Biz minnetle Singapur (ETRP 1201102) Ulusal Çevre Ajansı finansman desteği kabul, Singapur Ulusal Araştırma Vakfı (UÇK-CRP5-2009-03), Singapur Bilim, Teknoloji ve Araştırma Ajansı Rekabet Araştırmaları Programı ( 1324004108), Küresel Ar-Ge Proje Programı, Bilgi Ekonomisi Bakanlığı, Kore Cumhuriyeti (N0000677), Savunma Tehdit Azaltma Dairesi (DTRA, HDTRA1-13-1-0037) ve Sentetik Biyoloji Singapur Ulusal Üniversitesi Girişimi ( DPRT / 943/09/14).

Y 1. Üretim lipolytica KU70 Silme Gerilme <ol><li> bozulma kasetinin İnşaatı Not: polimeraz zincir reaksiyonu (PCR) amplifikasyonda kullanılan tüm primerler için Tablo 1&#39;e bakınız. <ol><li> LEU2 ekspresyon kaseti PCR kuvvetlendirmek için tasarım primerleri 20 Y&#39;den (Tablo 1 e bakınız) sırasıyla ve uzun ters primer (Tablo 1 # 2); lipolytica ifade vektörü 5 &#39;ve 3&#39; olarak LoxP yerleri sokacak uzun ileri primer (Tablo 1 # 1), LEU2 kasetinin sona erer. Klonlama işlemi sonraki adım için ilave bir sınırlama mevkisinin katılması için, primer # 1 &#39;de, bir BamHI sınırlandırma bölgesi ekleyin. </li><li> Tablo 2&#39;de ayrıntılı olarak yüksek doğrulukta bir DNA polimerazı kullanılarak PCR gerçekleştirin. </li><li> PCR purificatio kullanılarak loxp-promoteri-LEU2-terminatör-loxP kaseti PCR ürünü saflaştırmaküreticinin talimatlarına uygun olarak N kiti. Üreticinin talimatlarına uygun olarak 21 saflaştınlmış PCR ürününe 3&#39;-A çıkıntıları ekleyin. Kullanım T4 DNA ligazı, Tablo 3&#39;de belirtilen plazmid T-LEU2 vermek üzere, bir TA klonlama vektörüne A kuyruklu PCR ürünü bağlanması için. </li><li> PCR Y. astarları 3. / 4. kullanarak KU70 geninin 1 kb 5 &#39;yukarı serisini yükseltmek lipolytica Po1g Tablo 2&#39;deki gibi genomik DNA, 22., üreticinin talimatlarına uygun olarak bir PCR saflaştırma kiti kullanılarak PCR ürünü saflaştırmak. <ol><li> Çift. Tablo 4 uyarınca Sac II ile BamHI enzimleri ile saflaştınlmış PCR ürünü ve T-LEU2 plazmid hem özet üretici talimatlarına göre bir PCR saflaştırma kiti kullanılarak Sindirim karışımı saflaştırılır. Saflaştırılmış bağlanması için T4 DNA ligaz kullanma ve Sac II ile sindirilmiş PCR ürünü / T LEU2 Bam HI bölgelerine vermek üzereTablo 3&#39;de belirtilen plazmid T-LEU2-5E. </li></ol></li><li> PCR Y. astarları # 5 / # 6 kullanarak KU70 geninin 1 kb 3 &#39;aşağı serisini yükseltmek lipolytica Po1g Tablo 2&#39;deki gibi genomik DNA, 22., üreticinin talimatlarına uygun olarak bir PCR saflaştırma kiti kullanılarak PCR ürünü saflaştırmak. Çift saflaştırılmış PCR ürünü ve Tablo 4&#39;te göre değil ben ve Nde enzimlerle T-LEU2-5E plazmid hem sindirmek. <ol><li> üreticinin talimatlarına göre bir PCR saflaştırma kiti kullanılarak Sindirim karışımı saflaştırılır. Daha sonra, saflaştırılmış bağlanır ve Not I / nde Tablo 3&#39;de belirtilen, T4 DNA ligazı kullanılarak plazmid T-KO vermek üzere t-LEU2-5E I sitelerine PCR ürünü ile sindirilmiş. </li></ol></li><li> Bozulma kasetini üretmek için Tablo 4&#39;de gereğince Sac II ve nde I enzimleri ile plazmid T-KO sindirimi (Şekil 1 </strong&gt;). üreticinin talimatlarına göre bir PCR saflaştırma kiti kullanılarak sindirilmiş DNA fragmanlarının saflaştınlır. </li></ol></li><li> Yetkili Hücre hazırlama ve Y dönüşümü lipolytica Po1g suşu <ol><li> Yetkili Hücre hazırlama <ol><li> Y. bir koloni aşılamak YPD ortamı 10 ml (% 1 maya özütü,% 2 pepton,% 2 glukoz ve 50 mM sitrat tamponu, pH 4.0) 100 ml&#39;lik bir şişe içinde bir taze maya ekstresi-pepton-dekstroz (YPD) plakadan lipolytica Po1g soyu. Doyum noktasına kadar 20 saat (bir spektrofotometre kullanılarak ölçülen OD yaklaşık 15 600,) 225 rpm&#39;de 30 ° C çalkalama inkübatöründe inkübe edin. </li><li> Oda sıcaklığında 5000 x g&#39;de 5 dakika boyunca santrifüjlenerek pelet hücreleri. 