The funding for this research project was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) via a Discovery Grant to YL.

Reaktifler ve Tamponlar hazırlanması 1. <ol><li> 0.5 M etilendiamin tetraasetik asit (EDTA), <ol><li> 2 L&#39;lik bir beher içinde, deiyonize damıtılmış 800 ml su ile 186.1 gr EDTA ekleyin (GKD 2 O). NaOH peletleri ile 8.0 çözeltinin pH&#39;ının ayarlanması. 1.0 L&#39;lik son bir hacme O GKD 2 ekleyin ve 4 ° C&#39;de otoklav ve mağaza bir otoklav cam şişe çözüm aktarın. </li></ol></li><li> 10 x Tris-borat EDTA (10x TBE) <ol><li> 4 L&#39;lik bir plastik kabı içinde, tüm bileşenler kadar bir karıştırma çubuğu kullanılarak 4 L Karışım bir son hacme 432 g Tris-bazı, 200 g borik asit, 0.5 M EDTA (pH 8.0) ve GKD 2 O 80 ml ​​ilave çözündürülür. 4 ° C&#39;de otoklav ve mağaza 1 L cam şişe çözüm aktarın. </li></ol></li><li> % 10 denatüre edici poliakrilamid Hazır <ol><li> F kadar çözeltisi ve GKD 2 A: (1 29), 4 L&#39;lik bir plastik kap olarak, 1681,7 g üre 400 ml 10x TBE, 40% akrilamid / bisakrilamid 1 L eklemeNominal hacmi üre çözünene kadar bir karıştırma çubuğu kullanılarak 4 L bir karışımıdır. 4 ° C &#39;de 1 L&#39;lik amber cam şişe ve saklamak için bir çözüm aktarın. </li></ol></li><li> Yükleme Tamponu 2x Jel (2x GLB) <ol><li> 200 ml&#39;lik bir cam kabın içinde, 44 g üre, 8 g sakroz, 10 mg bromofenol mavi, 10 mg iksilen siyanol FF,% 10 sodyum dodesil sülfat 400 ul ve 10 x TBE ve 4 ml. 40 ml&#39;lik bir son hacme O GKD 2 ekleyin ve bir manyetik karıştırma çubuğu ile karıştırılarak 50 ° C&#39;de, hafif ısıtma ile bileşenleri çözülür. 4 ° C&#39;de Aktarım 1 mi, 1.5 ml mikrofüj tüplerine kısım saklayın. Not: Ön kullanımı herhangi bir katı yeniden çözünmesi için 90 ° C&#39;de kısa bir ısıtma gerektirir. </li></ol></li><li> 1 M Tris-hidroklorür (Tris-HCl) (pH 7.5) <ol><li> Bir cam şişe içerisinde, Tris-bazın 12.1 g ve GKD 2 O 70 ml ilave edilir ve katı madde çözülene kadar karıştırın. 1 M hidroklorik asit (HCI) ile pH&#39;ı 7.5&#39;e ayarlayın. 100 mi, otoklav ve taştan bir son hacme GKD 2 O eklemere 4 ° C&#39;de </li></ol></li><li> 5 M sodyum klorür (NaCI) <ol><li> Bir cam şişe içerisinde, GKD 2 O 150 ml NaCI 58.4 g çözülür GKD 2 O. ile 200 ml ses seviyesini ayarlamak 4 ° C&#39;de saklayın. </li></ol></li><li> DNA, Seyreltme Tampon maddesi <ol><li> bir cam şişe içinde, 1 M Tris-HCl (pH 7.5), 8.0 5 M NaCl mi ve 0.5 M EDTA, 0.4 mi (pH 8.0) içinde 2.0 ml karıştırılır. GKD 2 O, 4 ° C&#39;de otoklav ve mağaza ile 200 ml ses seviyesini ayarlayın. </li></ol></li><li> 1 M 4- (2-hidroksietil) -1-piperazinetansülfonik asit (HEPES) (pH 7,4) <ol><li> Bir cam şişe içerisinde, GKD 2 O 80 ml ​​HEPES, 2.38 g çözülür 5 N NaOH kullanılarak 7.4 pH ayarlamak ve 100 ml lik bir son hacme GKD 2 O ekleyin. </li></ol></li><li> 1 M magnezyum klorür (MgCI2) <ol><li> Bir cam şişe içerisinde, MgCl2 -6H 2 O 2.03 g ekleme ve GKD 2 O ile 100 ml&#39;ye ses getirmek </li></ol></li><li> Reaksiyon Tamponu (RB) <ol><li&gt; 50 ml konik bir tüp içinde, 1 M HEPES (pH 7.4), 5 M NaCI, 1.5 mL, 1 M MgCl2, 0.75 mi, ve Tween-20 5 ul 50 ul ekle. 50 ml&#39;lik bir son hacme O GKD 2 ekleyin. çözelti karıştırılır ve kullanılana kadar 4 ° C de, bir şırınga veren filtre (0.22 um) ve deposunu kullanan bir konik tüp içine filtre. </li></ol></li><li> Bağlama Tamponu (BB) <ol><li> 50 ml konik bir tüp içinde, bir son hacim için O GKD 2; 1 M Tris-HCI (pH 7.5), 500 ul, NaCI 8.8 g, MgCl2, 1 M, 50 ul, Tween 20 5 ul 50 ml. çözelti karıştırılır ve kullanılana kadar 4 ° C de, bir şırınga veren filtre (0.22 um) ve deposunu kullanan bir konik tüp içine filtre. </li></ol></li><li> Substrat solüsyonu (SS) <ol><li> 50 ml&#39;lik konik bir tüp içinde, NaCI 5.8 g, 1 M 3 mL MgCl2, 1.5 g üre ve GKD 2 O 40 ml ilave 10 mM HCI kullanılarak 5,0 çözeltinin pH&#39;ının ayarlanması. Çözelti tamponlu olduğundan, HCI kullanılarak pH ayarlamakdikkatle küçük HCI alikotları eklenmesi ile. 50 ml&#39;lik bir son hacme O GKD 2 ekleyin. </li><li> çözelti karıştırılır ve kullanılana kadar 4 ° C de, bir şırınga veren filtre (0.22 um) ve deposunu kullanan bir konik tüp içine filtre. </li></ol></li><li> Luria Bertani (LB) Broth <ol><li> Beher, GKD 2 O 1 L 20.0 g LB tozu çözmek 4 ° C&#39;de bir cam şişe, otoklav ve saklamak için aktarın. </li></ol></li><li> % 1.5 LB agar <ol><li> 250 ml&#39;lik bir şişe içinde, 1.5 g agar ve LB suyu 100 ml. 4 ° C&#39;de otoklav ve