我们非常感谢来自新加坡（ETRP 1201102）的国家环境局的资金支持，新加坡国立研究基金会（NRF-CRP5-2009-03），该机构对新加坡科学技术研究的竞争研究计划（ 1324004108），全球R＆D项目计划，知识经济部，韩国（N0000677），国防威胁降低局（DTRA，HDTRA1-13-1-0037）和合成生物学新加坡国立大学倡议（ DPRT / 943/09/14）。

1.新一代的Y的解脂 KU70删除应变<ol><li>中断盒建设 注:请参阅表1为聚合酶链反应（PCR）扩增使用的所有引物。 <ol><li>设计的引物20进行PCR扩增LEU2表达盒（ 见表1）从一个Y.和一个长的反向引物（＃2; 表1）分别; 解脂表达载体并引入LoxP位点到5&#39;和3&#39;LEU2启动盒与长正向引物（ 表1＃1）结束。引入用于克隆过程的后续步骤的额外限制性位点，加在引物＃1的巴姆 HI限制性位点。 </li><li>使用高保真DNA聚合酶如表2中详述的进行PCR。 </li><li>使用PCR purificatio纯化PCR产物loxP的启动子 - LEU2 - 终止-LoxP位盒根据制造商的说明Ñ套件。根据制造商的说明21 ...添加3&#39;-A突出的纯化的PCR产物。使用T4 DNA连接酶结扎A-尾PCR产物入TA克隆载体，产生质粒T-LEU2按表3。 </li><li>用PCR引物＃3/4＃从Y.扩增基因KU70的1 kb的5&#39;上游序列解脂 Po1g按表2的基因组DNA 22。纯化使用根据制造商的说明一个PCR纯化试剂盒将PCR产物。 <ol><li>双重消化两个纯化的PCR产物和T-LEU2质粒用Sac II和BamHⅠ酶按表4。使用PCR纯化试剂盒按照制造商的说明纯化消化混合物。使用T4 DNA连接酶，以结扎纯化和消化的PCR产物的囊 II / T-LEU2的巴姆 HI位点，得到质粒T-LEU2-5E按表3。 </li></ol></li><li>使用引物＃5 /＃6从Y.扩增KU70基因的1kb的3&#39;下游序列解脂 Po1g按表2的基因组DNA 22。纯化使用根据制造商的说明一个PCR纯化试剂盒将PCR产物。双重消化两个纯化的PCR产物和用Not I和的Nde I酶按表4的T-LEU2-5E质粒。 <ol><li>纯化使用PCR纯化试剂盒按照制造商的说明将消化混合物。然后，结扎纯化和消化的PCR产物的未 I / 的Nde I位点的T-LEU2-5E的，得到使用T4DNA连接酶按表3质粒的T-KO。 </li></ol></li><li>消化质粒T-KO用Sac II和NDE我酶按表4，产生中断盒（ 图1 </strong&gt;的）。纯化使用PCR纯化试剂盒按照制造商的说明将消化的DNA片段。 </li></ol></li><li>感受态细胞制备和Y改造解脂 Po1g应变<ol><li>感受态细胞制备<ol><li>接种Y.的殖民地从在10ml YPD培养基（1％酵母提取物，2％蛋白胨，2％葡萄糖和50mM柠檬酸盐缓冲液pH 4.0）中的100毫升烧瓶中新鲜酵母提取物-蛋白胨-葡萄糖（YPD）板解脂 Po1g菌株。孵育在30℃振荡培养箱中于225rpm下20小时，直到饱和度（的OD为约15 600，使用分光光度计测量）。 </li><li>通过在室温下以5000 xg离心离心5分钟沉淀细胞。清洗细胞用20ml的Tris-EDTA（TE）缓冲液（10mM的Tris，1mM EDTA中，pH值7.5），并如在步骤1.2.1.2所述细胞沉淀。重悬在1ml的0.1M乙酸锂（pH值6.0，用乙酸调节）的细胞中，并孵育在室温坦佩10分钟叉涂抹。 </li><li>分装感受态细胞（100微升）进入无菌1.5毫升管。进行到转化步骤下面马上或在-80℃下长期贮存添加甘油至25％的终浓度（体积/体积），并存储。 </li></ol></li><li>转型<ol><li>轻轻10微升变性的鲑精子DNA（10毫克/毫升）和1-5微克纯化的破坏盒的与100微升的感受态细胞混合在一起，并在30℃下孵育15分钟。 </li><li>加入700微升40％聚乙二醇（PEG）-4000（溶于0.1M醋酸锂pH值为6.0）中，拌匀，并在225转速在30℃摇床孵育60分钟。 注意:使用的PEG 4000平均分子量，代替PEG-3350（典型地在常规的酵母的转化中使用）是重要的。 </li><li>热火通过将管在39℃水浴60分钟震荡转化混合物。 </li><li>加入1 ml YPD中，恢复为在30℃下2小时，225转。离心机以9000rpm的XG在室温下1分钟，除去上清液和重悬沉淀在1ml TE缓冲液中。 </li><li> Repellet通过离心将细胞以9000rpm的XG的在室温下1分钟，并弃去上清液。重悬在100μlTE缓冲液中的沉淀和板到选择平板（ 例如 ，亮氨酸缺陷型板），并在30°C孵育2-3天。 </li><li>选择4至10个单菌落，并分别接种入2ml YPD培养基。在225rpm下在30℃振荡培养箱中孵育过夜。从转化22识别由基因组DNA的PCR分析阳性菌落按使用两组引物＃55 /＃56和＃56 /＃57 表5中 ，分别。 注意: 未 I线性pYLEX1载体转化到Y.