笔者要感谢我所有的本科独立研究学生的我谁在2010-2014年春季学期测试了我的转座子突变的想法微生物生理学研究生和一切。这项工作是由美国生物科学系的扬斯敦州立大学。

1.电穿孔感受态细胞的5,11 <ol><li>稀肠杆菌属的O / N LB培养。 YSU 1/20到50ml新鲜LB培养基中，并与以120rpm振荡及30℃至0.4和0.6之间的OD（600 nm）的生长。或者，种植其他细菌菌株在其最佳生长温度。 </li><li>寒意在冰上将细胞5分钟，并离心在4℃和7000×g离心5分钟。 </li><li>弃上清，重悬细胞于50ml无菌冰冷的水和离心机在4℃和7000×g离心5分钟。重复此步骤。 </li><li>弃去上清并将细胞重新悬浮在冰冷的水等于将细胞沉淀体积的体积。对于长期储存，在-80℃下，并解冻洗涤细胞，用10ml冰冷的10％（体积/体积）甘油，重悬在冰冷的10％（体积/体积）甘油，保存一个等于粒料体积上电前的冰。 </li><li>加入0.5微升的转座体的40微升细胞。市售的转座体溶液以0.33微克/微升的DNA浓度提供。尽管0.33微克建议，0.165微克的DNA是足够的。 </li><li>在冰冷，0.2毫米，电穿孔杯中放置细胞/转座体的混合物，然后点击该混合物以反应杯的底部。惊世骇俗的细胞在25μF，200Ω和2.5千伏。以确保细胞保持冷冻，在使用前保存在-20℃下冷冻的电试管。 </li><li>随即，加入960微升过滤灭菌代谢物阻遏（SOC）中的超最佳肉汤。通过上下吹打混匀，将细胞转移到一个无菌的1.5mL微管中。 注:该细胞开始震荡后死亡，但在SOC中的盐可以让他们恢复。节约，这是没有必要添加转座子或震荡阴性对照细胞。只需添加960微升无菌的SOC培养基它。 </li><li>孵育的细胞在30℃C 2 F或45-60分钟，以120rpm振荡。这提供了时间的转座到重组与宿主基因组，并为细胞表达卡那霉素抗性。 </li><li>传播100微升细胞在LB根琼脂平板上，孵育板O / N在30℃。保存剩余的转化混合物在冰箱中在4℃下。在稍后的日期长达2周的初始电穿孔后，如果需要扩展的附加量。 </li></ol> 2.网格化转化<ol><li>带的网格为空100×15mm的皮氏培养皿的盖子。从"A短期课程中的细菌遗传学"12复制一个网格。 </li><li>划在LB根琼脂板的底部的线。用一小片胶带将其固定到网格盖子，以便网格是通过琼脂可见。确保在板的底部的线，与所述网格的顶部，中心对齐。锚定板带滑动保持它，允许甚至是网格。副本电镀后的线路有利于菌落鉴定。 </li><li>挑取单个转化体用灭菌牙签并发现它在网格中的一个正方形的中心。刚刚接触的殖民地和地点。不掏牙签插入琼脂。为了避免污染板，只在一端处理牙签。 </li><li>现货在相邻的广场另一转化为步骤2.3。当板是满的，它孵化O / N在30℃。这是主盘。 </li></ol> 3.副本电镀以确定营养缺陷型表型的12 <ol><li>因为这是每个网格板，使一叠三间新鲜琼脂平板上编号1至三种:一为LB-根，两个用于M-9根和三为LB-根。使用前擦干板倒挂，O / N，在37℃。 </li><li>绘制在各板的顶部的线，如步骤2.2。 </li><li>擦拭副本电镀工具用70％乙醇，将一个无菌的平绒正方形上的副本电镀的顶部到OL和夹紧平绒广场下来。