saklayın. </li></ol></li><li> agar Tabaklar <ol><li> Bir mikrodalga LB agar tekrar çözündürülmesi ve çözeltiye ~ 50 ° C soğutulur. Petri kaplarına solüsyonu dökün ~ 5 plakaları yapmak ve onları katılaşmaya sağlar. </li></ol></li></ol> 2. sentezi ve E. saflaştırma coli- duyarlı DNAzim EC1 <ol><li> Şablon aracılığı Enzimatik Ligasyonu ile EC1 sentezi <ol><li> comm arındırınticari olarak uygundur, sentezlenmiş oligonükleotidler BS1, DE1 ve T1,% 10, standart protokollere göre poliakrilamid jel elektroforezi (dPAGE) denatüre edici ile (Tablo 1 &#39;de verilen sekansları). </li><li> BS1, DE1 ve T1 100 uM stok hazırlayın. kullanılıncaya kadar -20 ° C&#39;de saklayın. </li><li> 1.5 ml mikrofüj tüpüne, enzim ile ilgili olarak (500 mM Tris-HCI (pH 7.6), 100 mM MgCl2 sağladığı GKD 2 O 38.5 ul, DE1 5 ul ve 10 x T4 polinükleotid kinaz reaksiyon tamponu 5 ul 50 mM ditiotreitol (DTT), 1.0 mM spermidin). </li><li> adenosin trifosfat (ATP) (100 mM) 1 ul ekle. T4 polinükleotid kinaz 5 birim (10 U / ul) eklenir. reaksiyon karışımına dikkatli bir pipetleme karıştırın. </li><li> 30 dakika için 37 ° C &#39;de reaksiyon inkübe edin. </li><li> 5 dakika boyunca 90 ° C&#39;ye kadar ısıtılarak, reaksiyonun sönmesi. </li><li> GKD 2 O 118 ul, BS1 5 ul ve T1 5 ul ekleyin. 2 mi, 90 ° C tepkime ısısın, ve 10 dakika boyunca oda sıcaklığına kadar soğutulur. </li><li> Enzim, tedarikçi tarafından sağlanan 10 x T4 DNA ligaz reaksiyon tamponu 20 ul ekle (400 mM Tris-HCI (pH 7.8), 100 mM MgCl2, 100 mM DTT, 5 mM ATP). T4 DNA ligazı, 10 birim (5 U / ul) ilave edilir ve dikkatli bir şekilde pipetle karıştırılır. </li><li> 2 saat oda sıcaklığında inkübe edin. </li><li> 3 M sodyum asetat (NaOAc) (pH 5.2) 20 ul soğuk% 100 etanol 500 ul ekle. Vorteks ile çözüm karıştırın ve 30 dakika için -20 ° C dondurucu içinde bir tüp yerleştirmek. </li><li> 4 ° C&#39;de 20 dakika boyunca 20,000 x g&#39;de mikrofüj santrifüj. Dikkatle pipetleme süpernatant kaldırmak. </li><li> 4 ° C&#39;de 10 dakika boyunca 20,000 x g hızında tekrar soğuk% 70 etanol ve santrifüj ile pelet yıkayın. Bir kez daha süpernatant kaldırmak ve 10 dakika süre ile, vakum altında pelet kurutulur. </li><li> GKD 2 O 15 ul DNA tekrar ve daha sonra 2x GLB 15 ul ekleyin. </li><li> 90 ° C&#39;ye kadar kuvvetli bir şekilde karıştırın ve ıslak sertleştirilmesi için2 dak. Aşağıda tarif edildiği gibi,% 10 dPAGE tam uzunlukta DNA saflaştırılır. </li></ol></li><li> % 10 dPAGE Kurma <ol><li> Temiz iki cam levha (bir tam plaka ve bir çontuklu), iki 0.75 mm kalınlığında aralama ve 12 kuyu tarak. İki her iki tarafta spacers ve üstüne çentikli plaka ile düz bir yüzeye bir cam plaka yatıyordu. Birlikte tedarikçi tarafından sağlanan dört klipler kullanarak iki tabak Clip. </li><li> 150 ml&#39;lik bir plastik kabı içinde,% 10 dPAGE 40 ml, tetrametiletilendiamin 40 ul (TEMED) ve% 10 amonyum persülfat (APS), 400 ul dökün. bileşenleri karıştırın ve yavaş yavaş plakalar arasına çözüm dökün. </li><li> Tarak yerleştirin ve sonra jel üzerinde 10 dakika polimerize izin verir. Jel polimerize edildikten sonra, yavaş yavaş tarak kaldırmak ve GKD 2 O. ile kuyu yıkayın </li><li> jel elektroforezi aparat üzerine plakaları monte edin. aşırı ısınmayı önlemek için üretilen ısıyı dağıtmak için bir metal plaka kullanın. </li><li> Üst ve alt bölmelere üzerine 1x TBE dökünve kuyular iyi tampon batık emin olun. Bir pipet veya şırınga kullanarak 1x TBE ile kuyu yıkayın. </li><li> yükleme örnekleri önce 15 dakika için 35 mA&#39;da için cihaz ve önceden çalıştırılmış ayarlayın. </li></ol></li><li> % 10 dPAGE den Bağlanmış EC1 elüsyon <ol><li> Adım 2.2.6 ardından, alt boya (Bromophenol mavi) kadar 35 mA jel çalıştırmak cam plakanın alt ulaşır. Bu, yaklaşık 1.5 saat sürer. Gücü kapatın ve cihazdan plakaları çıkarın. </li><li> kağıt havlu birkaç yaprak tabak Lay ve dikkatle cam plakalar gelen aralayıcılar kaldırın. Dikkatli bir şekilde üst cam plaka kaldırmak ve plastik wrap ile jel sarın. </li><li> ikinci cam plakayı kaldırın ve tekrar plastik wrap ile karşılamak için jel çevirin. plastik wrap kırışıklıkları önlemek için özen gösterin. </li><li> 4 farklı DNA bantları (Tam bağlanan EC1, DE1, BS1 ve T1) üretecek UV gölgeleme (260 nm) kullanarak bağlandı ürünü gözünüzde canlandırın. </li><li&gt; Tüketim 1.5 ml mikrofuge&#39;de tüp steril bir jilet ve transferi ile üst bant (EC1). ince bir hamur haline dönene kadar steril bir pipet (200 ul uç boyutu) ile