解脂 Po1g感受态细胞，以便通过计数的数目来确定转化效率每微克质粒的菌落形成单位（cfu）的DNA用于23。 </li></ol></li></ol></li></ol> 2.标记救援<ol><li>华创表达载体的构建<ol><li> pYLEX1-CRE和pYLEX1-HPH建设<ol><li>设计的引物20进行PCR扩增CRE和HPH的开放阅读框。添加额外的腺嘌呤核苷酸的起始密码子上游的正向引物＃82和＃84，和插入在反向引物＃83和＃85的终止密码子的下游的PCR扩增CRE的开放读框的KpnI限制性位点和从pSH69（登录号HQ412578）24 HPH按表2。 </li><li>净化既CRE和使用根据制造商的说明一个PCR纯化试剂盒HPH片段。既消化酶我按照TABL与KPN纯化的综合招聘考试及HPH片段E 4。双酶切与PML和KpnI酶按表4中的pYLEX1质粒。用PCR纯化试剂盒按照制造商的说明纯化消化混合物。 </li><li>使用T4 DNA连接酶按表3结扎纯化和消化CRE和HPH片段至Y的PML的I / 的KpnI位点解脂表达载体，产生pYLEX1-CRE和pYLEX1-HPH分别。 </li></ol></li><li> pSL16-CRE-HPH建设<ol><li>使用引物＃86放大来自pYLEX1-CRE CRE的启动子-基因-终止子盒/＃87侧翼萨尔的I /PstⅠ限制位点按表2中 。根据制造商的说明纯化使用PCR纯化试剂盒将PCR产物。 <ol><li>双重消化两个纯化的PCR产物和pSL16-CEN1-1（227）的质粒25用Sal I和的PstⅠ酶按表4。纯化使用PCR纯化试剂盒按照制造商的说明将消化混合物。然后，在相应的位点结扎纯化和消化的PCR产品放入pSL16-CEN1-1（227），使用T4 DNA连接酶按表3来创建pSL16-CRE。 </li></ol></li><li> PCR使用引物扩增来自pYLEX1-HPH HPH的启动子-基因-终止子盒＃88 /＃89侧翼Xho I 位 /BglⅡ位限制性位点按表2中 。使用PCR纯化试剂盒根据制造商的说明纯化PCR产物。 <ol><li>双重消化两个纯化的PCR产物和用Xho I和BglⅡ酶切酶按表4 pSL16-CRE质粒。用PCR纯化试剂盒按照制造商的说明纯化消化混合物。然后，结扎纯化和消化的PCR产品放入pSL16-CRE在相应​​的网站使用T4DNA连接酶按表3，以产生pSL16-CRE-HPH。 注意:将所得的Cre表达的质粒，pSL16-CRE-HPH，怀有可选择潮霉素B的标记，是着丝粒和附加型复制载体（ 图2）。 </li></ol></li><li>验证所有通过消化适当的限制性内切酶（ 例如 ， 萨尔我/ PST我，切除从载体插入）按表4的结构。 </li></ol></li></ol></li><li> Cre重组酶介导的标记救援<ol><li>变换pSL16-CRE-HPH进入淘汰赛KU70菌株的感受态细胞按照上述第1.2节所述的改造方案。板转化细胞到YPD加潮霉素B（YPDH）板（含有400微克/毫升潮霉素B），并孵育在30℃下2-3天。 </li><li>进行菌落PCR按表5中使用引物＃84/85＃识别正面含改造后pSL16-CRE-HPH质粒的菌落。 注意:正向引物＃84和反向引物＃85位于潮霉素基因（ 表1）的编码区内部。只阳性克隆产生在PCR反应的特定PCR产物。 </li><li>挑选阳性克隆，接种于2ml YPDH培养基中，并在225rpm下在30℃振荡培养箱中孵育过夜饱和。测量用分光光度计细胞OD 600。收获细胞以约15的OD 600。离心机在5000 xg离心在室温下5分钟，并用2毫升无菌水中洗一次。 </li><li>再接种细胞于2ml YPD培养基具有初始OD为0.1 600和允许细胞在30℃振荡培养箱中于225rpm下生长过夜。条纹过夜细胞培养到YPD平板分离单菌落23。复制品板菌落到YPD，YPDH和亮氨酸缺陷型板23 </s了&gt;。 注:已经失去了LEU2标记，pSL16-CRE-HPH质粒只能生长在YPD平板（ 图3）菌落。 </li></ol></li></ol> 3.乙醇脱氢酶和醇氧化酶基因的缺失<ol><li>执行对Y.搜索使用基本局部比对搜索工具（BLAST）来识别要删除的26基因的候选解脂基因组数据库。输入S的蛋白质序列酵母的乙醇脱氢酶我到BLAST搜索工具（http://www.genome.jp/tools-bin/search_sequence?prog=blast&amp;db=yli&amp;seqid=aa）。 注:BLAST搜索工具将返回最相似的蛋白质序列中的Y.解脂基因组数据库。 </li><li>构建通过PCR缺失盒用引物＃7至＃56（ 表1），并执行酶切，并使用上文第1.1节描述的相同的方法连接反应。 </li><li>预备是KU70敲除菌株的感受态细胞，进行转化和进行PCR通过以下在上述1.2节中描述的协议用 ​​引物＃55，＃58〜＃81（ 表1），以确定阳性菌落。 注意:作为一个例子，对于YALI0E17787g基因缺失的方法在图4和5进行说明。报告的1 kb和2 kb的同源区域同源重组率的影响。 </li></ol>