双手甚至洗完后充满了微生物。防止引进由它的边缘处理平绒广场污染微生物。 </li><li>排列在主盘的线以描绘在夹具的线，然后将板在平绒正方形的顶部。轻轻推压，从板块转移细菌平绒广场。小心不要涂抹细菌。取下平绒广场的主盘。 </li><li>对齐画在板上的头号线与夹子的线，并将其放置在平绒广场之上。施加轻微的压力以从平绒方传送菌到板上。卸下车牌号码之一。 </li><li>丢弃平绒平方成水的烧杯中，并放置在复制品上电镀工具清洁，无菌的平绒方如在步骤3.3。此步骤防止过度接种导致突破生长和假阳性结果。 </li><li>对齐在车牌号码之一，在夹具上的一行行，并将其放置在新的平绒广场之上。轻轻推压，从板的头号转让细菌平绒广场。卸下车牌号码之一。 </li><li>对齐画在板上二号线与夹子的线，并将其放置在平绒广场之上。轻轻推压，从平绒广场转移到细菌车牌号码2。卸下车牌号码2。 </li><li>对于车牌号码3重复步骤3.8。第三板是用于从所述主板完全转移到另两个板的控制。放弃平绒广场到水的烧杯中。重用平绒广场，高压灭菌含有污染平绒广场烧杯，把它们洗干净，晾干，包装在铝箔和再高压灭菌。 </li><li>孵育所述板在30℃CO / N。生长在两个LB根琼脂平板上，但无法生长上的M-9-kan平板上的菌落被认为是营养缺陷型（ <st荣&gt;图2）。 </li><li>从板块的头号连胜了营养缺陷型到新鲜的LB根琼脂平板上，孵育板在30°CO / N。 </li><li>连胜走出隔离从平板划线步骤3.11 3个菌落到新鲜的LB-Kan平板。孵育板在30℃CO / N。这确保了该突变体是良好隔离。分板块分为三连胜，并从每个群体中一个部分。 </li><li>从步骤3.12导出到新鲜LB根板的划线出菌落斑菌落形成格栅与步骤2.1-2.4。每个突变体被一式三份重新测试。 </li><li>再次复制盘与步骤3.1-3.10以确认营养缺陷型表型。 </li></ol> 4.基因救援<ol><li>从步骤3.13中的液体LB根培养基中在30℃下生长的分离的突变体菌落的3ml的O / N培养。来自1毫升培养物中使用市售的基因组DNA纯化试剂盒纯化基因组DNA。 </li><li>成立了0.025 U A的混合物BFU CI限制性内切酶和14微升的1微克/微升基因组DNA在冰上20μl的反应。因为BFU CI切转座子在12不同的地点，它可以是更有效地使用限制性内切酶，BFA I或海第三，该切转座子在两个和三个不同的位点分别。 </li><li>孵育在37℃下反应25分钟，然后在80℃下进行20分钟，使酶失活。在0.8％琼脂糖凝胶上（ 图3）分析3微升的部分消化的DNA。一个成功的部分消化的结果在涂片来自上述10 kb的倒在凝胶的底部。 </li><li>结扎15微升用800单位的T4 DNA连接酶在500微升反应部分消化基因组DNA。孵育反应O / N在4℃。大反应体积最大化单个片段的连接反应，以自己和最小化两个或多个DNA片段之间的相互作用。 </li><li> Precipita德通过加入50μl的3M乙酸钠，pH 5.5，和1ml 95％的乙醇和孵育它在-20℃下20分钟后，连接的DNA。 </li><li>微量的DNA在4℃和13500×g离心10分钟，用70％乙醇洗涤沉淀，干燥它在离心真空浓缩，重悬它于10μl不含核酸酶的水。或者，可将DNA在空气中干燥，在室温15分钟。 </li><li>使用4微升悬浮的DNA转化大肠杆菌大肠杆菌菌株ECD100D PIR或ECD100D pir116通过电在第1 R6Kγ复制起点要求菌株与PIR基因复制6。 被动红外应变导致低拷贝质粒，并pir116菌株含有一个PIR突变基因导致的高拷贝质粒。每个转化体包含一个新的质粒，转座子，被中断的染色体（ 图1E）的一个区域。 </li><li> Inocul吃5ml的LB根培养基与单个菌落，它成长O / N振荡以120rpm和37℃，使用商业试剂盒从整个O / N培养纯化的质粒DNA。 </li><li>消化14微升质粒用限制性内切酶XhoI位一。确保反应的最终体积为20微升。这种酶能够识别在转座子在所述卡那霉素抗性基因的5&#39;端的中间部位。分析10微升的未消化的和消化的质粒在0.8％琼脂糖凝胶上（ 图3）。该质粒由2千转加侧翼宿主DNA中。 </li></ol> 5. DNA测序<ol><li>测序使用市售的测序试剂盒，引物同源的转座子的每一端，并且一毛细管测序分析系统的质粒。或者，该质粒可以被运到外部机构进行测序。 </li><li>使用分子量标准（1kb梯）的凝胶来估计该质粒的大小。然后，估计的质粒的所需50 fmol的纳克（毫微克）的数量。 </li><li>测定质粒DNA，用分光光度计在260纳米（A 260）和280nm（A 280）的波长的浓度。确保通过试管的光程长度为1cm。通过用50纳克/微升乘以260nm处的吸光度确定浓度。高品质的质粒DNA将有一个A 260 / A 280比值在1.8和2.0之间。 </li><li>要求每个测序反应的DNA的量是NG要求50 fmol的由质粒的浓度除以数。混合所需的质粒的量与水以使总体积达到10微升。 </li><li>在96℃下进行1分钟加热该质粒DNA /水混合物中，然后将其冷却至室温。 </li><li>加入8μl反应混合液的测序试剂盒和2微升1.6微米的测序引物之一。 </li><li> FOL较低的协议的测序试剂盒的热循环程序和反应清除。 </li><li>使用毛细管DNA分析系统中的每个样品进行分析。 </li><li>使用市售的软件程序包，以查看该DNA序列。搜索的顺序，"5&#39;-GAGACAG-3&#39;"，这是在过去的7碱基的转座子（ 图4）。这7 bp的片段的5&#39;末端之前的序列所属的转座子。这7 bp的片段的3&#39;末端之后的序列属于被中断的基因。 </li><li>确定使用基本局部比对搜索工具（BLAST）7,8-中断的基因的可能功能。使用以下link: <a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&amp;BLAST_PROGRAMS=megaBlast&amp;PAGE_TYPE=BlastSearch" target="_b;ank">http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&amp;BLAST_PROGRAMS=megaBlast&amp;PAGE_TYPE=BlastSearch</a> .粘贴中断的顺序ED基因到查询框中，选择数据库下的"其他"，并在页面底部点击"BLAST"。当结果出现后，向下滚动页面，查看比对结果（ 图5）。 </li></ol>