20 ml Tris-EDTA (TE) tampon çözeltisi (10 mM Tris, 1 mM EDTA, pH 7.5) ile hücrelerin yıkayın ve aşama 1.2.1.2 tarif edildiği gibi hücreler pelet. (Asetik asit ile pH değeri 6.0), 0.1 M lityum asetat, 1 ml hücrelerin tekrar ve oda Tempe 10 dakika süre ile inküberature. </li><li> Steril 1.5 ml tüpler kompetan hücreler (100 ul) kısım. hemen altındaki dönüştürme adımları geçin ya da uzun süreli depolama için -80 ° C&#39;da bir son% 25 konsantrasyonunda (h / h) ve saklamak için gliserol ekleyin. </li></ol></li><li> dönüşüm <ol><li> Yavaşça yetkin hücre 100 ul ile denatüre somon sperm DNA (10 mg / ml) ve saflaştırılmış bozulması kasetinin 1-5 ug, 10 ul karıştırın ve 15 dakika boyunca 30 ° C&#39;de inkübe edin. </li><li> (0.1 M lityum asetat pH 6.0 içinde çözülmüş)% 40 polietilen glikol (PEG) -4000 700 ul eklenir, iyice karıştırılır ve 60 dakika boyunca 225 rpm&#39;de 30 ° C çalkalama inkübatöründe inkübe edin. Not: yerine (tipik olarak geleneksel maya transformasyonu kullanılmaktadır) PEG-3350, 4000 ortalama molekül ağırlığına sahip PEG kullanılması önemlidir. </li><li> Isı 60 dakika için 39 ° C su banyosu içinde tüp yerleştirmek sureti ile transformasyon karışımı şok. </li><li> 1 ml YPD ortamı ilave edin ve30 ° C&#39;de 2 saat ve 225 rpm&#39;de için geri. Oda sıcaklığında 1 dakika boyunca 9.000 x g santrifüj, süpernatant kaldırmak ve TE tamponu 1 ml pelletini. </li><li> Oda sıcaklığında 1 dakika boyunca 9.000 x g&#39;de santrifüj ile hücreler repellet ve supernatant atın. TE tamponu, 100 | il pelletini ve seçici plakaları (örneğin, lösin-eksikli plakalar) üzerine plaka ve 2-3 gün boyunca 30 ° C&#39;de inkübe edin. </li><li> 10 tek koloniler 4 Pick up ve YPD orta 2 ml ayrı aşılamak. 225 rpm&#39;de 30 ° C&#39;de ve çalkalama gece boyunca inkübatör içinde inkübe edin. Sırasıyla primer # 55 / # 56, iki set ve # 56 / # 57 kullanılarak Tablo 5&#39;teki gibi transformantların 22 genomik DNA, PCR analizi ile pozitif kolonileri tanımlamak. Not: Ben pYLEX1 vektörü Y dönüştürülür doğrusallaştırılmış sayısını sayarak transformasyon etkinliğini belirlemek amacıyla lipolytica Po1g kompetan hücrelerug plazmid başına koloni oluşturan birim (CFU) DNA 23 kullanılır. </li></ol></li></ol></li></ol> 2. Marker Kurtarma <ol><li> Cre sentezleme plasmidinin yapımı <ol><li> pYLEX1-CRE ve pYLEX1-Eden Hastaneler İnşaat <ol><li> Tasarım primerleri 20 Cre ve Eden Hastaneler açık okuma çerçevelerini PCR kuvvetlendirmek için. Ileri primer # 82 ve # 84 başlangıç ​​kodonunun üst akışında bir ekstra bir adenin nükleotid ekleyin ve PCR CRE açık okuma çerçevelerini amplifiye ters primerler # 83 ve # 85, durdurma kodonunun aşağı Kpn I kısıtlama bölgelerinin sokulması ve Tablo 2&#39;deki gibi pSH69 (erişim numarası HQ412578) 24 dan hph. </li><li> Cre ve üreticinin talimatlarına göre bir PCR saflaştırma kiti kullanılarak HPH parçaları hem saflaştınlır. Digest hem ben Tabl başına enzim Kpn ile saflaştırılmış CRE ve hph fragmanlarıE 4.. Tablo 4 uyarınca Pml I ve Kpn I enzimleri ile pYLEX1 plazmid sindirimi Çift üreticinin talimatlarına göre bir PCR saflaştırma kiti kullanılarak Sindirim karışımı saflaştırılır. </li><li> Y. Pml I / Kpn I sitelerine saflaştırılmış ve sindirilmiş CRE ve hph parçalarını birbirine bağlamak için Tablo 3&#39;de göre T4 DNA ligaz kullanın sırasıyla