jel Crush. DNA elüsyon tamponu 550 ul ekle gömülü fluorofor korumak için alüminyum folyo ile tüp kapağı ve 15 dakika boyunca çalkalayın. </li><li> 5 dakika boyunca 20.000 xg&#39;de jel çözüm Santrifüj ve dikkatli bir 1.5 ml mikrofuge&#39;de tüp süpernatant 400 ul aktarın. pipetleme sırasında jel parçaları çekilmesi kaçının. </li><li> transfer süpernatan ile mikrofüj tüpe 40 3 M NaOAc (pH 5.2) içinde ul soğuk% 100 etanol içinde 1.0 ml. Vorteks ile çözüm karıştırın ve 30 dakika için -20 ° C dondurucu içinde bir tüp yerleştirmek. </li><li> 4 ° C&#39;de 20 dakika boyunca 20,000 x g&#39;de mikrofüj santrifüj. Dikkatle pipetleme süpernatant kaldırmak. </li><li> 4 ° C&#39;de 10 dakika boyunca 20,000 x g hızında tekrar soğuk% 70 etanol ve santrifüj ile pelet yıkayın. Bir kez daha, kaldırmakSüpernatan 10 dakika için vakum altında pelet kurutulur. </li><li> 260 nm&#39;de UV absorbansı ölçerek EC1 konsantrasyonunun belirlenmesi. 10 uM stok yapmak ve kullanılana kadar -20 ° C&#39;de örnek saklayın. </li></ol></li></ol> DNA üreazın 3. Konjugasyon <ol><li> Sukinimidil 4- (N-maleimidometil) sikloheksan-1-karboksilat (SMCC), hisse senedi hazırlanması <ol><li> DMSO 676 ul 1 mg SMCC çözülür. kullanılana kadar buz üzerinde girdap ve yer. </li></ol></li><li> Üreaz Stokta hazırlanması <ol><li> 1 x PBS (Mg + 2 veya Ca + 2), 1 ml 1 mg üreaz çözülür. Buz üzerinde kullanıma kadar yerleştirin. Not: Kristalize üreaz çözünmesi yavaştır ve yumuşak karıştırma denatürasyon önlemek için gereklidir. </li></ol></li><li> Üreaz-DNA sentezi (UrDNA) <ol><li> 2.5 mi mikrofüj tüpüne 100 uM LD1 10 ul ekle. GKD 2 O 140 ul 10x PBS 40 ul ve vorteks ekleyin. </li><li> SMCC s 80.5 ul ekleyintak, bir tezgah üstü santrifüj kullanılarak DMSO, girdap ve kısaca santrifüj 159.5 ul. </li><li> 60 dakika boyunca 37 ° C&#39;de inkübe edin. mikrofuge&#39;ye kapağın altında yoğuşmayı önlemek. </li><li> 1x PBS 200 ul 3 M NaOAc (pH 5.2), 60 ul soğuk% 100 etanol içinde 1.5 ml. Vorteks ile çözüm karıştırın ve 30 dakika boyunca -20 ° C&#39;de inkübe edin. </li><li> 4 ° C&#39;de 20 dakika boyunca 20,000 x g&#39;de santrifüjleyin çözeltisi. Süpernatantı ve vakum altında pelet kurulayın. aşırı kurumayı önlemek. </li><li> Kurutulmuş pelet, üreaz stokunun 400 ul, 5 saat oda sıcaklığında inkübe edin. </li><li> Önceden yıkanmış 100K MWCO santrifüj filtre kolonuna ham konjügat 200 ul aktarın. 5 dakika boyunca 14.000 x g&#39;de sütun santrifüjleyin. </li><li> 5 dakika boyunca 14.000 x g&#39;de kolon ve santrifüj ham konjügat kalan 200 ul aktarın. sütunu kaldırmak ve yeni bir 2.0 ml mikrofuge&#39;de tüp ( &quot;toplama tüpüne&quot;) olarak baş aşağı yerleştirin. </li><li> Santrifüjuge 2 dakika süreyle 1000 xg&#39;de (ters sütun ile) toplama tüpü. toplama tüpünü çıkarın ve konjugatlarının ek kurtarma için membran yıkamak için santrifüj sütuna 1x PBS 30 ul ekleyin. </li><li> Bir kez daha sütunu ters ve geri toplama tüpünün içine koyun. 2 dakika süreyle 1000 xg&#39;de (ters sütun ile) toplama tüpü santrifüj. Çıkarın ve sütun atmayın. </li><li> kullanılana kadar 4 ° C &#39;de UrDNA saklayın. </li></ol></li></ol> Manyetik boncuklar üzerine EC1 ve UrDNA 4. Montaj <ol><li> gibi manyetik boncuk (MB) stok karıştırın ve 1.5 ml mikrofüj tüpe MB süspansiyon 100 ul aktarın. MB izole bir manyetik raf tutucu mikrofuge&#39;ye tüpü yerleştirin. </li><li> pipetle ile süpernatantı ve tüp tampon (BB) bağlayıcı 150 ul ekleyin. tutucudan tüpü çıkarın ve dikkatlice homojen bir çözüm MB tekrar süspansiyon tüp dokunun. t süspansiyon sıçramasını önlemektüp ya da kap op. Böyle bir durumda, kısaca geri süspansiyona kalıntı dönmeye bir tezgah üstü santrifüj kullanın. </li><li> Adımı yineleyin 4.2 iki kez daha. </li><li> Bu süspansiyona 10 uM EC1 10 ul ekle. Dikkatle tüp dokunarak karıştırın. </li><li> Hafif 30 dakika boyunca çalkalayarak çözelti inkübe edin. MB çökmesini önlemek için tüpü her 2-3 dakikada bir dokunun. </li><li> MB izole ve pipetleme süpernatant kaldırmak için geri manyetik raf mikrofuge&#39;ye tüpü yerleştirin. (Adım 4.2 de tarif edildiği gibi) BB 150 ul MB üç kez yıkayın. </li><li> Yıkama işlemi tamamlandıktan sonra, BB 150 ul bir toplam MB askıya. Bu çözeltiye, UrDNA 15 ul ilave edin ve 2 dakika boyunca 45 ° C&#39;ye kadar çözelti ısıtılır. Solüsyon oda sıcaklığına kadar soğutulur ve 2 saat süreyle inkübe edin. </li><li> MB izole ve pipetleme süpernatant kaldırmak için geri manyetik