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用转座的转座子诱变是在肠杆菌属产生营养缺陷型的有效工具。 YSU和其他类型的革兰氏阴性和革兰氏阳性菌3,4。该过程是通过将Tn的5衍生的转座到宿主通过电启动。以确定菌落的插入，未转化的靶菌株必须是敏感的，以卡那霉素以选择用于通过转座子携带的抗性标记。有些主机产生限制性核酸内切酶降解转座子的DNA，才可以与宿主基因组重组。另外一个限制性内切酶抑制剂?...

原养细菌生长的培养基含有葡萄糖（M-9培养基）的M-9基本盐，通过中心碳代谢途径的葡萄糖转化为产生前体，如氨基酸，核酸和维生素，可以使用合成1。 M-9培养基中含有氯化铵作为氮源，钠和磷酸钾作为缓冲液和磷源，硫酸镁作为硫源和葡萄糖作为碳源和能源。卢里亚BERTANI（LB）培养基中含有丰富的蛋白胨，从和多种维生素和生长因子从酵母中提取的氨基酸。它支持营养缺陷型是不能合成的氨基酸，维生素和其它生长因子，生长所需的M-9培养基中的生长。因此，prototrophs将生长在LB和M-9培养基，而营养缺陷型将在LB培养基中，但不能生长在M-9培养基。通过将突变引入细菌的原养型群体和识别突变基因导致的营养缺陷型表型，它是口服ssible以获得细菌菌株更好地理解代谢的。 转座子诱变也可用于识别许多需要在M-9培养基生长于葡萄糖的基因。转座子随机插入 ​​自己到宿主基因组中2。通过发现在LB琼脂平板和副本电镀它们座子转化到M-9培养基琼脂平板上，有可能筛选营养缺陷型。中断的基因是通过基因救援标识。本研究中使用了市售的Tn的5衍生的转座子它随与转座酶蛋白混合的直链转座子的DNA片段的溶液中。该DNA片段缺少一个转座酶基因，但含有卡那霉素抗性的基因，一个R6Kγ型复制起点和两个镶嵌图案这对于转座在段3,4的各端结合的DNA序列。由于转座蛋白直接加入到该DNA，单独的DNA片段被定义为转座子，并且该DNA /转座蛋白复合物被定义为转座。所述转座子是通过电穿孔5转化为卡那霉素敏感的宿主（ 图1A）。在含卡那霉素（LB根）的LB琼脂平板上生长的菌落具有转座子插入（ 图1B），并复制铺板转化体对不生长在含有卡那霉素（M-9根）是营养缺陷型的M-9培养基琼脂平板（Figire 1C）。从突变体基因组DNA的纯化和部分消化的4-碱基切割型限制性内切核酸酶，BFU CI（ 图1D）。连接后的DNA转化到大肠杆菌 （ 大肠杆菌 ）的菌株，其中包含皮尔基因（ 图1E）。这个基因可以含有转座子和侧翼宿主染色体区域在大肠杆菌中复制的新的质粒大肠杆菌 6。卡那霉素抗性基因作为如候选资格的标记为新的质粒。最后，测序用引物互补于转座子和所得序列的基本局部比对搜索工具（BLAST）分析7,8的每一端用于确定的中断基因的身份。 这种转座突变策略提供了三大优势3。首先，由于转座蛋白直接结合到转座子，插入不依赖于宿主内的转座酶基因的表达。一旦转座子整合本身到宿主基因组中，转座酶被降解，从而防止转座子的额外运动。然而，额外的运动不能，如果主机具有一个内Tn的5易位元件防止。第二，引入转座子通过电穿孔的，能够用它在各种各样的宿主。它也消除了由细菌缀合或者通过vi来引入转座子RAL感染。这两个过程都需要宿主易感性。第三，夹杂物的卡那霉素抗性基因和R6Kγ型复制起点的转座体的可以更容易地识别被中断的基因。转座子中断区可以存储并测序质粒，省去了使用逆聚合酶链反应（PCR）技术进行基因鉴定。 在这段视频中介绍的协议描述了肠杆菌属的转座子突变的每一步。 YSU 9使用转座子从它引入到细菌细胞的推定的基因它被中断的识别。除了 ​​先前公布的协议3,4,10，详细的使用方法复制平板筛选营养缺陷型呈现。此诱变技术可以用于调查其他的表型，如抗生素和金属电阻，在不同类型的细菌，对的identifying需要确定培养条件下合成生物学研究下生长的基因的最小数量，或者教学的遗传学和微生物生理学课程实验室的组成部分。

