This work was supported by the European Commission project Indox-FP7-KBBE-2013-7-613549; a Cost-Action CM1303-Systems Biocatalysis; and the National Projects Dewry [BIO201343407-R] and Cambios [RTC-2014-1777-3].

1. Mutant Kütüphanesi İnşaatı <ol><li> Seçim bölgeler, mevcut kristal yapı ya da homoloji model 18 göre bilgisayar algoritmaları yardımıyla geçişin maruz bırakılması. <ol><li> Burada, rastgele mutagenez ve rekombinasyon (Met [α1] -Val109, Phe392-Gln566), yüksek sadakat PCR ile gen (844 bp) geri kalan yükseltme sırasında (Şekil 1) için Pleurotus eryngii gelen AAO iki bölgesini hedef. Not: Birkaç segmentler bağımsız veya kombine şekilde 16 geçişin ele alınabilir. </li></ol></li><li> mutajenik PCR ile hedeflenen bölgelere yükseltin. tanımlı bölgelerin PCR reaksiyonları atma kesimleri arasında alanları (~ 50 bp her) üst üste oluşturun. <ol><li> DNA şablonu ihtiva eden 50 ul (0.92 ng / | il), 90 nM oligo sens (kademeli bir MI ve kademeli bir M-II AAO-BP RMLN) içindeki bir nihai hacimde segmentler mutajenik PCR Hazırlama 90 nmntisense primeri (kademeli bir M-II için kademeli bir MI ve RMLC için AAO-92C), 0.3 mM dNTP (0.075 mM),% 3 (h / h) dimetilsülfoksit (DMSO), 1.5 mM MgCl2, 0.05 mM MnCl2 ve 0.05 U / ul Taq DNA polimerazı. Primerler dizileri Şekil 1&#39;de ayrıntılı olarak verilmiştir. </li><li> Aşağıdaki PCR programı kullanarak: 2 dakika boyunca (1 döngü) 95 ° C; 45 saniye, 45 saniye, 50 ° C, 45 sn için 74 ° C (28 döngü) 95 ° C; 10 dakika boyunca (1 döngü) 74 ° C. </li></ol></li><li> ultra-yüksek sadakat polimeraz olmayan mutajenik bölgeleri yükseltmek ve mutajenik segmentleri ve / veya doğrusallaştırılmış vektör çıkıntılar üst üste gelen alanları bulunmaktadır. <ol><li> ihtiva eden 50 ul&#39;lik bir son hacim içinde, reaksiyon karışımları hazırlayın: DNA numunesi (0.2 ng / | il), 250 nM oligo sens HFF, 250 nM oligo antisens HFR, 0.8 mM dNTP (0.2 mM her biri),% 3 (h / h) dimetilsülfoksit (DMSO) ve DNA polimerazı iproof 0.02 U / ul. Astarlar dizileri ayrıntılı olarak &lt;strong&gt; Şekil 1. </li><li> Aşağıdaki PCR programı kullanın: 30 sn (1 döngü) için 98 ° C; 10 saniye, 25 saniye boyunca 55 ° C, 45 saniye için 72 ° C (28 döngü) 98 ° C; 10 dakika boyunca (1 döngü) 72 ° C. Not: 1.2 açıklanan koşullara ve 43 bp (plazmid-M1 bölge) 1.3 örtüştüğü ile; 46 bp (M1 bölge HF bölge); 47 bp (HF bölge M2 bölge) ve 61 bp (M2 Bölge- plazmid) maya in vivo ekleme de lehine (Şekil 1) tasarlanmıştır. </li><li> imalatçının protokolüne göre, ticari bir jel özütleme kiti (mutajenik ve mutajenik) bütün PCR fragmanları arındırın. </li></ol></li><li> Yaklaşık 50 bp&#39;lik komşu bölgeleri hedef genin 5&#39;- ve 3&#39;-ucuna homolog olan oluşturulur, öyle ki vektör linearize. <ol><li> 2 ug DNA, 7.5 U Bam HI, 7.5 U Xho I, Tampon Ba 20 ug BSA ve 2 ul içeren bir doğrusallaştırma reaksiyon karışımı hazırlayın20 ul nihai hacim içinde m HI 10x. </li><li> 2 saat ve 40 dakika boyunca 37 ° C&#39;de reaksiyon karışımı inkübe edin. Daha sonra, 20 dakika boyunca 80 ° C &#39;de inaktivasyonu devam edin. </li></ol></li><li> Kalan dairesel plazmid (Şekil 2) ile kirlenmesini önlemek için agaroz jel ekstraksiyon tarafından lineerleştirilebilir vektörü arındırın. <ol><li> de