raf mikrofuge&#39;ye tüpü yerleştirin. </li><li> Reaksiyon tampon maddesi (RB), 100 ul ekle. t kaldırO manyetik raf tüp mikrofuge&#39;ye ve dikkatle MB tekrar süspansiyon. </li><li> adım 4.9 tekrarlayarak MB üç kez yıkayın. NOT: yıkanmış süpernatant hızla Melezleşmemiş UrDNA hala mevcut olup olmadığını bir yanlış pozitif sinyal neden olabilir, hangi belirlemek için test edilebilir. Test yıkama çözeltisine kırmızı 50 mM üre 10 ul ve% 0.04 fenol 10 ul ekleyerek yapılabilir. yıkama çözeltisi bir renk değişimine neden olmaz kadar MB yıkamak devam edin. 100 ul süspansiyon kullanilana kadar 4 ° C&#39;de saklanır. </li></ol> Bakteriyel hücreler 20 5. hazırlanması <ol><li> Kültürleme E. Stoklar gelen coli <ol><li> Plaka E. E. coli K12, bir alev altında veya biyolojik bir güvenlik kabini, bir gliserol stokundan LB agar kaplan üzerine (MG1655). </li><li> Steril bir pipet ucu kullanarak, yavaşça LB agar delinmesiyle önlemek için gliserol stok ve hafifçe çizgi bir agar plaka yüzeyi dokunun. </li><li> Kesikli invertPlaka ve 14 saat boyunca 37 ° C&#39;de inkübe edin. </li><li> 3 haftalık bir süre için 4 ° C sıcaklıkta Parafilm ile plaka ve mağaza kapatın. </li></ol></li><li> Kültürleme E. Hücre Sayımı için coli <ol><li> steril 14 ml kültür tüpünde, LB suyu 2 ml dağıtın. </li><li> Steril bir pipet ucu kullanarak, adım 5.1 hazırlanan çizgili agar plakasından tek bir koloni almak ve kültür tüpüne aktarın. </li><li> 37 ° C &#39;de kültür inkübe ve 14 saat boyunca 230 rpm&#39;de çalkalanır. </li><li> Seri olarak 10-misli aralıklarla bakteri kültürü seyreltin. </li><li> Her bir seyreltilmiş numune için eşit, ayrı LB agar plakaları üzerinde beş 100 ul alikotları plaka. plakalar ters çevirin ve 14 saat boyunca 37 ° C&#39;de inkübe edin. </li><li> Her bir seyreltme ortalama hücre konsantrasyonu elde etmek üzere her numune hücreleri sayın. NOT: 10 7 hücreleri genellikle E. için bir referans turnusol testi kurmak için kullanılan E. Bu seviyeye kadar coli E. coli hızlı bir renk chan tetikleyebilirge. Sonuçlar bölümünde tartışıldığı gibi Ancak, düzgün idam turnusol testi gibi düşük 500 hücreleri tespit edebilir. </li></ol></li><li> E. hazırlama Test coli hücreleri <ol><li> arzu edilen bir hücre süspansiyonu için, transfer 1.5 ml mikrofüj tüpüne kültürlendi, 1 mL stok. </li><li> 4 ° C&#39;de 10 dakika boyunca 6000 x g&#39;de santrifüjleyin hücreleri. Dikkatle hücre pelletini bozmadan süpernatant kaldırmak. </li><li> Hücre topağı, reaksiyon tampon maddesi 10 ul ilave edin ve tekrar süspansiyon hücreleri. 5 dakika boyunca bir hücre süspansiyonu sonikasyon. 5 dakika için bir buz kutusu hücre süspansiyonu aktarın. </li><li> 5 dakika daha hücre süspansiyonu sonikasyon. </li><li> 4 ° C&#39;de 10 dakika boyunca 13,000 x g&#39;de hücre süspansiyonu santrifüj. Test için supernatant (10 ul) kullanır. </li></ol></li></ol> 6. Litmus Testi <ol><li> 1.5 ml mikrofüj tüpüne, mikrofüj tüpüne reaksiyon tampon maddesi (RB) 100 ul ilave edildi ve vorteks yoluyla tüp ön yıkama vetampon atarak. </li><li> Aktarım yıkanmış mikrofüj tüpe monte EC1 (protokol 4) 15 ul. </li><li> Bir manyetik rafa mikrofuge&#39;ye tüp yerleştirerek manyetik boncuk yıkayın. pipetleme süpernatantı. , Raftan mikrofuge&#39;ye tüpünü çıkarın RB 100 ul ekleyin ve dikkatlice manyetik boncuklar tekrar süspansiyon. </li><li> adım 6.3 yineleyerek MB iki kez yıkayın. </li><li> , Manyetik rafa geri mikrofuge&#39;ye tüpü yerleştirin süpernatant kaldırmak ve 10 ul E. ekleyin E. coli örnek adım 5.3 hazırlanan. </li><li> yavaşça mikrofuge&#39;de tüp dokunarak dikkatlice örnek ve manyetik boncuk karıştırın. </li><li> 1 saat boyunca oda sıcaklığında reaksiyon inkübe edin. </li><li> Reaksiyona, GKD 2 O 90 ul ekleyin ve bir manyetik raf üzerine mikrofuge&#39;ye tüp yerleştirin. </li><li> Sonra manyetik ayırma yaklaşık 3 dakika, dikkatle 0.5 ml mikrofuge&#39;de tüp süpernatant 85 ul aktarın. yavaş yavaş süpernatant geri çekinherhangi bir manyetik boncuk toplama kaçının. </li><li> Yukarıdaki mikrofüj tüpe% 0,04 fenol kırmızısı 15 ul ve alt-tabaka çözeltisi 100 ul ilave edin. </li><li> renk değişikliği kaydetmek için belirli zaman aralıklarında bir fotoğraf çekmek. Not: pH değişikliği bir mikroelektrot içeren bir pH metre kullanılarak izlenebilir. Başlangıç ​​pH yaklaşık 5,2-5,5 olmalıdır (çözüm sarı renkte). Aksi takdirde, çözelti, 1 mM asetat tampon pH 5.0) ilavesiyle ayarlanabilir. </li></ol>