pYLEX1-CRE ve pYLEX1-hph elde lipolytica ifade vektörü. </li></ol></li><li> pSL16-CRE-Eden Hastaneler İnşaat <ol><li> PCR primerleri, 86. kullanılarak pYLEX1-CRE gelen CRE promotör-gen-sonlandırıcı kaseti amplifiye / 87. Tablo 2&#39;deki gibi Sal I / Pst I sınırlandırma bölgeleri çevreleyen. Üreticinin talimatlarına göre bir PCR saflaştırma kiti kullanılarak PCR ürünü saflaştırmak . <ol><li> Çift Sal I ile saflaştırılan PCR ürünü ve pSL16-CEN1-1 (227) plazmid 25 hem özetPst Tablo 4&#39;te göre ben enzimleri. Üreticinin talimatlarına göre bir PCR saflaştırma kiti kullanılarak sindirim karışımı arındırın. Daha sonra, Tablo 3&#39;de belirtilen, T4 DNA ligazı kullanılarak pSL16-CRE oluşturmak için karşılık gelen bölgelerde pSL16-CEN1-1 (227) saflaştırılmış ve sindirilmiş PCR ürünü ligate. </li></ol></li><li> PCR primerleri kullanılarak pYLEX1-Eden Hastaneler gelen Eden Hastaneler promotör-gen-sonlandırıcı kaseti amplifiye # 88 / # 89. Tablo 2&#39;deki gibi Xho I / Bgl II kısıtlama alanlarını yandan kuşatan, üretici talimatlarına göre bir PCR saflaştırma kiti kullanılarak PCR ürünü saflaştırmak . <ol><li> Çift. Saflaştınlmış PCR ürünü ve Tablo 4 uyarınca Xho I ve Bgl II enzimleri ile pSL16-CRE plazmid hem özet üretici talimatlarına göre bir PCR saflaştırma kiti kullanılarak Sindirim karışımı saflaştırılır. Daha sonra, karşılık gelen en pSL16-CRE saflaştırılmış ve sindirilmiş PCR ürünü ligateYer Tablo 3&#39;de belirtilen, T4 DNA ligazı kullanılarak pSL16-CRE-hph üretir. Not: Elde edilen Cre sentezleme plasmidi, pSL16-CRE-HPH, seçilebilir bir higromisin B işaretleyici taşımaktadır ve bir sentromer ve epizomal kopyalayan vektörü (Şekil 2). </li></ol></li><li> Tablo 4 uyarınca uygun kısıtlama enzimleri (örneğin, Sal I / Pst I, vektöründen inserti kesip çıkarmak için) ile sindirme ile bütün bileşenleri kontrol edin. </li></ol></li></ol></li><li> Cre rekombinaz aracılık ettiği işaretçi kurtarma <ol><li> Yukarıdaki Bölüm 1.2&#39;de açıklanan dönüşüm protokolü aşağıdaki KU70 nakavt suşu yetkili hücrelerine pSL16-CRE-HPH Transform. YPD artı higromisin B üzerine Plaka dönüştürülmüş hücreler (YPDH) plakaları (400 ug / ml higromisin B ihtiva eden) ve 2-3 gün boyunca 30 ° C&#39;de inkübe edin. </li><li> Tablo 5&#39;te göre koloni PCR gerçekleştirin primerler kullanılarak # 84 / # 85 pozitif tespit etmekdönüşümden sonra pSL16-CRE-HPH plazmidi içeren koloniler. Not: ileri primer 84. ve revers primer 85. hph geni (Tablo 1) kodlama bölgesi içinde yer alır. Sadece pozitif klonlar PCR reaksiyonunda spesifik bir PCR ürünü üretir. </li><li> Pozitif klon almak ve YPDH orta 2 ml inoküle ve doygunluk gece boyunca 225 rpm&#39;de 30 ° C çalkalama inkübatöründe inkübe edin. Spektrofotometre ile hücre OD 600 ölçün. ~ 15 bir OD 600 hücreleri Hasat. Oda sıcaklığında 5 dakika boyunca 5000 x g&#39;de santrifüj ve steril su, 2 ml ile bir kez yıkanır. </li><li> Bir ilk OD 0.1 600 YPD ortamı 2 ml hücreleri yeniden inoküle ve hücreler, gece boyunca 225 rpm&#39;de 30 ° C çalkalama inkübatöründe büyümeye olanak sağlar. Streak YPD plakalar üzerine bir gecede hücre kültürü tek koloniler 23 izole etmek. Replika plaka YPD, YPDH üzerine koloniler ve lösin eksik plakalar 23 </s&gt; Yukarı. Not: (Şekil 3), hem LEU2 işaretleyici kaybetmiş ve pSL16-CRE-STEH plazmid sadece YPD plakalar üzerinde büyüyebilir Koloni. </li></ol></li></ol> Alkol Dehidrogenaz ve Alkol Oksidaz Genleri 3. Silme <ol><li> Y üzerinde bir arama yapın silme 26 gen adaylarını belirlemek için Temel Yerel Hizalama Arama Aracı (BLAST) kullanarak lipolytica genom veritabanı. Giriş S. protein dizisidir ŞOK arama aracı haline cerevisiae alkol dehidrojenaz I (http://www.genome.jp/tools-bin/search_sequence?prog=blast&amp;db=yli&amp;seqid=aa). Not: BLAST arama aracı Y en benzer protein dizileri döndürür lipolytica genom veritabanı. </li><li> 56. (Tablo 1) primerleri 7. PCR ile yok etme kasetleri Construct ve kısıtlayıcı enzim dijestiyonu ve yukarıdaki bölüm 1.1&#39;de açıklandığı gibi aynı usuller kullanılarak ligasyon reaksiyonları gerçekleştirmek. </li><li> HazırlıkKU70 nakavt suşu yetkili hücreleri vardır dönüşümleri gerçekleştirmek ve yukarıdaki bölüm 1.2&#39;de açıklanan protokol takip ederek 81. (Tablo 1) 55. astar, # 58 ile pozitif koloniler tanımlamak için PCR gerçekleştirin. Not: Bir örnek olarak, YALI0E17787g gen silme işlemi, Şekiller 4 ve 5&#39;te tarif edilir. homolog rekombinasyon oranı 1 kb ve 2 kb uzunluğunda homolog bölgenin etkileri bildirilmiştir. </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+Chain+Reaction:+Basic+Protocol+Plus+Troubleshooting+and+Optimization+Strategies.">Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies.</a> J. Vis. Exp. (63), e3998 (2012).</li><li>Kobs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+blunt-end+DNA+fragments+into+the+pGEM-T+Vector+Systems.">Cloning blunt-end DNA fragments into the pGEM-T Vector Systems.</a> Promega Notes. 62, 15-18 (1997).</li><li>Sanders, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+laboratory+techniques:+plating+methods.">Aseptic laboratory techniques: plating methods.</a> J. Vis. Exp. (63), e3064 (2012).</li><li>Hegemann, J. H., Heick, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delete+and+repeat:+a+comprehensive+toolkit+for+sequential+gene+knockout+in+the+budding+yeast+Saccharomyces+cerevisiae.">Delete and repeat: a comprehensive toolkit for sequential gene knockout in the budding yeast Saccharomyces cerevisiae.</a> Methods Mol. Biol. 765, 189-206 (2011).</li><li>Yamane, T., Sakai, H., Nagahama, K., Ogawa, T., Matsuoka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+centromeric+DNA+from+yeast+Yarrowia+lipolytica+and+identification+of+protein-binding+site+required+for+plasmid+transmission.">Dissection of centromeric DNA from yeast Yarrowia lipolytica and identification of protein-binding site required for plasmid transmission.</a> J. Biosci. Bioeng. 105 (6), 571-578 (2008).</li><li>McGinnis, S., Madden, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLAST:+at+the+core+of+a+powerful+and+diverse+set+of+sequence+analysis+tools.">BLAST: at the core of a powerful and diverse set of sequence analysis tools.</a> Nucleic Acids Res. 32 ((Web Server issue)), W20-W25 (2004).</li><li>Madzak, C., Tr&#233;ton, B., Blanchin-Roland, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strong+hybrid+promoters+and+integrative+expression/secretion+vectors+for+quasi-constitutive+expression+of+heterologous+proteins+in+the+yeast+Yarrowia+lipolytica.">Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica.</a> J. Mol. Microbiol. Biotechnol. 2 (2), 207-216 (2000).</li><li>Nicaud, J. M., Le Clainche, A., Le Dall, M. T., Wang, H., Gaillardin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yarrowia+lipolytica,+a+yeast+model+for+the+genetic+studies+of+hydroxy+fatty+acids+biotransformation+into+lactones.">Yarrowia lipolytica, a yeast model for the genetic studies of hydroxy fatty acids biotransformation into lactones.</a> J. Mol. Catal. B-Enzym. 5 (1), 175-181 (1998).</li></ol>