bir muhabir bitişik reaksiyon karışımı ayrıca bir alikosu (5 ul) (v, w% 0.75), bir yarı-preparatif düşük erime noktalı agaroz jel mega kuyuya sindirim Reaksiyon karışımı yükleyin. </li><li> Run DNA elektroforezi (elektrotlar arasında 5 V / cm, 4 ᵒC) ve mega-kuyuya gelen agaroz jel ayırmak ve 1x TAE 4 ᵒC sıcaklıkta saklayın. </li><li> molekül ağırlığı merdiven ve raportör ile sokağın Leke. UV ışık altında bantları gözünüzde canlandırın. Nick pozisyon Lineerleştirilmiş vektör yerleştirir. Not: saflaştırılmıştır lineer vektör kalitesi great.Thanks olduğucal faktör maya başarılı rekombinasyon ve montaj, yarı hazırlayıcı DNA elektroforezi için jel boyama kaçının. Boyalar ve jel ekstraksiyonu için, UV&#39;ye maruz kalmadan kullanılması, in vivo rekombinasyon verimliliği ödün, DNA vektörünün stabilitesini etkileyebilir. Toksik EtBr boyalar alternatif olarak, jel Kırmızı ve SYBR boyalar genel olarak jel boyama için kullanılır. </li><li> UV ışık yokluğunda, bu izole edilebilir ve böylece lekeli raportör şeritte sıyrık yol göstericiliği kullanılarak Mega oyuklu fragmanında lineer vektör tanımlar. </li><li> agarozdan lineer vektör çıkarın ve üreticinin protokolüne uygun olarak, ticari bir jel özütleme kiti ile saflaştırılması. Not: antibiyotik ve oksotrofi işaretleri kullanan yüksek kopya epizomal mekik vektörleri: Bu maya GAL1 promoterinin kontrolü altında, urasil bağımsız ve ampisilin dirençli pJRoC30 vektör kullanılabilir Bu örnekte. </li></ol></li><li> eşmolar MI hazırlanmasıPCR parçalarının soğutun ve 2 de lineer vektör ile karıştırarak: doğrusallaştırılmış plazmidin en az 100 ng, 1 oranında (eşmolar kütüphanesi açık / vektör test farklı oranlarda iyi bir dönüşüm verimi elde etmek için). <ol><li> konsantrasyon ve saflığı belirlemek için PCR parçaları ve 260 nm ve 280 nm &#39;de lineer vektör absorbansı ölçülür. </li></ol></li><li> Bir ticari maya dönüştürme kiti kullanılarak DNA karışımı maya yetkin hücrelerini dönüştürmek, üreticinin talimatlarına uygun olarak (malzeme Tablo). <ol><li> Bağımlı S. - Burada bir yoksun proteaz ve URA3 kullanın cerevisiae suşu, BJ5465. (Aşağıya bakınız), tarama sırasında bir iç standart olarak ebeveyn daireselleşmiş vektörle hücrelerini dönüştürmek. Buna ek olarak, PCR parçalarının yokluğunda lineer vektör dönüştürerek arka plan kontrol edin. Not: ilk düşük salgılanmasını tespit durumunda, faydalanmaBJ5465 gibi cerevisiae proteaz eksikliği olan suşlarıdır kültür yüzey aktif protein birikimi geliştirmek için. Hedef enzimin hiperglikosilasyon glikosilasyon eksikliği cinslerinin kullanımını maruz (örneğin, sadece daha küçük manoz oligomerleri bağlama kapasitesine sahip Δ kre2), uygun bir seçenek olabilir. </li></ol></li><li> Plaka dönüştürdü SC bırakma plakalar üzerinde hücre ve üç gün boyunca 30 ° C&#39;de inkübe. Plakası (urasil ile desteklenmiş SC bırakma tabakalarına) URA3 - S. taranması için bir negatif kontrol olarak plazmid eksik S. cerevisiae hücreleri (aşağıya bakınız). </li></ol> Testi Tarama 2. Yüksek Verimli (Şekil 3) <ol><li> bir pipetleme robotu yardımıyla göz başına 50 | il minimum ortam maddesi ile (2.000 klon kütüphanesinin analiz 23 levha) steril 96 oyuklu plakalar bir uygun