This research was supported by grants from the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031944), the Next-Generation Biogreen 21 Program (PJ009524), NRF-2015M3D3A1A01064875 and the KRIBB Research Initiative Program.

1. PnP-gess ile Metagenomic Kütüphane Hazırlama <ol><li> E. bir metagenomic kütüphane inşa E. coli, bir Fosmid vektör ile üreticinin protokolüne 5&#39;e göre bir Fosmid kitaplığı üretimi kiti kullanılarak. </li><li> Kısım metagenomic kütüphanesi bir hücre kaynağıdır, -70 ° C&#39;de, önceki depolama için kütüphane 100 ul. Not: Bu kütüphane stokunun 600 nm (OD 600) arasında bir dalga uzunluğunda ölçülen bir numunenin optik yoğunluğu yaklaşık 100&#39;dür. </li><li> Buz üzerinde hazır metagenomic kütüphane 100 ul çözülme ve 50 ml Luria-Bertani (LB) ve 2 saat boyunca 37 ° C&#39;de kuluçkalandı ve ardından 12.5 ug / ml kloramfenikol içeren 500 ml&#39;lik bir şişe içinde inoküle. </li><li> 4 ° C&#39;de 20 dakika boyunca 1000 x g&#39;de santrifüj ile 50 ml konik bir tüp içinde hücreler hasat edilir. </li><li> Yine, 4 ° C&#39;de 20 dakika boyunca 1000 x g&#39;de 50 ml buz gibi soğuk damıtılmış su (DW) ve santrifüj hızlı pelletini. </li><li> Res% 10 (h / h) gliserol, 50 ul buz soğukluğunda DW pelet uspend. Elektroporasyon için, bu hücre kısım 50 ul kullanın. </li><li> Buz soğukluğundaki bir elektroporasyon küvetine ve elektroporasyona (1.8 kV / cm, 25 mF) karışım elektro uyumlu hücre 50 ul pGESS (E135K) 5 DNA (100ng) karışımı yerleştirin. </li><li> Hızla katabolik baskı (SOC) orta süper optimum suyu 1 ml ekleyin ve hafifçe hücreleri tekrar süspansiyon. </li><li> bir pipet kullanılarak 14 ml yuvarlak tabanlı bir tüpe hücreleri aktarın ve 1 saat 37 ° C&#39;de inkübe edin. </li><li> 12.5 ug / ml kloramfenikol ve 50 ug / ml ampisilin ihtiva eden LB 20 x 20 cm kare plaka elde edilen hücreler 500 ul yayıldı. 12 saat boyunca 30 ° C&#39;de inkübe plakası. </li><li> Bir hücre kazıyıcı kullanarak koloniler kazıyın ve buz gibi soğuk hücre depolama ortamı kullanarak bir 50 ml konik tüp içine hücreleri toplamak. </li><li> 4 ° C&#39;de 20 dakika boyunca 1000 x g&#39;de santrifüjleyin. 10 ml buz gibi soğuk hücre depolama ortamı pelletiniBir OD 100 600 ulaşmak için. </li><li> Kısım -70 ° C&#39;de depolama için hücreler 20 ul. </li></ol> 2. Metagenomic Kitaplığı&#39;ndan Yanlış Pozitif Çıkarma <ol><li> buz üzerinde metagenomic DNA fosmid ve pGESS (E135K) içeren (Kademe 1.13 den) stok metagenomic kütüphane hücreleri çözülme. </li><li> 14 ml&#39;lik yuvarlak tabanlı bir tüp içinde 50 ug / ml ampisilin ve 12.5 ug / ml kloramfenikol içeren 2 ml LB içinde hücre 10 ul inoküle. 4 saat 200 rpm&#39;de çalkalanarak 37 ° C&#39;de inkübe edin. </li><li> Bu arada, FACS makine açmak ve varsayılan FACS yazılımını açın. aşağıdaki ayarları kullanın: meme ucu çapı, 70 um; İleri Yayılım Alanı (FSC-A) duyarlılık, 300 V-logaritmik büyütme; Yan Dağılım Alanı (SSC-A) duyarlılık, 350 V-logaritmik büyütme; Floresein izotiosiyanat Konum (FITC-A) hassasiyeti, 450 V logaritmik amplifikasyonu; eşik parametresi, FSC değeri 5. Not: Normal ayarlar verilmiştirMalzemelerin Tablo belirtilen FACS cihaz için. Diğer FACS cihazlar için ayarları yapın. </li><li> 1 ml PBS ihtiva eden bir 5-ml&#39;lik yuvarlak tabanlı bir tüpe örnek 5 ul ekleyerek Aşama 2.2 metagenomic kütüphanesi hücreleri