<ol>
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Ed. 54, (2015).</li><li>He, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+specific+recognition+of+breast+tumors+by+an+RNA-cleaving+fluorogenic+DNAzyme+probe.">Highly specific recognition of breast tumors by an RNA-cleaving fluorogenic DNAzyme probe.</a> Anal. Chem. 87, 569-577 (2015).</li><li>Sumner, J. B., Hand, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoelectric+point+of+crystalline+urease.">Isoelectric point of crystalline urease.</a> J. Am. Chem. Soc. 51, 1255-1260 (1929).</li><li>Karplus, P. A., Pearson, M., Hausinger, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=70+Years+of+crystalline+urease:+What+have+we+learned.">70 Years of crystalline urease: What have we learned.</a> Acc. Chem. Res. 30, 330-337 (1997).</li><li>Omiccioli, E., Amagliani, G., Brandi, G., Magnani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+platform+for+Real-Time+PCR+detection+of+Salmonella+spp.,+Listeria+monocytogenes+and+Escherichia+coli+O157+in+milk.">A new platform for Real-Time PCR detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 in milk.</a> Food Microbiol. 26, 615-622 (2009).</li><li>Cui, S., Schroeder, C. M., Zhang, D. Y., Meng, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+sample+preparation+method+for+PCR-based+detection+of+Escherichia+coli+O157:H7+in+ground+beef.">Rapid sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef.</a> J. Appl. Microbiol. 95, 129-134 (2003).</li><li>Ibekwe, A. M., Watt, P. M., Grieve, C. M., Sharma, V. K., Lyons, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+fluorogenic+real-time+PCR+for+detection+and+quantification+of+Escherichia+coli+O157:H7+in+dairy+wastewater+wetlands.">Multiplex fluorogenic real-time PCR for detection and quantification of Escherichia coli O157:H7 in dairy wastewater wetlands.</a> Appl. Environ. Microbiol. 68, 4853-4862 (2002).</li><li>Strachan, N. J., Ogden, I. 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The authors have nothing to disclose.