sayıda doldurun. </li><li> plakalar dışında SC-açılan bireysel koloniler seçin ve 96-kuyucuğu aktarabilirsiniz. <ol><li> Her plaka olarak, ebeveyn bir iç standart olarak tip ve iyi URA3 ile H1 ile sütun numarasını 6 aşılamak - S. Bir negatif kontrol olarak hiçbir plasmid ile (urasil ile takviye SC ortamı içinde) cerevisiae hücreleri. Not: Kuyu H1 urasil ile desteklenmiş bırakma medya ile özel olarak doldurulur. hücre içermeyen boş bir de ihtiva eden ortam ayrıca bir sterilite kontrolü olarak hazırlanabilir. </li></ol></li><li> onların kapaklı tabak kapatın ve Parafilm onları sarın. Nemli bir çalkalayıcıda, 30 ° C&#39;de 48 saat, 225 rpm ve 80% bağıl nem için inkübe edin. </li><li> Parafilm kaldır pipetleme robotu yardımıyla her bir oyuğa sentezleme ortamı 160 ul, plakalar yeniden kapatın ve 24 saat daha inkübe. Not: Başka bir yerde 19 bildirildiği gibi Minimal orta ve ifade ortamı hazırlanır. Salgılama seviyeleri t, ve buna bağlı olarak çalışma altında genin bağlı olarak değişebilirO zaman tüm oyuklara hücre büyümesini senkronize etmek için her bir durumda, optimize edilmelidir kuluçkalama. </li><li> Santrifüj, 4 ° C&#39;de 10 dakika boyunca 2,800 x g plakaları (ana plakalar). </li><li> Aktarım robot çok istasyonlu işleme bir sıvı kullanarak çoğaltma plakasına ana plakadaki kuyulardan süpernatan 20 ul. Not: enzim salgılanmasını desteklemek amacıyla yaygın maya heterolog ekspresyonu (kullanılan sinyal peptidleri hedef proteinin doğal sinyal peptidi yerine tavsiye edilir, örneğin, α faktörü prepro-lideri, S. K 1. Öldürücü toksini lideri cerevisiae veya her iki peptit 13) daha uyumlu versiyonlarıdır. Seçenek olarak ise, doğal sinyal peptid sadece maya içinde salgılanması için açığa çıkabilir. </li><li> Pipetleme robot yardımı ile 100 mM sodyum fosfat tampon maddesi, pH 6.0 içinde 2 mM p -methoxybenzylalcohol 20 ul ekle. 96-biz kısaca plakaları karıştırınve kap mikseri ll, oda sıcaklığında 30 dakika boyunca inkübe. </li><li> Pipetleme robot ile, her çoğaltma plakasına FOX reaktifi 160 ul ve de FOX karışımının karıştırıcı (nihai konsantrasyon kısaca karıştırılmıştır: 100 uM ksilenol turuncu, 250 uM Fe (NH4) 2 (SO4) 2 ve 25 mM H 2 SO 4). <ol><li> Bu tür organik yardımcı solventlerin (DMSO, etanol, metanol) veya sorbitol 17 olarak duyarlılığını artırmak için bir reaktif için çeşitli katkı maddeleri ekleyin. Burada, 100 mm (Şekil 4) bir son konsantrasyona kadar sorbitol eklenerek cevap yükseltmek. </li></ol></li><li> Bir plaka okuyucusu üzerinde 560 nm&#39;de plakaları (uç noktası modu, t = 0) okuyun. </li><li> Renk gelişene kadar oda sıcaklığında inkübe ve tekrar (t 1) emilimini ölçmek. <ol><li> kuluçkadan sonra Abs değeri arasındaki farktan göreceli aktivitesini hesaplamak ve ilk ölçüm o pare normalizeHer bir plaka için ntal tipi (¨t 1 - t = 0). </li></ol></li><li> yanlış pozitif ekarte etmek iki ardışık yeniden gösterimleri için en iyi mutant hit Konu. Not: Genellikle, yeniden gösterimleri plazmid 19 yaş maya hücrelerinin dönüşümü, ardından, Escherichia coli içinde maya, amplifikasyon ve arıtma için plazmid izolasyon bulunmaktadır. Seçilen her bir klon pentaplicate yeniden elenir. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