seyreltilir. </li><li> FACS enstrüman yükleme noktası üzerine seyreltilmiş kütüphane örnek yerleştirin ve FACS yazılım kazanılması Dashboard&#39;daki Yükle düğmesini tıklayın. </li><li> tıklayıp gösterge paneli üzerinde Debisi düğmelerini kontrol ederek / sn 1,500 olaylar - 1000 olay hızını ayarlayın. </li><li> Günlük araç çubuğunda Dot Plot butonuna tıklayarak bir küresel çalışma sayfasında SSC-A dağılım arsa ölçekli vs günlüğü oluşturmak FSC-A ölçekli. araç çubuğundaki Poligon Kapısı düğmesini kullanarak tekli olayları (bakteriyel nüfus) kapsayacak şekilde dağılım kapısı R1 ayarlayın. </li><li> Günlük Histogram butonuna basarak çalışma sayfasındaki FITC-A ölçekli vs hücre sayımı ile bir histogram çizilir. Daha sonra, Cytome FITC gerilimini ayarlamakÇan-şekilli dağıtım pik FITC-A 10 daha az 2 olacak şekilde Müfettiş Kontrol penceresinden ter Ayarlar sekmesi. </li><li> Vs bir günlük ölçekli FSC-A oluşturun. Günlük FITC Küresel bir çalışma sayfasındaki Dot Plot butonuna tıklayarak arsa. kapı çan zirve etrafında dağılımının merkezine (toplam% 10 hücrelerinden +% 5 ve% -5 hücreleri arasında yer alır, böylece araç çubuğunda Poligon Kapısı düğmesini kullanarak arsa üzerinde bir sıralama kapısı R2 Set şeklinde eğri). </li><li> 1.2 mi LB FACS aletin çıkışında 50 ug / ml ampisilin ve 12.5 ug / ml kloramfenikol içeren ve R1 ve R2, kapılar hem tatmin sıralamak 10 6 hücre ihtiva eden bir toplama tüpü yerleştirin. </li><li> toplama tüpü, kap, ve yavaşça vorteks çıkarın. </li></ol> 3. Metagenomic Enzim Eleme <ol><li> 600 0,5 ulaşana OD kadar 200 rpm&#39;de çalkalanarak 37 ° C&#39;de (Kademe 2.11) kriteri hücreleri inkübe edin. </li><li> 1 ul hücre içi fosmid kopya sayısını yükseltmek için indüksiyon kopyalama çözümüdür ekleyin. 250 rpm&#39;de çalkalanarak 37 ° C&#39;de ek bir 3 saat inkübe hücreleri. </li><li> Nihai konsantrasyonda 14 ml&#39;lik bir yuvarlak tabanlı tüp içine 0.5 kültürlenmiş hücrelerin üzerine ilave edildi ve uygun bir alt tabaka (p-nitrofenil asetat, p-nitrofenil-β-D-selobiosidi ya da fenil fosfat) ekleyin, sıralama için hücrelerin hazırlanması için 100 uM. <ol><li> Kontrol grubu için, 14 ml&#39;lik yuvarlak dipli bir tüp içine Aşama 3.2 kültürlenmiş hücreler 0.5 ml ekleyin. Adım 3.3 substrat hacmi olarak aynı hacimde PBS ekleyin. </li></ol></li><li> 3 saat 200 rpm&#39;de çalkalanarak 37 ° C&#39;de iki örnek inkübe edin. </li><li> Bu arada, Adım 2.3 ile aynı konfigürasyona sahip FACS makineyi hazırlamak. </li><li> sırasıyla 1 ml PBS ihtiva eden iki adet 5 ml&#39;lik yuvarlak tabanlı tüpe (sıralama) hücreleri ve kontrol hücreleri 5 ul ekle. </li><li> Kontrol hücreleri t üzerine içeren tüp yerleştirinO FACS limanını yükleme ve FACS yazılım kazanılması Dashboard&#39;daki Yükle düğmesini tıklayın. Ön paneldeki Debisi düğmelerini kontrol ederek / sn 3000 olayları - 1000 olay Hızı ayarlayın. </li><li> Günlük yazılımının küresel çalışma sayfasındaki Dot Plot butonuna tıklayarak ve tekli olayları kapsayacak şekilde (bakteriyel nüfus) dağılım kapısı R1 ayarlamak üzerinde Poligon Kapısı düğmesini kullanarak SSC-A dağılım arsa ölçekli vs oluşturma günlük FSC-A ölçekli araç çubuğu. </li><li> Günlük araç çubuğunda Poligon Kapısı düğmesini kullanarak arsa üzerinde bir sıralama kapısı R2 çalışma sayfasında Nokta Arsa düğmesini tıklayarak ve set tarafından FITC bir