ve muramic asit (benzersiz bakteri hücre dan), canlı ve ölü mikrobiyal kütle hem tedbirler olarak hizmet edilmiştir , 4. Ancak, analiz konusunda bilgi eksikliği bilimsel emsalleri arasında geniş yaygınlaşması kısıtlar. Tüm exi arasındaaldononitrile için, türevlendirme analitik yöntemler sting GC tabanlı analizi takip asetatlar optimum hassas dengeleme açısından iyi bir seçenek olarak ortaya çıkmıştır, hassasiyet, basitlik, numune saklama 5 üzerinde iyi kromatografik ayırma ve kararlılık. Burada, aldononitrile asetatlar onların dönüşüm sonra topraktan glukozamin ve muramic asit güvenilir ve nispeten basit bir analizi için detaylı bir protokolü sunarız. Protokolü, dört adımdan oluşmaktadır: asit sindirim, örnek arıtma, türevlendirme ve GC belirlenmesi. Adım adım prosedürü eski yayınları 6, 7 göre ayarlanır. Buna ek olarak, yapısal olarak elektron iyonizasyon üzerinde oluşmuş türevi ve iyonu parçacıkları moleküler iyon doğrulamak için bir strateji sunulmaktadır. Biz aldononitrile asetat türevlendirilmiş glukozamin ve mur moleküler ağırlıkların belirlenmesi için GC-MS-EI-SİM, LC-ESI-TOF-MS ve izotopik olarak etiketlenmiş bir reaktif uygulananAmic asit; her türevleri 8 iyonu parçacıkları araştırmak için iyon spektrumunda izotop etiketli türevlerinin kütle kayma kullanılır. Teorik aydınlatılması ek olarak, türev ve iyonu parçacıkları moleküler iyon doğrulama δ 13 C ya da biyokimyasal çalışmalarda 9, 10, bu biyolojik iyon parçaları araştırmacılar kullanılarak yararlı olacaktır.

Watch this Scientific Journal Video about GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil at JoVE.com

GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Biz, glukozamin ve toprak ekstrakte muramic asit aldonitrile asetat türevlerinin GC-bazlı analizi için bir yöntem tarif protokolü. Kimyasal mekanizmasının aydınlatılması için, biz de türev ve elektron iyonizasyon halinde oluşan iyon parçalarının yapısını onaylamak için bir strateji sunmak.

Toprak Mikrobiyal Kalıntı Tayini için Aldononitrile Asetat türevlendirilmiş Glukozamin ve Muramic Asit GC tabanlı Algılama

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. ...

gc-based-detection-aldononitrile-acetate-derivatized-glucosamine

Research

JoVE Journal

Biology

16.8K Görüntüleme.  University of Wisconsin, Madison. Biz, glukozamin ve toprak ekstrakte muramic asit aldonitrile asetat türevlerinin GC-bazlı analizi için bir yöntem tarif protokolü. Kimyasal mekanizmasının aydınlatılması için, biz de türev ve elektron iyonizasyon halinde oluşan iyon parçalarının yapısını onaylamak için bir strateji sunmak.

Video: GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Microbial Communities in Nature and Laboratory - Interview

Doğa ve Laboratuvar Mikrobiyal Toplulukları - Röportaj

Biyoloji

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.
In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.
Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.

A method for the determination of acetate kinase activity is described. This assay utilizes a direct reaction for determining enzyme activity and kinetics of acetate kinase in the acetate-forming direction with different phosphoryl acceptors. Furthermore, this method can be utilized for assaying other acetyl phosphate or acetyl-CoA utilizing enzymes.

Enzim Asetat Kinaz Asetat oluşturan Faaliyet Doğrudan Algılama

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

Mikrobiyal Systems Biyoloji Chemostats Kullanımı

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Isolation of Native Soil Microorganisms with Potential for Breaking Down Biodegradable Plastic Mulch Films Used in Agriculture

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g. fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.

Plastic films labeled "biodegradable" are commercially available for agricultural use as mulches. Tillage represents an attractive disposal method, but degradation under field conditions is poorly understood. The purpose of this study was to develop methods for isolating native soil fungi and bacteria that colonize plastic mulch films after field burial.

Biyobozunur Plastik Malç Filmler Aşağı Breaking potansiyeli ile Yerli Toprak Mikroorganizmaların izolasyonu Tarımda Kullanılan

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

Mikrobiyal Tanımlama ve Karakterizasyonu için Pyrosequencing

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Determination of Fatty Acid Oxidation and Lipogenesis in Mouse Primary Hepatocytes

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid oxidation, or alternatively, synthesize new lipid via de novo lipogenesis. The net sum of these pathways is dictated by a number of factors, which in certain disease states leads to fatty liver disease. Excess hepatic lipid accumulation is associated with whole body insulin resistance and coronary heart disease. Tools to study lipid metabolism in hepatocytes are useful to understand the role of hepatic lipid metabolism in certain metabolic disorders.
In the liver, hepatocytes regulate the breakdown and synthesis of fatty acids via &#946;-fatty oxidation and de novo lipogenesis, respectively. Quantifying metabolism in these pathways provides insight into hepatic lipid handling. Unlike in vitro quantification, using primary hepatocytes, making measurements in vivo is technically challenging and resource intensive. Hence, quantifying &#946;-fatty acid oxidation and de novo lipogenesis in cultured mouse hepatocytes provides a straight forward method to assess hepatocyte lipid handling. 
Here we describe a method for the isolation of primary mouse hepatocytes, and we demonstrate quantification of &#946;-fatty acid oxidation and de novo lipogenesis, using radiolabeled substrates.

De novo lipogenesis and &#946;-fatty acid oxidation constitute key metabolic pathways in hepatocyte, pathways that are perturbed in several metabolic disorders, including fatty liver disease. Here we demonstrate isolation of mouse primary hepatocytes and describe quantification of &#946;-fatty acid oxidation and lipogenesis.

Fare İlköğretim Hepatositlerde Yağ Asidi Oksidasyon ve lipogenez Belirlenmesi

Lipid metabolism in liver is complex. In addition to importing and exporting lipid via lipoproteins, hepatocytes can oxidize lipid via fatty acid ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

Belirli Genomik Bölgeler ve enChIP tarafından İlişkili Moleküllerin Tanımlanması İzolasyonu

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

İzole Çalışma Sıçan Kalp Glukoz ve Yağ Asidi Oksidasyonu Oranlarının Belirlenmesine Yöntemleri

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against soilborne fungus Rhizoctonia solani. R. solani is a pathogen of a wide range of plant hosts worldwide. The fungus survives in soils as a saprophyte and grows rapidly on simple water agar media. The plate assay is a rapid method to compare composts for their ability to slow the growth of R. solani. The assay also correlates well with suppression of other soilborne fungal pathogens that survive as saprophytes in soils such as Alternaria early blights, Fusarium wilt, Phytophthora root rot, and Pythium root rot.

Presented is a protocol for a plate competition assay to identify whether a specific compost is likely to contain bacteria and fungi that suppress growth of Rhizoctonia solani.

Bir plaka rekabet tahlil olarak hastalık bastırma hızlı bir ön değerlendirme

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.