Bu yazıda, ipuçlarını çoğu S. yönlendirilmiş evrim tarafından enzimleri mühendisi laboratuarımızda istihdam özetledik cerevisiae (örnek olarak AAO kullanarak) onlar sadece burada açıklanan ortak bir yaklaşım izlenerek diğer ökaryotik enzim sistemleri ile kullanım için adapte edilebilir, böylece. Kütüphane oluşturulması açısından, biçim vermek ve 16 değiştirilmemiş proteinin geri kalan bölgeleri, bırakarak küçük protein uzan?...

Yönetmen moleküler evrim enzimleri 1, 2 tasarlamak için bir, sağlam, hızlı ve güvenilir bir yöntemdir. Doğal olmayan olarak, yeni reaksiyonlarda, yeni yüzeylerde hareket rastgele mutasyon, rekombinasyon ve tarama, enzimlerin geliştirilmiş versiyonları oluşturulabilir yinelenen mermi ile ortamlarda, hatta yeni metabolik hedeflere 3-5 ulaşmak için hücreyi yardımcı olmak. Yönlendirilmiş evrim kullanılan bilgisayarlar arasında, bira mayası Saccharomyces cerevisiae prokaryotik meslektaşları 6,7 aksi bulunmayan karmaşık ökaryotik proteinlerin fonksiyonel ifade çözümler bir repertuar sunuyor. Hücre biyolojisi çalışmalarında etraflıca kullanılan bu küçük ökaryot modeli yönlendirilmiş evrim 8 tarafından enzimleri mühendisi önemli özellikleri bütün bunlar post-translasyonel modifikasyonlar, manipülasyon ve dönüşüm verimliliği kolaylığı açısından birçok avantajı vardır. Ayrıca, yüksek frekanslıS. homolog DNA rekombinasyon onun etkin proof-okuma aygıtı bağlanmış cerevisiae karmaşık yapay yolların 9-12 tek enzimlerin farklı sistemlerin evrimi teşvik, in vivo kütüphane oluşturma ve gen montaj olanakları geniş bir yelpazeye açar. Laboratuvarımız maya farklı linyinaz moleküler evrim (doğal ahşap çürüme sırasında lignin parçalanması ile ilgili oksiredüktazlan) 13-14 için araçlar ve stratejiler tasarlanması son on harcadı. Bu iletişimde, biz hazırlamak ve S. ekran mutant kütüphaneleri için ayrıntılı bir protokol mevcut Model flavooxidase, -aril alkol oksidazı cerevisiae (AAO 15) -, kolayca diğer enzimlere tercüme edilebilir. Maya hücre aparatı 16, bir yardım: (Homolog in vivo Gruplama tarafından mutajenik Organize Rekombinasyon Süreci Morphing) protokolü odaklanmış yönlendirilmiş evrim yöntemini içerirKültür suyu 17 salgılanan AAO aktivitesi tespit etmek için Fenton tepkimesi göre da çok hassas bir tarama deneyi.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1. Culture media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>A0166</td>
			<td>CAS N&#186; 69-52-3 M.W. 371.39</td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>Difco</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C-0378</td>
			<td>CAS N&#186; 56-75-7 M.W. 323.13</td>
		</tr>
		<tr>
			<td>D-(+)-Galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G0750</td>
			<td>CAS N&#186; 59-23-4 M.W. 180.16</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td>CAS N&#186; 50-99-7 M.W. 180.16</td>
		</tr>
		<tr>
			<td>D-(+)-Raffinose pentahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>83400</td>
			<td>CAS N&#186; 17629-30-0 M.W. 594.51</td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Difco</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P0662</td>
			<td>CAS N&#186; 7778-77-0 M.W. 136.09</td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>Sigma Aldrich</td>
			<td>U1128</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Difco</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Nitrogen Base without Amino Acids</td>
			<td>Difco</td>
			<td>291940</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Synthetic Drop-out Medium Supplements without uracil</td>
			<td>Sigma-Aldrich</td>
			<td>Y1501</td>
			<td></td>
		</tr>
		