dağılım arsa ölçekli vs oluşturma günlük FSC-A ölçekli böylece daha az% 0.1 negatif hücrelerin R2 kapısı içinde tespit edilir. </li><li> sıralama örnek tüpü ile kontrol örneği tüpü değiştirin ve 1000 olay hızını ayarlamak - gösterge paneli üzerinde Debisi düğmelerini kontrol ederek / sn 3,000 olaylar. </li><li> 0.5 içeren bir toplama tüpü yerleştirinml FACS enstrüman çıkışında LB hem R1 ve R2 kapıları tatmin sıralamak 10 4 hücreleri. Not: sıralama kriteri R2 kapısı FITC-A&#39;nın üst% 0.1 ile% 5 arasında değişebilir. Bu protokolde, üst% 1 hücreleri pozitif olarak toplanmıştır. </li><li> toplama tüpü, kap, ve yavaşça vorteks çıkarın. </li><li> LB, 50 ug / ml ampisilin, 12.5 ug / ml kloramfenikol içeren agar plakaları, iki (90 mm&#39;lik Petri kaplara) üzerine 0.5 mL toplanan numuneleri yayıldı ve bir gece boyunca 37 ° C&#39;de inkübe plakası. </li><li> Gerekirse, adımları 3,1-3,14 yineleyerek FITC bir zenginleşme için sıralama ek mermi gerçekleştirin. </li></ol> 4. Hit Seçme ve Enzim Aktivitesi Onay <ol><li> Flow sitometri analizi <ol><li> Adım 3.14 inkübe plakadan, bir LED aydınlatıcı 488 nm dalga boyundaki gözetiminde bir koloni seçiciyi kullanarak hiçbir floresan gösteren bir koloni seçin. </li><li> 14 mi ro seçilen koloni inoküleund altlı 2 mi LB 600 0.5 ulaşana onun OD kadar 200 rpm&#39;de çalkalanarak 37 ° C&#39;de 50 ug / ml ampisilin ve 12.5 ug / ml kloramfenikol içeren içeren tüp. </li><li> 2 ul hücre içi fosmid kopya sayısını yükseltmek için indüksiyon kopyalama çözümüdür ekleyin. 250 rpm&#39;de çalkalanarak 37 ° C&#39;de ek bir 3 saat inkübe hücreleri. </li><li> 14 ml&#39;lik yuvarlak dipli bir tüp hazırlanır ve kültürlenmiş hücrelerde (Aşama 4.1.3) 1 ml ekleyin. 100 uM&#39;lik bir son konsantrasyonda uygun bir alt tabaka (p-nitrofenil asetat, s -nitrophenyl- β-D-selobiosidi ya da fenil fosfat) ekleyin. <ol><li> Bir kontrol için, bir 14 ml yuvarlak tabanlı tüp içine Adım 4.1.3 den 1 ml kültür hücreleri ekleyin ve Adım 4.1.4 substrat hacmi olarak aynı hacimde PBS ekleyin. </li></ol></li><li> 3 saat 200 rpm&#39;de çalkalanarak 37 ° C&#39;de iki örnek inkübe edin. </li><li> Bu arada, Adım 2.3 ile aynı konfigürasyona sahip FACS makinesi hazırlamak. </li><li> sırasıyla 1 ml PBS ihtiva eden 5 mL&#39;lik yuvarlak dipli bir tüplere kontrolü ve alt-tabaka ile muamele edilmiş hücreler içinde 5 ul ekle. </li><li> FACS cihazın yükleme noktası üzerinde tüp içeren kontrol hücrelerini koyun ve FACS yazılım kazanılması Dashboard&#39;daki Yükle düğmesini tıklayın. Ön paneldeki Debisi düğmelerini kullanarak 3000 olayları / sn - 1000 olay hızını ayarlayın. </li><li> FITC-A ölçmek için Edinme butonuna tıklayın. </li><li> Alt tabaka tedavi numune tüpü ile kontrol numunesi tüpünü değiştirin. Ön paneldeki Debisi düğmelerini kullanarak 3000 olayları / sn - 1000 olay hızını ayarlayın. </li><li> FITC-A ölçmek için Edinme butonuna tıklayın. </li><li> Günlük FITC-A ölçekli vs hücre sayımı bir histogram çizerek hücrelerin iki grup floresan karşılaştırın. </li></ol></li><li> Diğer deneyler, enzim etkinliğini doğrulamak için <ol><li> Seçilen enzim adayların daha tanımlaması için, standart ekstraksiyon pro kullanarak fosmid DNA ayıklamakTicari ekstraksiyon kitleri prosedürleri ve nükleotid sekansı analiz veya in vitro enzim aktivitesi 6,7 test edin. </li></ol></li></ol>