The authors have nothing to disclose.

Bu çalışma için Amacımız Y. hedefli gen tırnakları hızlı ve etkin üretimini sağlamaktır Bunu başarmak için lipolytica Po1g suşu. Çeşitli düşünceler ele alınması gerekmektedir. İlk olarak, yüksek bir dönüşüm etkinliği gerekir. Y için Böylece, etkili ve uygun bir kimyasal dönüşümü protokolü lipolytica Po1g suşu Bu çalışmada tanımlanmıştır. PEG-4000 kullanımı, bu soyun başarılı bir transformasyonu için önemli bir faktördür. ...

Saccharomyces cerevisiae, Yarrowia lipolytica, geleneksel olmayan bir maya farklı çevresel koşullar 1,2 değişikliklere yanıt olarak maya ya da misel şeklinde büyüyebilir. Böylece, bu iki şekilli maya mantar farklılaşma, morfolojilerinden ve taksonomi 3,4,5 çalışma için iyi bir model olarak kullanılabilir. Genellikle yaygın olarak organik asitler, polialkoller, aroma maddeleri, emülsiyon yapıcılar ve yüzey aktif maddeler 6,7,8,9 gibi gıda katkı maddelerinin çeşitli üretmek için kullanılan bir kasa (GRAS) maya türleri, olarak kabul edilir. Bu kadar hücre kuru ağırlığının 10% 70, zorunlu bir aerobe ve doğal olarak yüksek miktarlarda lipitleri biriken yeteneğine tanınmış yağlı maya, yani. Ayrıca besinlerin 11,12,13 atık kaynaklarının kalıntıların farklı türde de dahil olmak üzere, büyüme için karbon kaynakları geniş bir yelpazede yararlanabilirler. Bu eşsiz özelliklerin tümü Y. yapmak için çok cazip lipolyticaçeşitli biyoteknolojik uygulamalar. Y&#39;nin tüm genom sekansı, ancak lipolytica 14,15 yayınlandı, bu sıradışı maya genetik manipülasyon diğer maya türleri daha karmaşıktır. İlk olarak, bu maya türlerinin dönüşümü nedeniyle istikrarlı ve verimli bir genetik transformasyon sisteminin 16,17 olmaması çok daha az etkilidir. Doğal epizomal plazmid sistemi bu maya 18 bulunmuştur İkinci olarak, lineer ekspresyon kasetlerinin zahmetli genomik entegrasyon yaygın ilgili genlerin ekspresyonu için kullanılır. Bu maya doğru homolog rekombinasyon yoluyla verimliliği hedefleyen gen düşüktür ve çoğu entegrasyon olayları homolog olmayan sonunda katılmadan (NHEJ) 19 içinden gelmesi nedeniyle genetik knock-out ve knock-ins Üçüncü nesil sınırlıdır. Bu çalışmada, Y için optimize edilmiş bir transformasyon protokolü rapor lipolytica </em&gt; kolay, hızlı, verimli ve tekrarlanabilir Po1g suşu. Hassas homolog rekombinasyon frekansını artırmak için, NHEJ yolunun önemli bir enzimi kodlayan KU70 geni silinmiş. Optimize edilmiş dönüşüm protokolü kullanılarak ve 1 kb Y. KU70 geni homoloji bölgeleri kuşatan içeren doğrusal bir nakavt kaset dönüştürerek lipolytica Po1g başarıyla silindi. Bu gen silinmesi metodoloji sağlamlığı daha sonra Po1g KU70 Δ suşu alkol dehidrogenaz ve alkol oksidazı genlerini hedef alan ile gösterilmiştir. Bu KU70 delesyon suşu doğal tipte Po1g soyunun daha hedefleme homolog rekombinasyon aracılığıyla gen bir ölçüde daha yüksek bir verimlilik sergilemiş olduğu gözlemlenmiştir. Buna ek olarak, higromisin B direnç taşıyan bir yineleme Cre ifade plazmid işaretleyici kurtarma gerçekleştirmek için inşa edilmiştir. işaretleyici kurtarma th hedef genin birden fazla mermi kolaylaştırırE gen silinmesi Elde edilen mutantlar. Gen silme yanı sıra, burada tarif edilen genetik transformasyonu ve gen silinmesi için protokol özel bir lokus genlerin eklemek için, ve Y alana özgü mutasyonların katılması için uygulanabilir lipolytica genomu.