<table><tbody><tr><td>EZ-Tn5 R6K&amp;gamma;ori/KAN-2 Tnp Transposome Kit</td><td>Epicentre (Illumina)</td><td>TSM08KR</td><td></td></tr><tr><td>EC100D pir&lt;sup&gt;+&lt;/sup&gt; Electrocompetent E. coli</td><td>Epicentre (Illumina)</td><td>ECP09500</td><td>Capable of replicating plasmids with an R6K&amp;gamma; replication origin at a low copy number</td></tr><tr><td>EC100D pir-116 Electrocompetent E. coli</td><td>Epicentre (Illumina)</td><td>EC6P095H</td><td>Capable of replicating plasmids with an R6K&amp;gamma; replication origin at a high copy number</td></tr><tr><td>5X M-9 Salts</td><td>Thermo Fisher</td><td>DF048517</td><td></td></tr><tr><td>Lennox LB Broth</td><td>Thermo Fisher</td><td>BP1427-2</td><td></td></tr><tr><td>Agar</td><td>Amresco, Inc.</td><td>J637-1KG</td><td>Solid media contained 1.6% (w/v) agar</td></tr><tr><td>Kanamycin Sulfate&amp;nbsp;</td><td>Amresco, Inc.</td><td>0408-25G</td><td>When required, media contained 50 &amp;mu;g/ml kanamycin sulfate</td></tr><tr><td>D-Glucose Monohydrate</td><td>Amresco, Inc.</td><td>0643-1KG</td><td></td></tr><tr><td>Yeast Extract</td><td>Amresco, Inc.</td><td>J850-500G</td><td></td></tr><tr><td>Tryptone</td><td>Amresco, Inc.</td><td>J859-500G</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P4504-500G</td><td></td></tr><tr><td>NaCl</td><td>Amresco, Inc.</td><td>X190-1KG</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>BP214-500</td><td></td></tr><tr><td>MgSO&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>BP213-1</td><td></td></tr><tr><td>Super Optimal Broth with Catabolite Repression (SOC) medium</td><td></td><td></td><td>0.5% (w/v) yeast extract, 2% (w/v) tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 20 mM MgSO&lt;sub&gt;4&lt;/sub&gt; and 20 mM Glucose</td></tr><tr><td>BfuC I</td><td>NEB</td><td>R0636S</td><td>Partial digestion of genomic DNA</td></tr><tr><td>Xho I</td><td>NEB</td><td>R0146S</td><td>Plasmid digestion</td></tr><tr><td>T4 DNA Ligase</td><td>NEB</td><td>M0202S</td><td></td></tr><tr><td>Nuclease Free Water</td><td>Amresco, Inc.</td><td>E476-1L</td><td>For dissolving precipitated DNA and restriction endonuclease reactions</td></tr><tr><td>GenomeLab DTCS - Quick Start Kit</td><td>Beckman Coulter</td><td>608120</td><td>DNA sequencing</td></tr><tr><td>Wizard Genomic DNA Purification Kit</td><td>Promega</td><td>A1120A</td><td></td></tr><tr><td>Wizard Plus SV Minipreps DNA Purification System</td><td>Promega</td><td>A1460</td><td>Plasmid purification</td></tr><tr><td>Replica Plating Block</td><td>Thermo Fisher</td><td>09-718-1</td><td></td></tr><tr><td>Velveteen Squares</td><td>Thermo Fisher</td><td>09-718-2</td><td>Replica plating</td></tr><tr><td>PetriStickers</td><td>Diversified Biotech</td><td>PSTK-1100</td><td>Grid for replica plating</td></tr><tr><td>Petri Dishes</td><td>Thermo Fisher</td><td>FB0875712</td><td></td></tr><tr><td>Gene Pulser II</td><td>BioRad</td><td></td><td>Electroporation</td></tr><tr><td>Electroporation Cuvettes &amp;ndash; 2 mm</td><td>BioExpress</td><td>E-5010-2</td><td></td></tr><tr><td>CentriVap DNA Vacuum Concentrator</td><td>Labconco</td><td>7970010</td><td>Drying DNA</td></tr><tr><td>CEQ 2000XL DNA Analysis System</td><td>Beckman Coulter</td><td></td><td>DNA sequencing</td></tr><tr><td>Vector NTI Advance 11.5.0</td><td>Life Technologies</td><td>12605099</td><td>DNA sequence analysis</td></tr></tbody></table>