<ol>
	<li>Eggeling, L., Bott, M., Marienhagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+screening+methods-biosensors.">Novel screening methods-biosensors.</a> Curr. Opin. Biotech. 35, 30-36 (2015).</li><li>Uchiyama, T., Abe, T., Ikemura, T., Watanabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate-induced+gene-expression+screening+of+environmental+metagenome+libraries+for+isolation+of+catabolic+genes.">Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes.</a> Nat. Biotechnol. 23 (1), 88-93 (2005).</li><li>Van Sint Fiet, S., van Beilen, J. B., Witholt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+biocatalysts+for+chemical+synthesis.">Selection of biocatalysts for chemical synthesis.</a> Proc. Natl. Acad. Sci. U. S. A. 103 (6), 1693-1698 (2006).</li><li>Binder, 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=A+high-throughput+approach+to+identify+genomic+variants+of+bacterial+metabolite+producers+at+the+single-cell+level.">A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level.</a> Genome Biol. 13 (5), R40 (2012).</li><li>Choi, 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=Toward+a+generalized+and+High-throughput+Enzyme+Screening+System+Based+on+Artificial+Genetic+Circuits.">Toward a generalized and High-throughput Enzyme Screening System Based on Artificial Genetic Circuits.</a> ACS Synthetic Biology. 3 (3), 163-171 (2014).</li><li>Lee, D. H., 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=A+novel+psychrophilic+alkaline+phosphatase+from+the+metagenome+of+tidal+flat+sediments.">A novel psychrophilic alkaline phosphatase from the metagenome of tidal flat sediments.</a> BMC Biotechnology. 15 (1), (2015).</li><li>Warnecke, F., 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=Metagenomic+and+functional+analysis+of+hindgut+microbiota+of+a+wood-feeding+higher+termite.">Metagenomic and functional analysis of hindgut microbiota of a wood-feeding higher termite.</a> Nature. 450 (7169), 560-565 (2007).</li><li>Kim, U. J., Shizuya, H., de Jong, P. J., Birren, B., Simon, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+propagation+of+cosmid+sized+human+DNA+inserts+in+an+F+factor+based+vector.">Stable propagation of cosmid sized human DNA inserts in an F factor based vector.</a> Nucleic Acids Research. 20 (5), 1083-1085 (1992).</li><li>Jemli, S., Ayadi-Zouari, D., Hlima, H. B., Bejar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocatalysts:+application+and+engineering+for+industrial+purposes.">Biocatalysts: application and engineering for industrial purposes.</a> Crc. Cr. Rev. Biotechn. 36 (2), 246-258 (2014).</li><li>Lorenz, P., Eck, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metagenomics+and+industrial+applications.">Metagenomics and industrial applications.</a> Nat. Rev. Microbiol. 3 (6), 510-516 (2005).</li><li>Uchiyama, T., Miyazaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Product-induced+gene+expression,+a+product+responsive+reporter+assay+used+to+screen+metagenomic+libraries+for+enzyme+encoding+genes.">Product-induced gene expression, a product responsive reporter assay used to screen metagenomic libraries for enzyme encoding genes.</a> Applied and environmental microbiology. 76 (21), 7029-7035 (2010).</li><li>Mohn, W. W., Garmendia, J., Galvao, T. C., De Lorenzo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveying+bio-transformations+with+&#224;+la+carte+genetic+traps:+translating+dehydrochlorination+of+lindane+(gamma-hexachlorocyclohexane)+into+lacZ-based+phenotypes.">Surveying bio-transformations with &#224; la carte genetic traps: translating dehydrochlorination of lindane (gamma-hexachlorocyclohexane) into lacZ-based phenotypes.</a> Environmental microbiology. 8 (3), 546-555 (2006).</li><li>Tang, S. Y., Cirino, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+application+of+a+mevalonate-responsive+regulatory+protein.">Design and application of a mevalonate-responsive regulatory protein.</a> Angew Chem. Int. Ed. 50 (5), 1084-1086 (2011).</li><li>Jha, R. K., Kern, T. L., Fox, D. T., Strauss, C. E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+an+Acinetobacter+regulon+for+biosensing+and+high-throughput+enzyme+screening+in+E.+coli+via+flow+cytometry.">Engineering an Acinetobacter regulon for biosensing and high-throughput enzyme screening in E. coli via flow cytometry.</a> Nucleic Acids Res. 42 (12), 8150-8160 (2014).</li></ol>