Şekil 1 &#39;de gösterildiği gibi bir turnusol test etmek için bir bakteri duyarlı DNAzıme RNA bölünme etkinliğinin etki yaptı, üreaz ve manyetik ayırma kullanımı ile mümkün olmaktadır. Bakteriyel tespiti için modifiye test edeceği gösteri rağmen E ile yapılan E. coli bağımlı RNA-yaran DNAzim, 5,19,20 tasarım genellikle herhangi RNA-yaran DNAzıme için uzatılabilir. Yeni hedefler için rasgele dizi havuzlarından yeni RNA-yarma DNAzımler izole et...

Bacterial pathogens are one of the major causes of global morbidity and mortality. Outbreaks from hospital-acquired infections, food-borne pathogens, and bacterial contaminants in the environment pose serious and on-going threats to public health and safety. To prevent these outbreaks, effective tools are needed that permit pathogen detection in a timely fashion under a variety of settings. Simple but still effective tests that are portable and cost-effective are greatly coveted, especially in regions that are susceptible to outbreaks but cannot afford expensive testing facilities.1-3 Although there exists a multitude of methods to detect bacteria, many of them are not suitable as screening or on-site testing tools because they require long test times, expensive instruments and complicated testing procedures. 
Colorimetric tests are particularly attractive for point-of-care or field applications as color changes can be easily detected by the naked eye. The litmus test for pH is simple, quick, and effective. Although it is a very old technology, it is still widely used today because of its simplicity and effectiveness. Surprisingly, this simple test had never been modified to achieve the detection of other analytes before we recently developed an approach of modifying this test for E. coli testing.4
The expanded litmus test for E. coli employs three additional components: an E. coli activated RNA-cleaving DNAzyme (EC1), 5 urease, and magnetic beads. DNAzymes refer to synthetic single-stranded DNA molecules with catalytic activity.6 They can be isolated from random-sequence DNA pools using in vitro selection.7,8 They are highly stable and can be produced cost-effectively using high-efficiency automated DNA synthesis.9 For these reasons, DNAzymes, particularly RNA-cleaving DNAzymes, have been widely examined for biosensing applications.6,10,11 RNA-cleaving DNAzyme sensors have been developed to detect metal ions,12-16 small molecules,17,18 bacterial pathogens5,19-21 and cancer cells.22 Given the great availability of target-induced RNA-cleaving DNAzymes, any assay that utilizes a DNAzyme can be potentially expanded to detect a diverse range of analytes.
Urease is chosen for its ability to hydrolyze urea into ammonia,23,24 resulting in a pH increase. Urease is also highly efficient, stable and amenable for conjugation to other biomolecules. Therefore, we postulated that a conjugate of an RNA-cleavage DNAzyme with urease would allow the use of litmus test for the detection of other targets.5
The action of the RNA-cleaving DNAzyme is relayed to urease-mediated increase of pH through the use of magnetic beads that are immobilized with the DNAzyme-urease conjugate. Because the activity of the DNAzyme under investigation is strictly dependent on E. coli, the presence of this bacterium in the test solution will result in the release of urease from the magnetic beads to the solution, which is then taken and used to hydrolyze urea in a reporter solution that contains a pH-sensitive dye. The final outcome of this procedure is a color change that can be conveniently reported by the dye or pH paper.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="24">
			<td height="24" style="height:24px;width:275px;">Ethylenediaminetetraacetic acid (EDTA)</td>
			<td style="width:183px;">VWR AMRESCO</td>
			<td style="width:163px;">0105</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Hydroxide (NaOH) pellets</td>
			<td>BIO BASIC CANADA INC.</td>
			<td>SB6789</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Tris-base</td>
			<td>VWR AMRESCO</td>
			<td>0497</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Boric acid</td>
			<td>AMRESCO</td>
			<td>0588</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Urea</td>
			<td>VWR AMRESCO</td>
			<td>M123</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">40% acrylamide/bisacrylamide (29:1) solution</td>
			<td>BIO BASIC CANADA INC.</td>
			<td>A0007</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sucrose</td>
			<td>Bioshop Canada inc.</td>
			<td>SUC507</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Bromophenol blue</td>
			<td>Bioshop Canada inc.</td>
			<td>BRO777</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Xylenecyanol FF</td>
			<td>SIGMA-ALDRICH</td>
			<td>X-4126</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10% sodium dodecyl sulfate</td>
			<td>Bioshop Canada inc.</td>
			<td>SDS001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Hydrochloric Acid (HCl)</td>
			<td>CALEDON LABORATORIES LTD</td>
			<td>6026</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Chloride (NaCl)</td>
			<td>Bioshop Canada inc.</td>
			<td>SOD001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Bioshop Canada inc.</td>
			<td>HEP001</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Magnesium Chloride (II) hexahydrate</td>
			<td>VWR AMRESCO</td>
			<td>0288</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Tween 20</td>
			<td>Bioshop Canada inc.</td>
			<td>TW508</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Adenosine Triphospahte (ATP)</td>
			<td>AMRESCO</td>
			<td>0220</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Acetate trihydrate (NaOAc)</td>
			<td>SIGMA-ALDRICH</td>
			<td>S8625</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Ethanol</td>
			<td>Commercial Alcohols</td>
			<td>P016EAAN</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Tetramethyleneethylenediamine (TEMED)</td>
			<td>AMRESCO</td>
			<td>0761</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10% Ammonium persulfate (APS)</td>
			<td>BIO BASIC CANADA INC.</td>
			<td>AB0072</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC)</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>22360</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dimethyl sulfoxide (DMSO)</td>
			<td>CALEDON LABORATORIES</td>
			<td>803540</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Urease</td>
			<td>SIGMA-ALDRICH</td>
			<td>U0251</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">1&#215; Phosphate Buffered Saline (PBS)</td>
			<td>ThermoFisher SCIENTIFIC</td>
			<td>70011-069</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">0.04% Phenol red</td>
			<td>SIGMA-ALDRICH</td>
			<td>P3532</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10&#215;T4 polynucleotide kinase reaction buffer</td>
			<td>Lucigen</td>
			<td>30061-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10&#215; T4 DNA ligase reaction buffer</td>
			<td>Bio Basics Canada</td>
			<td>B1122-B</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">T4 DNA ligase (5 U/uL)</td>
			<td>Thermo Fischer Scientific</td>
			<td>B1122</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Luria Bertani (LB) Broth</td>
			<td>AMRESCO</td>
			<td>J106</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Agar</td>
			<td>AMRESCO</td>
			<td>J637</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">T4 polynucleotide kinase (10 U/uL)</td>
			<td>Lucigen</td>
			<td>30061-1</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">E. coli K12 (MG1655)</td>
			<td>ATCC</td>
			<td>ATCC700926</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Centrifuge</td>
			<td>Beckman Coulter, Inc.</td>
			<td>392187</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Glass plates</td>
			<td>CBS scientific</td>
			<td>ngp-250nr</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">0.75 mm thick spacers</td>
			<td>CBS scientific</td>
			<td>VGS-0725r</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">12-well comb</td>
			<td>CBS scientific</td>
			<td>VGC-7512</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">UV Lamp</td>
			<td>UVP</td>
			<td>95-0017-09</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Spectrophotometer (NanoVue)</td>
			<td>GE Healthcare</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Metal plate</td>
			<td>CBS scientific</td>
			<td>CPA165-250</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Vortex</td>
			<td>VWR International</td>
			<td>58816-123</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Gel electrophoresis apparatus</td>
			<td>CBS scientific</td>
			<td>ASG-250</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Petri dishes</td>
			<td>VWR International</td>
			<td>25384-342</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">100 kDa MWCO centrifugal filters</td>
			<td>EMD Millipore</td>
			<td>UFC510024</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Magnetic Bead (BioMag)</td>
			<td>Bangs Laboratories Inc</td>
			<td>BM568</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Magnetic Seperation Rack</td>
			<td>New England BioLabs</td>
			<td>S1506S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Microfuge tubes</td>
			<td>Sarstedt</td>
			<td>72.69</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Syringe filter (0.22 um)</td>
			<td>VWR International</td>
			<td>28145-501</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">14 mL culture tube</td>
			<td>VWR International</td>
			<td>60818-725</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Cell culture incubator</td>
			<td>Eppendorf Scientific</td>
			<td>M13520000</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Branson Ultrasonic cleaner</td>
			<td>Branson</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Camera (Canon Powershot G11)</td>
			<td>Canon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">50 mL conical tube</td>
			<td>VWR International</td>
			<td>89004-364</td>
			<td></td>
		</tr>
	</tbody>
</table>