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>2. PCR Reactions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Mix</td>
			<td>Agilent genomics</td>
			<td>200415-51</td>
			<td>25 mM each</td>
		</tr>
		<tr>
			<td>iProof High-Fidelity DNA polymerase</td>
			<td>Bio-rad</td>
			<td>172-5301</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese(II) chloride tetrahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M8054</td>
			<td>CAS N&#186; 13446-34-9 M.W. 197.91</td>
		</tr>
		<tr>
			<td>Taq DNA Polymerase</td>
			<td>Sigma-Aldrich</td>
			<td>D4545</td>
			<td>For error prone PCR</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>3. Plasmid linearization</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI restriction enzyme</td>
			<td>New England Biolabs</td>
			<td>R0136S</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>New England Biolabs</td>
			<td>B9001S</td>
			<td></td>
		</tr>
		<tr>
			<td>XhoI restriction enzyme</td>
			<td>New England Biolabs</td>
			<td>R0146S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Red</td>
			<td>Biotium</td>
			<td>41003</td>
			<td>For staining DNA</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>4. FOX assays</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium iron(II) sulfate hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>F3754</td>
			<td>CAS N&#186; 7783-85-9 M.W. 392.14</td>
		</tr>
		<tr>
			<td>Anysil Alcohol</td>
			<td>Sigma Aldrich</td>
			<td>W209902</td>
			<td>CAS N&#186; 105-13-5 M.W. 138.16</td>
		</tr>
		<tr>
			<td>D-Sorbitol</td>
			<td>Sigma-Aldrich</td>
			<td>S1876</td>
			<td>CAS N&#186; 50-70-4 M.W. 182.17</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide 30%</td>
			<td>Merck Millipore</td>
			<td>1072090250</td>
			<td>FOX standard curve</td>
		</tr>
		<tr>
			<td>Xylenol Orange disodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>52097</td>
			<td>CAS N&#186; 1611-35-4 M.W. 716.62</td>
		</tr>
		<tr>
			<td>Agarose gel stuff</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Norgen</td>
			<td>28035</td>
			<td>CAS N&#186; 9012-36-6</td>
		</tr>
		<tr>
			<td>Gel Red</td>
			<td>Biotium</td>
			<td>41003</td>
			<td>DNA analysis dye</td>
		</tr>
		<tr>
			<td>GeneRuler 1Kb Ladder</td>
			<td>Thermo Scientific</td>
			<td>SM0311</td>
			<td>DNA M.W. standard</td>
		</tr>
		<tr>
			<td>Loading Dye 6x</td>
			<td>Thermo Scientific</td>
			<td>R0611</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-melting temperature agarose</td>
			<td>Bio-rad</td>
			<td>161-3112</td>
			<td>CAS N&#186; 39346-81-1</td>
		</tr>
	