The authors have nothing to disclose.

Biyokatalizörler üretim verimliliğinin arttırılması en doğal enzim kaynaklarından biri olarak kabul edilir sanayi 9 ve metagenome bazlı biyo-kimyasal başarısı için bir anahtardır. Bu anlamda, bu genetik kaynakları çoğunluğu 10 keşfedilmemiş olan metagenome elde edilen yeni enzimler tarama için gereklidir. Çeşitli tarama yöntemleri doğrudan kopyalama etkinleştiricileri 11, 12 kullanılarak enzim ürünlerinin tespit geliştirilmiştir, fakat bu teknikler, belirli...

Son yıllarda geliştirilmiş olan, yüksek verimli tek hücreli deney tekniği yeni bir enzim işlevsel etkinlikler 1&#39;e göre büyük ölçekli genetik kütüphaneden taranabilir sağlar. tek hücre düzeyinde, transkripsiyon düzenleyici proteinler, bir hedef enzim etkinliğinin bir sonucu olarak üretilen küçük molekülleri algılayarak raportör gen ekspresyonunu başlatmak için kullanılır. Buna bir yaklaşım, alt-tabaka bir raportör protein 2 ekspresyonunu indükler formül (SIGEX) yöntemini tarama alt-tabaka ile indüklenen gen ekspresyonunu kullanarak, Ralstonia eutropha E2 bir fenol-bozucu operonun izole çıkıyor. Pseudomonas putida NhaR benzaldehid dehidrojenazı 3 seçmek için kullanılır, ve Corynebacterium glutamicum LysG çeşitli mutant kütüphaneler 4 yeni bir L-Lizin üreten soyun yüksek verimli tarama kullanılmıştır. Daha önce, bir genetik enzim screening sistemi (GESS) bir genel uygulanabilir bir tarama platformu 5 olarak önerilmiştir. Bu sistem P., DmpR fenol-tanıma dimetilfenol regülatör kullanır putida. DmpR (E135K) ve DmpR bir mutant, S. nitrofenolu (PNP) saptanması için gess (PNP-GESS) &#39;de kullanılabilmektedir. Fenolik bileşikleri üreten hedef enzimlerin varlığında, GESS E. coli hücreleri bir floresan-aktive edilmiş hücre ayırıcı (FACS) kullanılarak, tek hücrelerin hızlı izole edebilecek, bir floresan sinyali verir. Ancak metagenomic enzimin ekspresyonu geleneksel rekombinant enzimlerin daha zayıf görünmektedir; Bu nedenle, GESS uygun çalışma durumunda 5 ile birlikte ribozomal bağlanma yeri (RBS) ve terminatör dizileri ve kombinasyonunu araştırırken en fazla hassasiyet fenolik bileşikleri tespit etmek için tasarlanmıştır. Gess temel avantajlarından biri bu tek yöntem teorik olarak ov taranmasını izin vermesidirBRENDA veritabanında enzimlerin er 200&#39;den fazla farklı türleri (Tablo 1, http: // www.brenda-enzymes.info, 2013,7) sadece farklı substratlar kullanılarak. O selülaz lipaz gördü ve metil paration hidrolaz (MPH) p-nitrofenil butirat, p-nitrofenil-cellotrioside ve metil paration, sırasıyla, 5, uygun substratlann PNP-gess kullanılarak tespit edilebilir. Son zamanlarda, bu PnP-gess kullanılarak tanımlanan yeni enzim biri bir alkalin fosfataz (AP), soğuğa uyarlanmış deniz metagenomes 6&#39;da bulunan ilk termal olarak AP olduğu kanıtlanmıştır. Burada tarama işleminin detayları PnP-GESS bir metagenomic kütüphane 5,6 arasında yeni aday enzimleri belirlenmesi hızlı enzimleri, lipaz, selülaz ve alkalin fosfataz, üç farklı türde aktivitelerini tespit -ve sunulmuştur. süreçler metagenome PnP-gess ile önişleme ve bir fl işletim dahilsitometri sıralayıcı ow. Elde edilen isabetleri fazla tanımlama için dizilenmiştir gerekir ise, bu protokol akış sitometrisi kullanılarak enzim aktivitesi onay işlemine prosedürünü kapsamaktadır.