<table><tbody><tr><td>&lt;strong&gt;Reagent/Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Oligonucleotide primers</td><td>Integrated DNA Technologies</td><td></td><td>25 nmole DNA oligos</td></tr><tr><td>&lt;em&gt;Y. lipolytica&lt;/em&gt; strain Po1g</td><td>Yeastern Biotech</td><td></td><td>leucine auxotrophic derivative of the wild-type strain W29 (ATCC 20460)</td></tr><tr><td>Vector pYLEX1</td><td>Yeastern Biotech</td><td>FYY203-5MG</td><td>&lt;em&gt;Y. lipolytica&lt;/em&gt; expression vector</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; TOP10</td><td>Invitrogen</td><td></td><td>For cloning and propagation of plasmids</td></tr><tr><td>pGEM-T vector&amp;nbsp;</td><td>Promega</td><td>A3600</td><td>TA cloning vector</td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27106</td><td>For plasmid isolation</td></tr><tr><td>Wizard SV Gel and&lt;br/&gt; PCR Clean-Up System</td><td>Promega</td><td>A9282</td><td>Extract DNA fragments from agarose gels&lt;br/&gt; and purify PCR products from an amplification reaction</td></tr><tr><td>The iProof high-fidelity&lt;br/&gt; DNA polymerase</td><td>Bio-Rad</td><td>172-5302</td><td>High-fidelity DNA polymerase</td></tr><tr><td>BamHI&amp;nbsp;</td><td>New England Biolabs</td><td>R0136S</td><td>Restriction enzyme</td></tr><tr><td>BglII&amp;nbsp;</td><td>New England Biolabs</td><td>R0144L</td><td>Restriction enzyme</td></tr><tr><td>KpnI</td><td>New England Biolabs</td><td>R0142S</td><td>Restriction enzyme</td></tr><tr><td>NdeI&amp;nbsp;</td><td>New England Biolabs</td><td>R0111S</td><td>Restriction enzyme</td></tr><tr><td>NotI</td><td>New England Biolabs</td><td>R0189L</td><td>Restriction enzyme</td></tr><tr><td>PmlI</td><td>New England Biolabs</td><td>R0532S</td><td>Restriction enzyme</td></tr><tr><td>PstI&amp;nbsp;</td><td>New England Biolabs</td><td>R0140S</td><td>Restriction enzyme</td></tr><tr><td>SacII</td><td>New England Biolabs</td><td>R0157S</td><td>Restriction enzyme</td></tr><tr><td>SalI</td><td>New England Biolabs</td><td>R0138S</td><td>Restriction enzyme</td></tr><tr><td>XhoI</td><td>New England Biolabs</td><td>R0146L</td><td>Restriction enzyme</td></tr><tr><td>T4 DNA ligase</td><td>New England Biolabs</td><td>M0202L</td><td></td></tr><tr><td>Taq DNA polymerase&amp;nbsp;</td><td>Bio-Rad</td><td>M0267L</td><td></td></tr><tr><td>Ampicillin</td><td>Gibco-Life Technologies</td><td>11593-027</td><td>Antibiotics</td></tr><tr><td>Hygromycin B</td><td>PAA</td><td>P21-014</td><td>Antibiotics</td></tr><tr><td>GeneRuler 1 kb DNA ladder</td><td>Thermo Scientific</td><td>SM0312</td><td>1 kb DNA ladder</td></tr><tr><td>PEG4000</td><td>Sigma</td><td>95904-F</td><td></td></tr><tr><td>Tris</td><td>Promega</td><td>H5135</td><td></td></tr><tr><td>EDTA</td><td>Bio-Rad</td><td>161-0729</td><td></td></tr><tr><td>Salmon Sperm DNA</td><td>Invitrogen</td><td>15-632-011</td><td></td></tr><tr><td>Lithium Acetate</td><td>Sigma</td><td></td><td></td></tr><tr><td>Acetic acid</td><td>Sigma</td><td></td><td></td></tr><tr><td>Glass beads (425-600 &amp;micro;m)</td><td>Sigma</td><td>G8772</td><td></td></tr><tr><td>RNAse A</td><td>Thermo Scientific</td><td>EN0531</td><td></td></tr><tr><td>DNA Loading Dye</td><td>Thermo Scientific</td><td>R0611</td><td></td></tr><tr><td>Bacto Yeast Extract</td><td>BD</td><td>212750</td><td></td></tr><tr><td>Bacto Peptone</td><td>BD</td><td>211677</td><td></td></tr><tr><td>D-Glucose</td><td>1st Base</td><td>BIO-1101</td><td></td></tr><tr><td>YNB without amino acids</td><td>Sigma</td><td>Y0626</td><td></td></tr><tr><td>DO Supplement-Leu</td><td>Clontech</td><td>630414</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>Difco LB Broth</td><td>BD</td><td>244620</td><td></td></tr><tr><td>Difco LB Agar</td><td>BD</td><td>244520</td><td></td></tr><tr><td>Bacto Agar</td><td>BD</td><td>214010</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PCR machine</td><td>Biorad</td><td>T100 Thermal Cycler</td><td></td></tr><tr><td>Water bath</td><td>Memmert</td><td>WNB 14</td><td></td></tr><tr><td>Stationary/Shaking Incubator</td><td>Yihder</td><td>LM-570RD</td><td></td></tr><tr><td>Thermo-shaker</td><td>Allsheng</td><td>MS-100</td><td></td></tr><tr><td>Micro centrifuge</td><td>Eppendorf</td><td>5424R</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Spectrophotometer</td><td>Eppendorf&amp;nbsp;</td><td>BioPhotometer plus</td><td></td></tr><tr><td>Gel imager</td><td>GE</td><td>Amersham Imager 600</td><td></td></tr></tbody></table>