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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.

我们对这项研究的目的是来实现快速，高效的新一代靶向基因敲除在Y.解脂 Po1g应变。有好几个因素需要加以解决，以实现这一目标。首先，需要一个高转化效率。因此，高效，便捷的化学转化协议为Y.解脂 Po1g菌株在本研究中所描述。使用的PEG-4000的是该菌株的成功转化的关键因素。用PEG-3350时（为酿酒酵母的转化）代替PEG-4000质粒转化获得没有转化体。此外，需要使用?...

不像酿酒酵母 ， 解脂耶氏，一个非常规酵母，可以响应于环境条件1,2-变化在酵母或菌丝的形式生长。因此，这种二形酵母可用作真菌分化，形态发生和分类​​3,4,5的研究的良好模型。它通常被认为是安全的（GRAS）的酵母物种，它被广泛用于生产各种食品添加剂如有机酸，多元醇，芳香化合物，乳化剂和表面活性剂6,7,8,9这样。它是专性需氧菌和能够以高含量自然积累脂质的公知的含油酵母， 即高达细胞干重10的70％。它也可以利用碳源的广泛增长，其中包括浪费资源的养分11,12,13不同种类的残留物。所有这些独特的功能使Y.为解脂非常有吸引力各种生物技术应用。 虽然Y的全基因组序列解脂已发表​​14,15，这种非常规酵母的基因操作比其它酵母种更加复杂。第一，该酵母物种的转化是有效的要少得多，由于不存在稳定高效的遗传转化系统16,17的。第二，线性表达盒的费力的基因组整合通常用于感兴趣基因的表达，因为没有天然游离质粒系统已经在该酵母18被发现。第三，代遗传敲奏和敲造访受到限制，因为该基因通过准确同源重组在该酵母靶向效率是低的，并通过非同源末端连接（NHEJ）19最积分事件发生。 在这项研究中，我们报道了Y.优化改造协议解脂 </青霉&gt; Po1g应变，这是很容易，快速，高效和可重复的。以提高精确同源重组的频率，我们删除KU70基因，其编码在NHEJ途径的关键酶。通过使用优化的改造方案和改造含有侧翼的1 kb的同源地区，Y的KU70基因的敲除线性磁带解脂 Po1g被成功删除。这种基因缺失的方法的稳健性然后通过在Po1g KU70Δ应变靶向乙醇脱氢酶和醇氧化酶基因的证明。据观察，在KU70缺失菌株表现出同源重组介导的基因的一个相当高的效率比野生型Po1g应变的定位。此外，携带潮霉素B抗性标记的复制的Cre表达质粒构建以执行标记救援。标记救援促进多轮基因在针对第Ë获得的基因缺失突变体。除了 ​​基因缺失，我们对这里描述的遗传转化和基因缺失协议可应用到插入基因的具体位点，并引入位点特异性突变引入Y.解脂基因组。

<table border="0" cellpadding="0" cellspacing="0" width="605">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:140px;">Reagent/Material</td>
			<td colspan="3" style="width:465px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oligonucleotide primers</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>25 nmole&#160; DNA oligos</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Y. lipolytica strain Po1g</td>
			<td>Yeastern Biotech</td>
			<td></td>
			<td style="width:207px;">leucine auxotrophic derivative of the wild-type strain W29 (ATCC 20460)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vector pYLEX1</td>
			<td>Yeastern Biotech</td>
			<td>FYY203-5MG</td>
			<td>Y. lipolytica expression vector&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E. coli TOP10&#160;</td>
			<td>Invitrogen</td>
			<td></td>
			<td>For cloning and propagation of plasmids</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pGEM-T vector&#160;</td>
			<td>Promega</td>
			<td>A3600</td>
			<td>TA cloning vector</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>For plasmid isolation</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:140px;">Wizard SV Gel and 
			PCR Clean-Up System</td>
			<td>Promega</td>
			<td>A9282</td>
			<td style="width:207px;">Extract DNA fragments from agarose gels 
			and purify PCR products from an amplification reaction</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:140px;">The iProof high-fidelity 
			DNA polymerase</td>
			<td>Bio-Rad</td>
			<td>172-5302</td>
			<td>High-fidelity DNA polymerase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BamHI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0136S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BglII&#160;</td>
			<td>New England Biolabs</td>
			<td>R0144L</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KpnI</td>
			<td>New England Biolabs</td>
			<td>R0142S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NdeI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0111S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NotI</td>
			<td>New England Biolabs</td>
			<td>R0189L</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PmlI</td>
