Biz Ron Davis, Adam Deutschbauer, ve tüm HIP HOP laboratuar Toronto Üniversitesi&#39;nde tartışmalar ve öneriler için teşekkür ederim. CN, Ulusal İnsan Genomu Araştırma Enstitüsü (Grant Numarası HG000205), RO1 HG003317 CIHR MOP-84.305, ve Kanada Kanser Derneği (020.380) hibe ile desteklenmektedir. JO Stanford Genom Eğitim Programı (Hibe Numarası T32 HG00044 Ulusal İnsan Genomu Araştırma Enstitüsü) ve Ulusal Sağlık Enstitüleri (Hibe Numarası P01 GH000205) tarafından desteklenmiştir. GG, Kanadalı Dr Yenilik Vakfı hibe tarafından kısmen desteklenen NHGRI RO1 HG003317 ve Kanada Kanser Derneği, Grant # 020.380, TD ve Donnelly Sıralama Merkezi tarafından desteklenmektedir. Brenda Andrews ve Jack Greenblatt. AMS, Toronto açın Bursu bir Üniversitesi tarafından desteklenmektedir.

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Çıkar çatışması ilan etti.

Burada, biz Etiketli mutant koleksiyonları oluşturmak için farklı mikroorganizmaların barkodlu mutant koleksiyonları mevcut koleksiyonları geniş bir yelpazede kolayca adapte edilebilir, mütevazı bir değişiklik ile bir protokol sıraladı. Patojenik maya C etiketli transposon mutagenez bir protokol bildirdin Biz vurgulamak albicans, çok benzer bir protokol, tek hücreli mantarlar geniş bir yelpazede adapte olabilir . Modifiye Bu protokol, bakteri 13 iyi çalışıyor ve şu anda ek fungal ve bakteriyel genomların bir dizi koleksiyonları yapım aşamasındadır . Şu anda, bu testte, gen-küçük molekül etkileşimleri için tek kapsamlı, genom tarafsız ekran sağlar. Testinin özellikle ikna edici bir özelliği gen ya da küçük molekül hiçbir ön bilgi gerekli olmasıdır. Bu testlerin kapsam ve gücüne rağmen, diğer laboratuarlara aktarılabilirliği sonuçların analizi için başlangıç ​​sermayesi yatırım ve bilişim araçları ile biraz engellemiştir. Analizi için güçlü araçlar ile birlikte bir sonraki nesil dizisi okuma kabulü ile, onların evlat edinme artmasını bekliyoruz.