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>5. Kits and cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>S. cerevisiae strain BJ5465</td>
			<td>LGC Promochem, Spain</td>
			<td>ATTC 208289</td>
			<td>Protease deficient strain with genotype: MAT&#945; ura3-52 trp1 leu2-delta1 his3-delta200 pep4::HIS3 prb1-delta1.6R can1 GAL</td>
		</tr>
		<tr>
			<td>E. coli XL2-Blue competent cells</td>
			<td>Agilent genomics</td>
			<td>200150</td>
			<td>For plasmid purification and amplification</td>
		</tr>
		<tr>
			<td>NucleoSpin Gel and PCR Clean-up Kit</td>
			<td>Macherey-Nagel</td>
			<td>740,609,250</td>
			<td>DNA gel extraction</td>
		</tr>
		<tr>
			<td>NucleoSpin Plasmid Kit</td>
			<td>Macherey-Nagel</td>
			<td>740,588,250</td>
			<td>Column miniprep Kit</td>
		</tr>
		<tr>
			<td>Yeast Transformation Kit</td>
			<td>Sigma-Aldrich</td>
			<td>YEAST1-1KT</td>
			<td>Included DNA carrier (Salmon testes)</td>
		</tr>
		<tr>
			<td>Zymoprep yeast plasmid miniprep I</td>
			<td>Zymo research</td>
			<td>D2001</td>
			<td>Plasmid extraction from yeast</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>6. Plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Greioner Bio-One</td>
			<td>655101</td>
			<td>Clear, non-sterile, Polystyrene (for activity measurements)</td>
		</tr>
		<tr>
			<td>96-well plates</td>
			<td>Greioner Bio-One</td>
			<td>655161</td>
			<td>Clear, sterile, Polystyrene (for microfermentations)</td>
		</tr>
		<tr>
			<td>96-well plate lid</td>
			<td>Greioner Bio-One</td>
			<td>656171</td>
			<td>Clear, sterile, Polystyrene (for microfermentations)</td>
		</tr>
	</tbody>
</table>

directed evolution method in saccharomyces cerevisiae: mutant library creation and screening

In vivo yüksek frekans homolog DNA rekombinasyon bağlanmış inşaat, klonlama ve mutant kütüphanelerin ifadesini içeren bir süreç biyoteknolojik uygulamalar için enzimler tasarlarken Saccharomyces cerevisiae yönlendirilmiş evrim çok çekici avantajlar sunuyor. Burada, toplam etkinliğini arttırmak için bir mantar aril-alkol oksidaz (AAO) örneğine dayalı olarak maya ve ekran mutant kütüphaneleri oluşturmak için bir protokol mevcut. İki protein kesimleri rastgele mutagenezi ile ve in vivo DNA yeniden birleştirmesinden odaklı yönelik evrim tabi tutuldu. Her bir segment kuşatan 50 bp çıkıntıları tam kendiliğinden kopyalanan plazmidin sebebiyet veren bir doğrusallaştırılmış vektörde AAO-füzyon geninin, doğru yeniden birleştirme sağladı. Fonksiyonel AAO türevleri ile zenginleştirilmiş mutant kütüphaneler S. tarandı Fenton tepkimesi göre hassas bir yüksek verimli bir deney ile cerevisiae süpernatanlar. genel prosesS. kütüphane inşaat cerevisiae ilave PCR reaksiyonları, in vitro DNA rekombinasyon ve ligasyon adımlarından kaçınmak, burada açıklanan kolaylıkla diğer ökaryotik genlerin gelişmeye uygulanabilir.