			<td>New England Biolabs</td>
			<td>R0532S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PstI&#160;</td>
			<td>New England Biolabs</td>
			<td>R0140S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SacII</td>
			<td>New England Biolabs</td>
			<td>R0157S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SalI</td>
			<td>New England Biolabs</td>
			<td>R0138S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">XhoI</td>
			<td>New England Biolabs</td>
			<td>R0146L</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T4 DNA ligase</td>
			<td>New England Biolabs</td>
			<td>M0202L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taq DNA polymerase&#160;</td>
			<td>Bio-Rad</td>
			<td>M0267L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ampicillin</td>
			<td>Gibco-Life Technologies</td>
			<td>11593-027</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hygromycin B</td>
			<td>PAA</td>
			<td>P21-014</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GeneRuler 1 kb DNA ladder</td>
			<td>Thermo Scientific</td>
			<td>SM0312</td>
			<td>1 kb DNA ladder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PEG4000</td>
			<td>Sigma</td>
			<td>95904-F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris</td>
			<td>Promega</td>
			<td>H5135</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Bio-Rad</td>
			<td>161-0729</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Salmon Sperm DNA</td>
			<td>Invitrogen</td>
			<td>15-632-011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lithium Acetate</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass beads (425-600 &#181;m)</td>
			<td>Sigma</td>
			<td>G8772</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNAse A</td>
			<td>Thermo Scientific</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA Loading Dye</td>
			<td>Thermo Scientific</td>
			<td>R0611</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Yeast Extract</td>
			<td>BD</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D-Glucose</td>
			<td>1st Base</td>
			<td>BIO-1101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">YNB without amino acids</td>
			<td>Sigma</td>
			<td>Y0626</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DO Supplement-Leu</td>
			<td>Clontech</td>
			<td>630414</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Difco LB Broth</td>
			<td>BD</td>
			<td>244620</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Difco LB Agar</td>
			<td>BD</td>
			<td>244520</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bacto Agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR machine</td>
			<td>Biorad</td>
			<td>T100 Thermal Cycler</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water bath</td>
			<td>Memmert</td>
			<td>WNB 14</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stationary/Shaking Incubator</td>
			<td>Yihder</td>
			<td>LM-570RD</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermo-shaker</td>
			<td>Allsheng</td>
			<td>MS-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro centrifuge</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spectrophotometer</td>
			<td>Eppendorf&#160;</td>
			<td>BioPhotometer plus</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel imager</td>
			<td>GE</td>
			<td colspan="2">Amersham Imager 600</td>
		</tr>
	</tbody>
</table>