<table border="1"><tbody><tr><td> Antibiyotikler </td><td> Satıcı ve katalog numaraları </td></tr><tr><td> Carbenicillin </td><td> Sigma, part # C1613 </td></tr><tr><td> Kanamisin </td><td> Sigma, part # K1876 </td></tr><tr><td> spectinomycin </td><td> Sigma, part # S0692 </td></tr><tr><td> Kloramfenikol </td><td> Sigma, part # C0378 </td></tr><tr><td> DNA Temizlik-up ve konsantrasyon kitleri </td><td></td></tr><tr><td> QIAprep Spin Miniprep Kiti </td><td> Qiagen, part # 27.106 </td></tr><tr><td> HiSpeed ​​Plazmid Maxi Seti </td><td> Qiagen, part # 12.663 </td></tr><tr><td> QIAquick PCR Arıtma Kiti </td><td> Qiagen, parçası # 28106 </td></tr><tr><td> QIAquick Jel Ekstraksiyon Seti </td><td> Qiagen, part # 28.704 </td></tr><tr><td> PCR ve elektroforez reaktifler </td><td></td></tr><tr><td> Taq DNA Polimeraz (Mg-serbest) Tampon </td><td> New England Biolabs, part # M0320L </td></tr><tr><td> Deoksinükleotid Çözüm Mix </td><td> New England Biolabs, part # N0447L </td></tr><tr><td> 25 mM MgCl 2 </td><td> Sigma, part # 63.036 </td></tr><tr><td> Agaroz yükleme, boya, ve nükleik asit jel elektroforezi için uygun leke </td><td> Çeşitli </td></tr><tr><td> 10X TAE tampon </td><td> Sigma, part # T8280 </td></tr><tr><td> 1 Kb Plus DNA Ladder </td><td> Invitrogen, parçası # 10787026 </td></tr><tr><td> YPD suyu </td><td></td></tr><tr><td> 10 g maya özü </td><td> Sigma, part # Y1625 </td></tr><tr><td> 20 g Bacto pepton </td><td> BD Biosciences, part # 211.677 </td></tr><tr><td> 20 g dekstroz </td><td> Sigma, part # D9434 </td></tr><tr><td> Laboratuvar </td><td></td></tr><tr><td> Çok kanallı pipetler (1000, 200, ve 20 mcL) </td><td> Çeşitli </td></tr><tr><td> Tek kullanımlık pipetleme rezervuarları </td><td> Çeşitli </td></tr><tr><td> 15 ve 50 ml santrifüj tüplerine </td><td> Çeşitli </td></tr><tr><td> 96 - ve 384-Eh Derin Şey Tabaklar </td><td> Axygen Bilimsel parçası # P-2ml-SQ-CS &amp; P-384240SQCS </td></tr><tr><td> 96-ve 384-iyi PCR plakaları ve mühür filmi </td><td> Çeşitli </td></tr><tr><td> Plaka silindir, conta çok iyi plakaları için </td><td> Sigma, part # R1275 </td></tr><tr><td> 30 ° C ve 37 ° C plakalar üzerinde bakteri ve maya büyüyen ve tüpler sallayarak inkübatörler </td><td> Çeşitli </td></tr><tr><td> In vitro transpozon mutagenez </td><td></td></tr><tr><td> EZ-Tn5 Transposase </td><td> Epicentre Biyoteknoloji, part # TNP92110 </td></tr><tr><td> Yüksek verim dönüşüm </td><td></td></tr><tr><td> Seqprep 96 HT Plazmid Hazırlık Seti </td><td> Kenar Biosystems, part # 84.359 </td></tr><tr><td> polietilen glikol, moleküler ağırlığı 3350 </td><td> Çeşitli </td></tr><tr><td> Lityum asetat </td><td> Sigma, part # 517.992 </td></tr><tr><td> Steril, tabak 6-iyi </td><td> Corning, part # 3335 </td></tr><tr><td> 50 mg / ml üridin </td><td> Sigma, part # U3750 </td></tr><tr><td> 