P. AAO eryngii lignin saldıran başlamak için H 2 O 2 ile mantar peroksidazlar sağlayan bir hücre dışı flavooxidase olduğunu. AAO iki segment etkinliğini ve S ifadesini arttırmak için geçişin tarafından odaklanmakta-yönlendirilmiş evrim tabi tutuldu cerevisiae 19. S. tarafından barındırdığı yabancı enzimlerin bağımsız olarak cerevisiae, maya mutant kütüphaneler inşa fragman...

Watch this Scientific Journal Video about Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening at JoVE.com

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 ...

directed-evolution-method-saccharomyces-cerevisiae-mutant-library

Research

JoVE Journal

Biology

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

10.8K Görüntüleme.  Institute of Catalysis, CSIC. We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae. 

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

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Diseksiyonu Saccharomyces cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Biyoloji

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

Yağ Asidi Kantitatif Phosphoproteomics Uyarılmış Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Proteinlerin Yönetmen Evolution Mutagenez ve Fonksiyonel Seçim Protokolleri E. coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

Mikroarray Analizi için Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Maya İzleme Mitophagy Floresan Mikroskopi Testi Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

MISSION LentiPlex Pooled shRNA Library Screening in Mammalian Cells

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to knockdown gene expression levels in loss of gene function studies.
There are two main methods for performing RNAi. The first is the use of small interfering RNAs (siRNAs) that are chemically synthesized, and the second utilizes short-hairpin RNAs (shRNAs) encoded within plasmids 1. The latter can be transfected into cells directly or packaged into replication incompetent lentiviral particles. The main advantages of using lentiviral shRNAs is the ease of introduction into a wide variety of cell types, their ability to stably integrate into the genome for long term gene knockdown and selection, and their efficacy in conducting high-throughput loss of function screens. To facilitate this we have created the LentiPlex pooled shRNA library.
The MISSION LentiPlex Human shRNA Pooled Library is a genome-wide lentiviral pool produced using a proprietary process. The library consists of over 75,000 shRNA constructs from the TRC collection targeting 15,000+ human genes 2. Each library is tested for shRNA representation before product release to ensure robust library coverage. The library is provided in a ready-to-use lentiviral format at titers of at least 5 x 108 TU/ml via p24 assay and is pre-divided into ten subpools of approximately 8,000 shRNA constructs each. Amplification and sequencing primers are also provided for downstream target identification.
Previous studies established a synergistic antitumor activity of TRAIL when combined with Paclitaxel in A549 cells, a human lung carcinoma cell line 3, 4. In this study we demonstrate the application of a pooled LentiPlex shRNA library to rapidly conduct a positive selection screen for genes involved in the cytotoxicity of A549 cells when exposed to TRAIL and Paclitaxel. One barrier often encountered with high-throughput screens is the cost and difficulty in deconvolution; we also detail a cost-effective polyclonal approach utilizing traditional sequencing.

Here we use a human LentiPlex pooled library and traditional sequencing methods to identify gene targets promoting cell survival. We demonstrate how to set up and deconvolute a LentiPlex screen and validate the results.

MİSYON LentiPlex Memeli Hücreleri shRNA Kütüphane Tarama Pooled

RNA interference (RNAi) is an intrinsic cellular mechanism for the regulation of gene expression. Harnessing the innate power of this system enables us to ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

Yılında Kistik Fibrozis Transmembran İletkenlik Regülatörü Protein İfadesi ve Arıtma Saccharomyces cerevisiae

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Floresan kullanarak mRNA Moleküllerin Görselleştirme ve Analiz<em&gt; Yerinde</emIn&gt; Hibridizasyon<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

İzlenmesi protein refolding için birleştiğinde Tahliller<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

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