genetic engineering of an unconventional yeast for renewable biofuel and biochemical production

Yarrowia lipolytica bir, patojen olmayan dimorfik ve kesinlikle aerobik maya türüdür. kendine özgü fizyolojik özellikleri ve metabolik özellikleri nedeniyle, bu sıradışı maya sadece mantar farklılaşma temel doğası çalışma için iyi bir model değil, aynı zamanda biyokimyasal üretim ve kapsamlı genetik manipülasyonlar gerektiren çeşitli biyoteknolojik uygulamalar için umut verici bir mikrobik bir platformdur. Y&#39;nin Ancak genetik manipülasyonlar lipolytica nedeniyle başta KU70 geni isnat edilebilir verimli ve istikrarlı bir genetik transformasyon sisteminin eksikliği yanı sıra homolog olmayan rekombinasyon çok yüksek oranlarda sınırlı olmuştur. Burada, verimli genetik transformasyonu için ve Y gen silinmesi için kolay ve hızlı bir protokol rapor lipolytica Po1g. Y dışsal DNA etkin transformasyonu için İlk olarak, bir protokol lipolytica Po1g kurulmuştur. Second, hedef genlerin daha silinmesi için geliştirilmiş çift çaprazlama homolog rekombinasyon oranı elde etmek için, KU70 gen 1 kb homoloji silah taşıyan bir bozulma kaset dönüştürerek tarafından silindi. Üçüncü olarak, KU70 geninin silindikten sonra gelişmiş gen silme verimliliğini göstermek için, biz tek tek KU70 nakavt platformu zorlanma aynı prosedürler kullanılarak alkol dehidrogenaz ve alkol oksidaz kodlayan 11 hedef genleri silindi. Kesin homolog rekombinasyon oranı silinmesi için en az% 0.5 önemli bir artış gözlenmiştir Po1g KU70 ö 11 hedef genlerin tek gen silinmesi için% 33 -71% ile Po1g içinde KU70 geninin. higromisin B direnç işareti, CRE / LoxP taşıma sistemi bir replikatif plazmit ve maya nakavt suşlarında seçim marker geni, sonunda targe birden fazla tur kolaylaştırmak için Cre rekombinazın ifadesinin uzaklaştırıldıted genetik manipülasyonlar. Ortaya çıkan tek gen silme mutantlar biyoyakıt ve biyokimyasal üretimde potansiyel uygulamalar var.

Lineerleştirilmiş Y. lipolytica ifade vektörü Y genomunda pBR yerleştirme platformu yerleştirildi Tek bir geçit rekombinasyona 27 gerçekleştirerek lipolytica Po1g süzün. Bu çalışmada kurulan hızlı kimyasal dönüşüm prosedürü, doğrusallaştırılmış Y. kullanarak lipolytica ifade vektörü başarıyla&gt; 100 CFU / ug DNA bir transformasyon verimi yabani tip Po1g suşuna transforme edildi. 1 kb homolog diziler...