<table><tbody><tr><td>CopyControl</td><td>Epicentre</td><td>CCFOS110</td><td>Fosmid library production kit&amp;nbsp;</td></tr><tr><td>CopyControl Induction Solution</td><td>Epicentre</td><td>CCIS125</td><td>Fosmid copy induction solution</td></tr><tr><td>EPI300</td><td>Epicentre</td><td>EC300110</td><td>Electrocompetent cell</td></tr><tr><td>pCC1FOS</td><td>Epicentre</td><td>CCFOS110</td><td>Fosmid vector</td></tr><tr><td>Gene Pulser Mxcell</td><td>Bio-Rad</td><td></td><td>Electroporation cuvette and electroporate system</td></tr><tr><td>FACSAria III</td><td>Becton Dickinson</td><td></td><td>Flow Cytometry (FACS machine)</td></tr><tr><td>AZ100M</td><td>Nikon</td><td></td><td>Microscope&amp;nbsp;</td></tr><tr><td>UltraSlim&amp;nbsp;</td><td>Maestrogen</td><td></td><td>LED illuminator</td></tr><tr><td>50-ml conical tube</td><td>BD Falcon</td><td></td><td></td></tr><tr><td>14-ml round-bottom tube&amp;nbsp;</td><td>BD Falcon</td><td></td><td></td></tr><tr><td>5-ml round-bottom tube</td><td>BD Falcon</td><td></td><td></td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl phosphate</td><td>Sigma-Aldrich</td><td>N7653</td><td>Substrate</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl &amp;beta;-D-cellobioside</td><td>Sigma-Aldrich</td><td>N5759</td><td>Substrate</td></tr><tr><td>&lt;em&gt;p&lt;/em&gt;-nitrophenyl butylate</td><td>Sigma-Aldrich</td><td>N9876&amp;nbsp;</td><td>Substrate</td></tr><tr><td>Luria-Bertani (LB)</td><td>BD Difco</td><td>244620</td><td>Tryptone 10 g/L, Yeast extract 5 g/L, Sodium Chloride 10 g/L</td></tr><tr><td>Super Optimal broth (SOB)</td><td>BD Difco</td><td>244310</td><td>Tryptone 20 g/L, Yeast extract 5 g/L, Sodium Chloride 0.5 g/L, Magnesium Sulfate 2.4 g/L, Potassium Chloride 186 mg/L</td></tr><tr><td>Super Optimal broth with Catabolite repression (SOC)</td><td></td><td>SOB, 0.4 % glucose</td><td></td></tr><tr><td>2x Yeast Extract Tryptone (2xYT)</td><td>BD Difco</td><td>244020</td><td>Pancreatic digest of Casein 16 g/L, Yeast extract 10 g/L, Yeast extract 5 g/L</td></tr><tr><td>Cell storage media</td><td></td><td></td><td>2x YT broth, 15% Glycerol, 2% Glucose</td></tr><tr><td>pGESS(E135K)</td><td></td><td></td><td>A DNA vector containing dmpR, egfp genes with their appropriate promoters, RBS, and terminator.&lt;br/&gt; See the reference 5 in the manuscript for more details.</td></tr><tr><td>Chloramphenicol</td><td>Sigma</td><td>C0378</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A9518</td><td></td></tr><tr><td>BD FACSDiva</td><td>Becton Dickinson</td><td></td><td>Flow Cytometry Software Version 7.0</td></tr><tr><td>PBS</td><td>Gibco</td><td>70011-044</td><td>0.8% NaCl, 0.02% KCl, 0.0144% Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.024% KH&lt;sub&gt;2&lt;/sub&gt;OP&lt;sub&gt;4, &lt;/sub&gt;pH 7.4</td></tr></tbody></table>