100X Tris-EDTA tampon çözelti </td><td> Sigma, part # T9285 </td></tr><tr><td> 1x TE/0.1M LiOAc </td><td> Çeşitli </td></tr><tr><td> somon testis DNA </td><td> Sigma, part # 1626 </td></tr><tr><td> Barkodlu koleksiyonları Büyüme </td><td></td></tr><tr><td> 48 kuyucuğu; plakalar kültürlerin büyüyen eğer </td><td> Greiner, part # M9437 </td></tr><tr><td> Yapışkan levha contaları </td><td> ABgene, kısmen # AB-0580 </td></tr><tr><td> 200 mL kültür matara </td><td> Çeşitli </td></tr><tr><td> Absorbans yetenekli Spektrofotometre </td><td> Çeşitli </td></tr><tr><td> 250 ml&#39;lik şişeler veya sallayarak spektrofotometre ısı kontrollü çalkalayıcı </td><td> Çeşitli </td></tr><tr><td> Güvenli-Lock Mikrosantrifüj Tüp, 2 mL </td><td> Eppendorf, kısmen # 0030 120.094 </td></tr><tr><td> Hibridizasyon ekipman </td><td></td></tr><tr><td> Hibridizasyon Fırını 640 </td><td> Affymetrix, part # 800.138 </td></tr><tr><td> GeneChip akışkan İstasyonu 450 </td><td> Affymetrix, parça # 00-0079 </td></tr><tr><td> GeneArray Tarayıcı 3000 </td><td> Affymetrix, parça # 00-0212 </td></tr><tr><td> Kayan kabinle birlikte Kaynar su banyosu </td><td> Çeşitli </td></tr><tr><td> Hibridizasyon sarf </td><td></td></tr><tr><td> Genflex Etiket 16K Dizi v2 </td><td> Affymetrix, part # 511.331 </td></tr><tr><td> Denhardt Çözüm 50X konsantre </td><td> Sigma, part # D2532 </td></tr><tr><td> Streptavidin, Ar-fikoeritrin konjuge (SAPE) </td><td> Invitrogen, part # S866 </td></tr><tr><td> Güvenli-Lock Mikrosantrifüj Tüp, 0,5 mL </td><td> Eppendorf, kısmen # 0030 123.301 </td></tr><tr><td> Ufacık Tough-Spotlar </td><td> Çeşitlendirilmiş Biotech, part # LTTM-1000 </td></tr><tr><td> 0.5 M EDTA </td><td> BioRad, part # 161-0729 </td></tr><tr><td> 10% Tween </td><td> Sigma, part # T2700 </td></tr><tr><td> MES serbest asit monohidrat </td><td> Sigma, part # M5287 </td></tr><tr><td> MES sodyum tuzu </td><td> Sigma, part # M5057 </td></tr><tr><td> 5 M NaCl </td><td> Sigma, part # 71.386 </td></tr><tr><td> 20X SSPE </td><td> Sigma, part # S2015 </td></tr><tr><td> Moleküler biyoloji notu suyu </td><td> Sigma, part # W4502 </td></tr><tr><td> Hibridizasyon Astarlar </td><td> Çeşitli tedarikçileri (standart tuzsuzlaştırma) </td></tr><tr><td> Uptag </td><td> 5 &#39;GATGTCCACGAGGTCTCT 3&#39; </td></tr><tr><td> Buptagkanmx4 </td><td> 5 &#39;biotin GTCGACCTGCAGCGTACG 3&#39; </td></tr><tr><td> Dntag </td><td> 5 &#39;CGGTGTCGGTCTCGTAG 3&#39; </td></tr><tr><td> Bdntagkanmx4 </td><td> 5 &#39;biotin GAAAACGAGCTCGAATTCATCG 3&#39; </td></tr><tr><td> B213 </td><td> 5 &#39;biotin CTGAACGGTAGCATCTTGAC 3&#39; </td></tr><tr><td> Uptagkanmx </td><td> 5 &#39;3 GTCGACCTGCAGCGTACG&#39; </td></tr><tr><td> Dntagkanmx </td><td> 5&#39;GAAAACGAGCTCGAATTCATCG 3 &#39; </td></tr><tr><td> Uptagcomp </td><td> 5&#39;AGAGACCTCGTGGACATC 3 &#39; </td></tr><tr><td> Dntagcomp </td><td> 3 5&#39;CTACGAGACCGACACCG &#39; </td></tr><tr><td> Upkancomp </td><td> 5&#39;CGTACGCTGCAGGTCGAC 3 &#39; </td></tr><tr><td> Dnkancomp </td><td> 5&#39;CGATGAATTCGAGCTCGTTTTC 3 &#39; </td></tr><tr><td> Sıralama Astarlar: Illumina Platformu </td><td> Çeşitli tedarikçilerden </td></tr><tr><td> UpTag İleri (100uM) </td><td> 5 &#39;CAA GCA GAA GAC GGC ATA CGA GCT CTT CCG ATC T GAT GTC CAC GAG GTC TCT 3&#39; </td></tr><tr><td> UpTag Ters (100uM) </td><td> 5 &#39;AAT GAT ACG GCG ACC ACC GAC ACT CTT TTK CTA CAC GAC GCT CTT CCG ATC T nnnnn GTC GAC CTG CAG CGT ACG 3&#39; </td></tr><tr><td> DownTag İleri (100uM) </td><td> 5 &#39;CAA GCA GAA GAC GGC ATA CGA GCT CTT CCG ATC T GAA AAC GAG CTC GAA TTC ATC G 3&#39; </td></tr><tr><td> DownTag Ters (100uM) </td><td> 5 &#39;AAT GAT ACG GCG ACC ACC GAC ACT CTT TTK CTA CAC GAC GCT CTT CCG ATC T nnnnn CGG TGT CGG TCT CGT AG, 3 ` </td></tr><tr><td> 1 okuyun UP-tag sıra astar (100uM) </td><td> 5 &#39;GTC GAC CTG CAG CGT ACG 3&#39; </td></tr><tr><td> 1 AŞAĞI-etiket sıra astar (100uM) </td><td> 3 5 &#39;CGG TGT CGG TCT CGT AG&#39; </td></tr><tr><td> 2 Endeksi Sıralama astar (standart Illumina R1 astar) (100uM) </td><td> 5 &#39;AC ACT CTT TTK CTA CAC GAC GCT CTT CCG ATC T 3&#39; </td></tr><tr><td> Ek Sıralama Reaktifler / Ekipman </td><td></td></tr><tr><td> Qiagen MinElute 96 UF PCR arıtma Takımı </td><td> Qiagen, part # 29.051 </td></tr><tr><td> Vakum pompası </td><td> Herhangi bir satıcı </td></tr><tr><td> Macherey-Nagel Vakum Manifold </td><td> Macherey-Nagel, kısmen # 740 681 </td></tr><tr><td> Invitrogen Quant-iT dsDNA BR Assay Kit </td><td> Invitrogen, part # Q32853 </td></tr><tr><td> Invitrogen Qubit tahlil tüpleri </td><td> Invitrogen, part # Q32856 </td></tr><tr><td> % 40 akrilamid artı% 1 N, N&#39;-metilen-bis-akrilamid, 37.5:1 </td><td> Bio Rad, part # 161-0148 </td></tr><tr><td> Tris Base </td><td> Sigma, part # T1503-1KG </td></tr><tr><td> Borik Asit </td><td> Sigma, part # B6768-500G </td></tr><tr><td> 0,5 M EDTA, pH 8.0 </td><td> Teknova, part # E0306 </td></tr><tr><td> Amonyum persulfate </td><td> Sigma, part # A3678-25G </td></tr><tr><td> TEMED </td><td> Bioshop, part # TEM001.25 </td></tr><tr><td> Ethidium Bromür Çözümü </td><td> Bioshop, part # ETB444.10 </td></tr><tr><td> 0,5 M amonyum asetat </td><td> Teknova, part # A2000 </td></tr><tr><td> 10mM Magnezyum asetat tetrahidrat </td><td> Sigma, part # M0631-100G </td></tr><tr><td> 1mm EDTA, pH 8.0 </td><td> 0,5 M EDTA, pH 8.0 </td></tr><tr><td> Etanol </td><td> Çeşitli </td></tr><tr><td> Sodyum asetat, pH 5.2 </td><td> Teknova, part # S0297 </td></tr><tr><td> Hız vakum </td><td> Çeşitli </td></tr><tr><td> Tek-Read Küme Üretimi Kiti </td><td> Illumina, kısmen # GD-300-1001 </td></tr><tr><td> V4 36C Sıralama Seti </td><td>Illumina, part # FC-104-4002 </td></tr><tr><td></td><td></td></tr></tbody></table> 10x TBE tarifi <table border="1"><tbody><tr><td> Tutarlar </td><td> Reaktifler </td></tr><tr><td> 108 gram </td><td> Tris Base </td></tr><tr><td> 55 gram </td><td> Borik Asit </td></tr><tr><td> 40ml </td><td> 0,5 M EDTA (pH 8.0) </td></tr><tr><td colspan="2"> DH 2 O 1L işareti </td></tr></tbody></table> % 12 Poliakrilamid jel tarifi <table border="1"><tbody><tr><td> Hacimleri </td><td> Reaktifler </td></tr><tr><td> 5.8 ml </td><td> % 40 akrilamid artı% 1 N, N&#39;-metilen-bis-akrilamid, 37.5:1 </td></tr><tr><td> 12 ml </td><td> dH 2 O </td></tr><tr><td> 2 ml </td><td> 10X TBE </td></tr><tr><td> 140 ul </td><td> % 10 amonyum persülfat </td></tr><tr><td> Toplam hacmi: 20ml </td><td></td></tr></tbody></table> Uptag astar karışımı: Süspanse edin Uptag ve GKD 2 O Buptagkanmx4 her 100 mcM, daha sonra her biri 50 mcM nihai bir konsantrasyon 1:1 oranında karıştırın . -20 ° C&#39;de muhafaza Downtag astar karışımı: Süspanse edin Dntag ve Bdntagkanmx4 her biri 100 mcM GKD 2 O, sonra 50 mcM her son bir konsantrasyon için 1:1 oranında karıştırın . -20 ° C&#39;de muhafaza Karışık oligonükleotidler: 100 mcM GKD 2 O her aşağıdaki sekiz oligos (standart desalted) süspanse edin: Uptag,,, Dntag, Uptagkanmx, Dntagkanmx, Uptagcomp, Dntagcomp, Upkancomp, Dnkancomp. 12.5 mcM her son bir konsantrasyon için sekiz oligonükleotidler bir eşit miktarda karıştırın. 12X MES stok: 10 ml için 0.7 g MES serbest asit monohidrat ve 1.93 g MES sodyum tuzu, 8 ml moleküler biyoloji notu su çözülür. Iyi karıştırıldıktan sonra, pH ve pH 6,5-6,7 gerekirse ayarlayabilirsiniz. Toplam hacim 10 ml su ekleyin. Sterilize Filtre ve 4 saklamak ° C ışığı (örneğin, folyo tüp sarın) karşı korumalıdır. Çözüm, gözle görülür bir şekilde sarı veya 6 ay sonra hale gelmesi durumunda değiştirin. 2X Hibridizasyon tamponu: 50 ml, 8.3 ml (2.9.14) 12X (MES) hisse senetleri, 5 M NaCl, 4.0, 0.5 M EDTA, 0.1 ml% 10 Tween 20 (hacim / hacim) ml, ve 19.9 ml GKD süzülmüş 17.7 ml karıştırmak 2 O. Sterilize Filtresi. A Yıkama: Mix 300 ml 20X SSPE, 1 ml% 10 Tween (hacim / hacim), 699 ml GKD 2 O. Sterilize Filtresi. Yıkama B: Mix 150 ml 20X SSPE, 1 ml% 10 Tween (hacim / hacim), 849 ml GKD 2 O. Sterilize Filtresi. Barkod sıralama primerler UP-etiket astar dizileri 5 &#39;kuyruk (kalın), F ve R astar dahil Illumina belirli bir bağdaştırıcı dizileri . Değişken dizisi (italik), dizin / çoğullama okuma kullanılan 5-mer indeksleme etiketi temsil eder . 3 &#39;kuyruk (altı çizili) uptag barkod kuşatan ortak astar temsil eder ve maya barkod yükseltmek için gereklidir. AŞAĞI-tag astar 5 Ancak, 3 &#39;kuyruk (altı çizili) yükseltmek için kullanılan ortak primerler ile değiştirilir, UP-tag primerler (Illumina dizisi) kuyruk kuyruk 5 özdeş&#39; dizileri AŞAĞI etiketi barkod.