colorimetric detection of bacteria using litmus test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

Bakteriyel test edeceği prensibi Şekil 1 &#39;de açıklanmıştır Test üç temel malzeme kullanır. Belirli bir bakteri, üreaz ve manyetik boncuklar ile aktive olan bir RNA klivaj DNAzim. DNAzim ilgi konusu bir bakterinin yüksek düzeyde spesifik tespit elde etmek için moleküler tanıma elemanı olarak kullanılır. Üreaz ve manyetik boncuk DNAzıme RNA klivaj aktivitesinin sinyal transdüksiyonunu elde etmek için kullanılır. Bu üreaz-DNAzim konjugatların?...

Watch this Scientific Journal Video about Colorimetric Detection of Bacteria Using Litmus Test at JoVE.com

Colorimetric Detection of Bacteria Using Litmus Test

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

Turnusol Testi kullanarak Bakterilerin Kolorimetrik Tespiti

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

colorimetric-detection-of-bacteria-using-litmus-test

Research

JoVE Journal

Biology

9.1K Görüntüleme.  McMaster University. We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads. 

Video: Colorimetric Detection of Bacteria Using Litmus Test

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Protein Ubikitinasyon Tespiti

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Biyoloji

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

Biyolojik Tahliller Manyetik Modülasyon Biyoalgılayıcı Sistemi kullanarak hızlı Homojen Algılama

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any two individuals is greater than previously thought3. In contrast, array-based genotyping has failed to identify a significant contribution of common sequence variants to the phenotypic variability of common disease4,5. Taken together, these observations have led to the evolution of the Common Disease / Rare Variant hypothesis suggesting that the majority of the "missing heritability" in common and complex phenotypes is instead due to an individual's personal profile of rare or private DNA variants6-8. However, characterizing how rare variation impacts complex phenotypes requires the analysis of many affected individuals at many genomic loci, and is ideally compared to a similar survey in an unaffected cohort. Despite the sequencing power offered by today's platforms, a population-based survey of many genomic loci and the subsequent computational analysis required remains prohibitive for many investigators.
To address this need, we have developed a pooled sequencing approach1,9 and a novel software package1 for highly accurate rare variant detection from the resulting data. The ability to pool genomes from entire populations of affected individuals and survey the degree of genetic variation at multiple targeted regions in a single sequencing library provides excellent cost and time savings to traditional single-sample sequencing methodology. With a mean sequencing coverage per allele of 25-fold, our custom algorithm, SPLINTER, uses an internal variant calling control strategy to call insertions, deletions and substitutions up to four base pairs in length with high sensitivity and specificity from pools of up to 1 mutant allele in 500 individuals. Here we describe the method for preparing the pooled sequencing library followed by step-by-step instructions on how to use the SPLINTER package for pooled sequencing analysis (<a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a>). We show a comparison between pooled sequencing of 947 individuals, all of whom also underwent genome-wide array, at over 20kb of sequencing per person. Concordance between genotyping of tagged and novel variants called in the pooled sample were excellent. This method can be easily scaled up to any number of genomic loci and any number of individuals. By incorporating the internal positive and negative amplicon controls at ratios that mimic the population under study, the algorithm can be calibrated for optimal performance. This strategy can also be modified for use with hybridization capture or individual-specific barcodes and can be applied to the sequencing of naturally heterogeneous samples, such as tumor DNA.