multi-enzyme screening using a high-throughput genetic enzyme screening system

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

Üç fenolik yüzeyler önerilen protokol takip ederek Taean&#39;ın, Güney Kore okyanus gelgit düz çökellerin bir metagenome kütüphanesinden yeni metagenomic enzimleri belirlemek için incelenmiştir. Kütüphane yapımı için ortalama 30-40 kb metagenome dizileri E. dayanmaktadır fosmids içine yerleştirildi E. coli K faktörü replikon ve bir hücrenin tek bir kopya olarak sunulmuştur. Fosmids yaygın nedeniyle sabit bir yayılma 8 karmaşık gen...

Watch this Scientific Journal Video about Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System at JoVE.com

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

Çok enzim Yüksek verim Genetik Enzim Eleme Sisteminin Kullanılması Eleme

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

multi-enzyme-screening-using-high-throughput-genetic-enzyme-screening

Research

JoVE Journal

Biology

8.7K Görüntüleme.  Korea Research Institute of Bioscience and Biotechnology. This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, and phenyl phosphate). 

Video: Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

Yeni çözünmez Kromojenik altyapı deneyiyle kitleri kullanılarak karbonhidrat parçalayıcı Enzimlerin yüksek verimli tarama

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Biyokimya

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Kültür Hücreler Kanser ile ilişkili Sinyal İletimi sorgulanması için bir Multiplexed Lusiferaz tabanlı Tarama Platformu

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Tıp

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

Yüksek Verimli Ekran Metagenomic Kütüphaneler selülaz Aktivitesi Biomining

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

Biyoloji

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

Yüksek verim Maya Plazmid overekspresyonu Ekran

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Mantar Endoglucanase Faaliyet Yüksek Verimli Tarama Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

Bir ev yapımı Çift kızdırma Lusıferaz Testi kullanarak yüksek verim Fonksiyonel Taraması

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Post-transkripsiyonel ayarlı genlerin kimyasal Modülatörler için yüksek verimli tarama

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Yüksek Verimli Tarama kullanma Kinaz-substrat Çiftlerinin Kimlik


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

Genetic Screens

Genetic screens are critical tools for defining gene function and understanding gene interactions. Screens typically involve mutating genes and then assessing the affected organisms for phenotypes of interest. The process can be &#8220;forward&#8221;, where mutations are generated randomly to identify unknown genes responsible for the phenotypes, or it can be &#8220;reverse&#8221;, where specific genes are targeted for mutation to observe what phenotypes are produced.Here, JoVE reviews various types of genetic screens, including those that depend on either loss-of-function or gain-of-function mutations, which respectively decrease or increase the activity of genes. We then explore general protocols for forward and reverse screens in a popular model organism, the nematode worm. Finally, we highlight how screens are applied in research today, for example to better understand gene interactions that may contribute to neurodegenerative diseases.

Forward and Reverse Genetic Screens

Genetic screens are critical tools for defining gene function and understanding gene interactions. Screens typically involve mutating genes and then ...

Education

JoVE Science Education

İleri Biyoloji

Genetik

Insoluble Chromogenic Biomass-Based Enzyme Screening: An Assay to Evaluate the Degradation Activity of Plant Biomass-Degrading Enzymes

Source: Sch&#252;ckel, J., et al. <a href="https://www.jove.com/v/54286">High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits.</a> J. Vis. Exp. (2016).
This video demonstrates an assay to screen enzymes effective in degrading complex polysaccharides in plant biomass using insoluble chromogenic biomass or ICB. The enzymes hydrolyze ICB to release soluble dye-linked oligosaccharides, and the enzyme activity is assessed by colorimetric analysis of the reaction product.

Çözünmeyen Kromojenik Biyokütle Bazlı Enzim Taraması: Bitki Biyokütlesini Parçalayan Enzimlerin Bozunma Aktivitesini Değerlendirmek İçin Bir Test

Source: Sch&#252;ckel, J., et al. High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits. J. ...