competitive genomic screens of barcoded yeast libraries

Yeni nesil sıralama teknolojilerindeki gelişmeler sayesinde, neredeyse her gün yeni gen dizilimlerini erişimi var. Bu gelişmelerin temposu hızlanıyor, daha fazla derinlik ve genişlik umut verici. Bu olağanüstü gelişmelerin ışığında, genin işlevini tanımlamak için hızlı, paralel yöntemleri için ihtiyaç her zamankinden daha önemli hale gelir. Maya ve E. genom silme mutantlar koleksiyonları coli gen fonksiyon fonksiyonel karakterizasyonu için beygirleri olarak hizmet etmiş, ancak bu yaklaşım ölçeklenebilir değildir, mevcut gen silme yaklaşımları silinir ve doğrulanabilir bir genomu oluşturan genlerin her biri binlerce gerektirir. Bu çalışma tamamlandıktan sonra, biz yüksek verim fenotipleme takip edebilirsiniz. Geçtiğimiz on yıl boyunca, laboratuar, rekabetçi, minyatür, yüksek verimli genomu paralel olarak yapılabilir testleri geniş bir portföy rafine vardır. Bu paralelizasyon barkod mutasyonu için bir vekil olarak hizmet, çünkü DNA &#39;etiketler&#39;, ya da her bir mutant haline &#39;barkod&#39; dahil edilmesi mümkündür ve bir mutant spor değerlendirmek için barkod bolluk ölçebilirsiniz. Bu çalışmada, DNA dizisi ve barkodlu mutant koleksiyonları arasındaki boşluğu doldurmak için çalışırlar. Bunu başarmak için yeni dizisi paralel barkod deneyleri kadar açılır, ancak zayıf mikropların karakterize bir kombine transposon kesintisiz barkodlama yaklaşımı tanıtmak. Bu yaklaşım göstermek için yeni bir Candida albicans barkodlu bozulması koleksiyonunu sergilemek ve hem mikroarray tabanlı ve yeni nesil sıralama tabanlı platformlar 10,000 toplamak için nasıl kullanılabileceğini tarif - 1.000.000, gen-gen ve ilaç-gen etkileşimleri tek bir deney .

Watch this Scientific Journal Video about Competitive Genomic Screens of Barcoded Yeast Libraries at JoVE.com

Competitive Genomic Screens of Barcoded Yeast Libraries

Biz gen-ilaç ve gen-çevre etkileşimleri anlamak için kapsamlı, tarafsız genom ekranlar geliştirdik. Bu mutant koleksiyonlarını tarama yöntemleri sunulmaktadır.

Barkodlu Maya Kütüphaneler Rekabet Genomik Ekranlar

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

competitive-genomic-screens-of-barcoded-yeast-libraries

Research

JoVE Journal

Biology

18.1K Görüntüleme.  University of Toronto. Biz gen-ilaç ve gen-çevre etkileşimleri anlamak için kapsamlı, tarafsız genom ekranlar geliştirdik. Bu mutant koleksiyonlarını tarama yöntemleri sunulmaktadır.