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

解脂耶氏是一种非致病的，二相性和严格的有氧酵母物种。由于其独特的生理功能和代谢特征，这非常规酵母不仅对真菌分化的基本性质的研究的良好模型，但也用于生化生产和各种生物技术应用，其需要大量的基因操作一个有希望的微生物的平台。不过，Y的遗传操作解脂一直有限由于缺少一种有效的和稳定的遗传转化系统以及可主要归因于KU70基因的非同源重组率很高。在这里，我们报告一个简单而快速的协议为高效遗传转化并为Y.基因缺失解脂 Po1g。首先，外源DNA的高效转化为Y.协议解脂 Po1g成立。山高次，以达到增强的双交换同源重组速率为靶基因的进一步缺失，所述KU70基因通过转化中断箱运送1kb的同源臂删除。第三，以展示KU70基因的缺失后增强的基因缺失的效率，我们单独删除11编码上使用KU70淘汰赛平台菌株的相同程序乙醇脱氢酶和醇氧化酶的靶基因。据观察，精确的同源重组的速率从低于0.5％大幅增加为删除 在Po1g的KU70基因到33％-71％，为单基因缺失Po1g KU70Δ11靶基因。复制型质粒携带潮霉素B抗性标记和酶Cre / loxP系统构建，并最终被Cre重组酶表达去除酵母基因敲除菌株的选择标记基因便于多轮圆盾的特德遗传操作。所得单基因缺失突变体具有在生物燃料和生化生产的应用潜力。

线性Y.解脂表达载体插入到PBR对接平台在Y的基因组解脂 Po1g应变通过执行一个交叉重组27。通过使用本研究建立的快速化学转化过程中，线性Y.解脂表达载体在&gt; 100个/微克DNA的转化效率被成功转化到野生型Po1g菌株。由1kb的同源序列侧接敲除盒转化到Po1g菌株和KU70基因被成功删除（ 图1）。这里，正向引物＃56退火到位?...

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.

可再生生物燃料及生化生产的非常规酵母基因工程

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

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13.5K 次观看.  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.

视频: 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.

机器人平台的高通量原生质体分离和转型

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 ...

科研

遗传学

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

一种从任意 DNA 源进行大规模 gRNA 库生产的通用协议

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

Production and Purification of Baculovirus for Gene Therapy Application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

杆状病毒基因治疗应用的生产与纯化

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

细菌结合频率的高分辨率比较

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

人牙小泡干细胞的分离、鉴定及微 rna 遗传修饰

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

基于细菌选择和生长的盘状细胞基因工程

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

一种在裂变酵母中的深度固序辅助自发抑制屏

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60&#8722;80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2&#8722;4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

用于抗倍体植入前基因测试的半导体测序

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

哺乳动物细胞中基于CRISPR的聚集基因屏幕

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.