generation of enterobacter sp. ysu auxotrophs using transposon mutagenesis

原养细菌生长上的M-9基本盐培养基中添加葡萄糖（M-9培养基），它被用作碳源和能源。营养缺陷型可以使用转座体来生成。在这个协议中使用的可商购的，Tn的5衍生的转座体由含有R6Kγ型复制起点，卡那霉素抗性和两个镶嵌序列结尾的基因，该基因作为转位结合位点的DNA的线性段。的转座子，提供作为DNA /转座蛋白复合物，通过电穿孔导入到原养型菌株， 肠杆菌属。 YSU，并随机整合到自己这台主机的基因组。转化体复制铺板到的Luria-BERTANI琼脂含有卡那霉素，（LB根）平板上并在含卡那霉素（M-9根）M-9培养基琼脂平板上。生长在LB-kan平板上，但尚未上的M-9-kan平板上的转化体被认为是营养缺陷型。纯化的基因组从营养缺陷型的DNA进行部分消化，连接并转化到一个皮尔+大肠埃希氏菌 （ 大肠杆菌 ）菌株。该R6Kγ复制起点允许质粒在PIR + E.复制大肠杆菌菌株，和卡那霉素抗性标记允许质粒的选择。每个转化具有包含由中断染色体区域侧翼的转座子的新质粒。桑格测序和基本局部比对搜索工具（BLAST）建议被中断的基因的推定的身份。有三个优势，使用这种转座突变的策略。首先，它不依赖于转座酶基因的由宿主中的表达。其次，转座子被导入到目标主机通过电穿孔，而不是通过接合或转导，因此是更有效的。三，R6Kγ复制起点可以很容易地识别突变克烯是将一部分回收的重组质粒。该技术可用于研究参与其他特征肠杆菌属的基因。 YSU或更多种类的细菌菌株。

转型肠杆菌属的。 YSU通过电穿孔用的转座子启动的随机基因组插入到宿主基因组（ 图1A，B）。一个成功的电产生几千它转化生长在LB-kan平板。为了获得300-400，良好的间距每个平板菌落，对每个LB-根琼脂平板上转化混合物传播量进行了优化。每个转化体中包含至少一种转座子插入，但它是不清晰的，如果为增长的重要基因上的M-9培养基，通过影印打断未经筛选。转化体转移至?...

Watch this Scientific Journal Video about Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis at JoVE.com

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

肠杆菌属。 YSU生长在最小的葡萄糖盐培养基。通过用转座子其中随机插入本身入宿主基因组转化它产生营养缺陷型。突变体是通过影印从复合培养基到基本培养基中。打断了基因鉴定的基因救援和测序。

的产生<em&gt;肠杆菌</em&gt; SP。 YSU营养缺陷型使用转座子突变

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

generation-enterobacter-sp-ysu-auxotrophs-using-transposon

Research

JoVE Journal

Biology

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

13.8K 次观看.  Youngstown State University.  肠杆菌属。 YSU生长在最小的葡萄糖盐培养基。通过用转座子其中随机插入本身入宿主基因组转化它产生营养缺陷型。突变体是通过影印从复合培养基到基本培养基中。打断了基因鉴定的基因救援和测序。 

视频: Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

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

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70&#916;. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in 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 ...

科研

遗传学

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

基因靶向随机诱变在裂变酵母中选择染色质稳定蛋白酶突变体

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

使用酵母毒性和抑制器屏幕识别细菌效应蛋白的宿主通路

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

免疫与感染

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

使用细菌共轭的 Enterobacterial 菌株 (如大肠杆菌或志贺菌福氏) 创建稠密的转插入库

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

高通量并行测序在金黄叙利亚仓鼠感染期间测量钩端螺旋体螺旋体转插入突变体的适应性

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

复制的有序, 非冗余图书馆的铜绿假单胞菌应变 PA14 座子插入突变体

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

使用模块化金门克隆的多基因结构快速组装

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

生物工程

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.

绿色怪物承载多基因缺失的酵母菌株的产生过程

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

的产生

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