Video: Competitive Genomic Screens of Barcoded Yeast Libraries

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

Metagenomic Kütüphaneler Büyük Ölçekli Ekranlar

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

Biyoloji

Isolation of Genomic DNA from Mouse Tails

Fare Tails Genomik DNA izolasyonu

Large Insert Environmental Genomic Library Production

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent environmental genomic (i.e. metagenomic) approaches have emerged, enabling researchers to bridge this cultivation gap by capturing the genetic content of indigenous microbial communities directly from the environment. To this end, genomic DNA libraries are constructed using standard albeit artful laboratory cloning techniques. Here we describe the construction of a large insert environmental genomic fosmid library with DNA derived from the vertical depth continuum of a seasonally hypoxic fjord. This protocol is directly linked to a series of connected protocols including coastal marine water sampling [1], large volume filtration of microbial biomass [2] and a DNA extraction and purification protocol [3]. At the outset, high quality genomic DNA is end-repaired with the creation of 5 -phosphorylated blunt ends. End-repaired DNA is subjected to pulsed-field gel electrophoresis (PFGE) for size selection and gel extraction is performed to recover DNA fragments between 30 and 60 thousand base pairs (Kb) in length. Size selected DNA is purified away from the PFGE gel matrix and ligated to the phosphatase-treated blunt-end fosmid CopyControl vector pCC1 (EPICENTRE <a href="http://www.epibio.com/item.asp?ID=385">http://www.epibio.com/item.asp?ID=385</a>). Linear concatemers of pCC1 and insert DNA are subsequently headfull packaged into phage particles by lambda terminase, with subsequent infection of phage-resistant E. coli cells. Successfully transduced clones are recovered on LB agar plates under antibiotic selection and archived in 384-well plate format using an automated colony picking robot (Qpix2, GENETIX). The current protocol draws from various sources including the CopyControl Fosmid Library Production Kit from EPICENTRE and the published works of multiple research groups [4-7]. Each step is presented with best practice in mind. Whenever possible we highlight subtleties in execution to improve overall quality and efficiency of library production. The whole process of fosmid library production and automated colony picking takes at least 7-10 days as there are many incubation steps included. However, there are several stopping points possible which are mentioned within the protocol.

Construction of a fosmid library with environmental genomic DNA isolated from the vertical depth continuum of a seasonally hypoxic fjord is described. The resulting clone library is picked into 384-well plates and archived for downstream sequencing and functional screening by the application of an automated colony picking system.

Büyük Ekle Çevre Genomik Kütüphane Üretim

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Maya hücreleri Mitokondri saflaştırılması

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

Zebrafish kimyasal ekranlar için otomatik görüntüleme ve analiz geliştirilmesi.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

Maya Colony Gömme Yöntemi

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

MR Genomik - Sıra Desenler Genomik DNA içinde incelenmesi için Kamu Kaynak

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

Picoeukaryote genomik Dönüşüm<em&gt; Ostreococcus tauri</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

Genomik DNA 5-hydroxymethylcytosine seçici Yakalama

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Assessing Differences in Sperm Competitive Ability in Drosophila

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the number of successful mating events rather than on maximizing survival and viability 1. Sperm competition represents the competition between males after copulating with the same female 2, in which their sperm are coincidental in time and space. This phenomenon has been reported in multiple species of plants and animals 3. For example, wild-caught D. melanogaster females usually contain sperm from 2-3 males 4. The sperm are stored in specialized organs with limited storage capacity, which might lead to the direct competition of the sperm from different males 2,5.
Comparing sperm competitive ability of different males of interest (experimental male types) has been performed through controlled double-mating experiments in the laboratory 6,7. Briefly, a single female is exposed to two different males consecutively, one experimental male and one cross-mating reference male. The same mating scheme is then followed using other experimental male types thus facilitating the indirect comparison of the competitive ability of their sperm through a common reference. The fraction of individuals fathered by the experimental and reference males is identified using markers, which allows one to estimate sperm competitive ability using simple mathematical expressions 7,8. In addition, sperm competitive ability can be estimated in two different scenarios depending on whether the experimental male is second or first to mate (offense and defense assay, respectively) 9, which is assumed to be reflective of different competence attributes.
Here, we describe an approach that helps to interrogate the role of different genetic factors that putatively underlie the phenomenon of sperm competitive ability in D. melanogaster.

Assessing Differences in Sperm Competitive Ability in Drosophila

Differential sperm competitive ability among Drosophila males with distinct genotypes can be ascertained through double-mating experiments. Each of these experiments involves one of the males of interest and a reference male. Readily identifiable markers in the progeny allow inference of the fraction of individuals fathered by each male.

Sperm Rekabet Yeteneği in değerlendirilmesi farklılıklar<em&gt; Drosophila</em

Competition among conspecific males for fertilizing the ova is one of the mechanisms of sexual selection, i.e. selection that operates on maximizing the ...