Pooled DNA sequencing is a fast and cost-effective strategy to detect rare variants associated with complex phenotypes in large cohorts. Here we describe the computational analysis of pooled, next-generation sequencing of 32 cancer-related genes using the SPLINTER software package. This method is scalable, and applicable to any phenotype of interest.

SPLINTER kullanarak Pooled Sıralama gelen Nadir Genomik Variantların Algılama

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic 1-3. One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis 4. Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects 5. For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage 6-8. Recently, several other nematode species have been shown to utilize bacteria to kill insects 9-13, and investigations have begun examining the interactions between the nematodes and bacteria in these systems 9.
We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila 14. Similar methods have been used to investigate other nematode-bacterium associations 9,15-18and the approach therefore is generally applicable.
The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization 14,16,19. Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues 14,16,19-21. This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication 22or grinding 23, which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24. Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18, and is less laborious than other methods, including sonication 22,25-27and individual nematode dissection 28,29.

To study the mutualism between Xenorhabdus bacteria and Steinernema nematodes, methods were developed to monitor bacterial presence and location within nematodes. The experimental approach, which can be applied to other systems, entails engineering bacteria to express the green fluorescent protein and visualizing, using fluorescence microscopy bacteria within the transparent nematode.

Floresan Mikroskobu kullanılarak Nematodlar Bakteriler görselleştirme

 
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Yüksek çözünürlüklü Melt Analizi ile PCR kullanarak hızlı ve verimli Zebra Genotiplemesi

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Üzerinde Bakterilerin Etkilerinin Ölçme<em&gt; C. Elegans</emBir Yumurta Tutma Testi Kullanma&gt; Davranış

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

3&#39;LIFE Test kullanma Yüksek throughput miRNA Hedefleri tespiti

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to differentiate the myoglobin (Mb) of cetaceans from that of seals and other animals. The strip provides rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. Two monoclonal antibodies (mAbs) with reactivity toward the Mb of cetaceans were developed. The amino acid sequences of Mb antigenic reactive regions from various animals were analyzed in order to design two synthetic peptides (a general peptide and a specific peptide) and thereafter raise the mAbs (subclass IgG1). The mAbs were selected from hybridomas screened by indirect ELISA, western blot and dot blot. CGF5H9 was specific to the Mbs of rabbits, dogs, pigs, cows, goats, and cetaceans while it showed weak to no affinity to the Mbs of chickens, tuna and seals. CSF1H13 can bind seals and cetaceans with strong affinity but showed no affinity to other animals. Cetacean samples from four families (Balaenopteridae, Delphinidae, Phocoenidae and Kogiidae) were used in this study, and the results indicated that these two mAbs have broad binding ability to Mbs from different cetaceans. These mAbs were applied on a sandwich-type colloidal gold immunochromatographic test strip. CGF5H9, which recognizes many species, was colloid gold-labeled and used as the detection antibody. CSF1H13, which was coated on the test zone, detected the presence of cetacean and seal Mbs. Muscle samples from tuna, chicken, seal, five species of terrestrial mammals and 15 species of cetaceans were tested in triplicate. All cetacean samples showed positive results and all the other samples showed negative results.

This protocol describes the development of two IgG class monoclonal antibodies (mAbs) strongly reactive to myoglobin of cetaceans. These mAbs are applied on a colloidal gold immunochromatographic test strip based on the sandwich format to differentiate the Mb of cetaceans from seal and other animals.

Deniz Memelileri Miyoglobin Tespiti için bir Kolloidal Altın merkezli İmmunokromatografik Testi Strip Gelişimi

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Hassas immünohistokimyasal sitozin Modifiye Formlar Algılama

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

Detection of Tissue-resident Bacteria in Bladder Biopsies by 16S rRNA Fluorescence In Situ Hybridization

Visualization of the interaction of bacteria with host mucosal surfaces and tissues can provide valuable insight into mechanisms of pathogenesis. While visualization of bacterial pathogens in animal models of infection can rely on bacterial strains engineered to express fluorescent proteins such as GFP, visualization of bacteria within the mucosa of biopsies or tissue obtained from human patients requires an unbiased method. Here, we describe an efficient method for the detection of tissue-associated bacteria in human biopsy sections. This method utilizes fluorescent in situ hybridization (FISH) with a fluorescently labeled universal oligonucleotide probe for 16S rRNA to label tissue-associated bacteria within bladder biopsy sections acquired from patients suffering from recurrent urinary tract infection. Through use of a universal 16S rRNA probe, bacteria can be detected without prior knowledge of species, genera, or biochemical characteristics, such as lipopolysaccharide (LPS), that would be required for detection by immunofluorescence experiments. We describe a complete protocol for 16S rRNA FISH from biopsy fixation to imaging by confocal microscopy. This protocol can be adapted for use in almost any type of tissue and represents a powerful tool for the unbiased visualization of clinically-relevant bacterial-host interactions in patient tissue. Furthermore, using species or genera-specific probes, this protocol can be adapted for the detection of specific bacterial pathogens within patient tissue.

This protocol is for the unbiased detection of tissue-associated bacteria in patient biopsies by 16S rRNA in situ hybridization and confocal microscopy.

Situ Hibridizasyonda 16S rRNA Floresan ile Mesane Biyopsilerinde Doku Dağı Bakterilerin In Salgılanması

Visualization of the interaction of bacteria with host mucosal surfaces and tissues can provide valuable insight into mechanisms of pathogenesis. While ...