Watch this Scientific Journal Video about Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production at JoVE.com

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

Yenilenebilir Biyoyakıt ve Biyokimyasal Üretim için bir Sıradışı Maya Genetik Mühendisliği

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

genetic-engineering-an-unconventional-yeast-for-renewable-biofuel

Research

JoVE Journal

Genetics

13.5K Görüntüleme.  National University of Singapore. We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

Video: Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Yüksek verim Protoplast İzolasyonu ve Dönüşüm için Robotik Platformu

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Genetik

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is particularly relevant for survival in fluctuating environments such as those found in beer fermentation. However, sugar transitions are an environmental variable that is challenging to predict and detect, impacting the outcome of beer fermentations. This protocol describes an in vivo system to monitor transcriptional activation associated with the glucose-to-maltose metabolic shift in Saccharomyces eubayanus that applies to different wild Saccharomyces yeast strains. 
The system employs an episomal bioluminescent transcriptional reporter for maltose metabolism, focusing on MAL32, since it provides a good readout for metabolic shifts, as studied in S. cerevisiae. For this, yeast strains were transformed with plasmids containing the MAL32 regulatory region from S. eubayanus, controlling the expression of a gene encoding for a destabilized version of firefly luciferase1, and a hygromycin resistance gene used exclusively during transformation to ensure plasmid acquisition. Following selection, transformed yeast cells can be cultured under non-selective conditions, as the episomal plasmid remains stable in culture conditions for up to 7 days. 
This system was validated under a complex sugar environment in microfermentation assays, confirming the effectiveness of the luciferase reporter in informing metabolic transitions. Samples were collected regularly and analyzed with a luminometer, providing continuous insights into yeast responses. While broadly applicable, this protocol is particularly valuable for assessing yeast performance under fermentation conditions, where metabolic changes pose a significant challenge. Additionally, this methodology can be adapted by selecting alternative promoters to explore a broader range of responses to environmental changes, allowing characterization as well as optimization of wild yeast strains for diverse industrial applications.

This protocol employs a bioluminescent reporter, allowing measurements of transcriptional activity in Saccharomyces eubayanus to monitor the glucose-to-maltose transition, enabling real-time analysis of metabolic adaptations and supporting strain optimization for industrial fermentation under diverse conditions.

In vivo (İn canlı) Mayada Biyolüminesan Raportör Kullanılarak Metabolik Geçiş Sırasında Transkripsiyonel Aktivitenin İzlenmesi

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

Etanol ile lignoselüloz biyo dönüşümünü için Sağlam Pentoz-fermente Maya Evrimi Teknikleri

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Biyomühendislik

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

Mikrobiyal optogenetic Uygulamaları Tasarım ve Otomatik Aydınlatıcı, kültürleme ve Örnekleme Sistemi Uygulaması

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

Farelerde oral sonda ardından Probiyotik Maya Dönüşümü ve Gastrointestinal Bağışıklık Dokular Onların Kurtarma

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

İmmünoloji ve Enfeksiyon

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

Damlacık Tabanlı RNA Sıralamaile Yüksek EldeLi Maya Zorlanma Fenotilik

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

Çoklu Gen Delesyonlar Taşıma Maya Suşlarında Nesil için Yeşil Canavar Süreci

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Biyoloji

Light-Controlled Fermentations for Microbial Chemical and Protein Production

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Optogenetic control of microbial metabolism offers flexible dynamic control over fermentation processes. The protocol here shows how to set up blue light-regulated fermentations for chemical and protein production at different volumetric scales.

Mikrobiyal Kimyasal ve Protein Üretimi için Işık Kontrollü Fermantasyonlar

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Evrim Yöntemi yönettiği<em&gt; Saccharomyces cerevisiae</em&gt;: Mutant Kütüphane Oluşturma ve Tarama

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

Biofuels: Producing Ethanol from Cellulosic Material

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
In this experiment, cellulosic material (such as corn stalks, leaves, grasses, etc.) will be used as a feedstock for the production of ethanol.&#160;The cellulosic material is first pretreated (ground and heated), digested with enzymes, and then fermented with yeast.&#160;Ethanol production is monitored using an ethanol probe.&#160;The experiment can be extended to optimize ethanol production by varying the feedstock used, pretreatment conditions, enzyme variation, yeast variation, etc.&#160;An alternative method of monitoring the reaction is to measure the carbon dioxide produced (using a gas sensor) instead of the ethanol.&#160;As a low-tech alternative, glucose meters (found in any drug store) can be used to monitor the glucose during the process, if an ethanol probe or carbon dioxide gas sensor is not available.
With an increased emphasis on &#8216;inquiry-based learning&#8221;, scientific probes are becoming more popular.&#160;Handheld devices like the Vernier Lab Quest used in conjunction with a variety of probes (such as those for conductivity, dissolved oxygen, voltage, and more) allow for less focus on collecting data and/or making graphs and more on analyzing the data and making predictions.&#160;Another advantage is that these are small and lightweight and can be taken into the field for measurements.

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University
In this experiment, cellulosic material (such as corn stalks, leaves, ...