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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笔者有没有透露。

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 border="0" cellpadding="0" cellspacing="0" width="707">
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			<td height="21" style="height:21px;width:216px;">Name of Reagent</td>
			<td style="width:145px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EZ-Tn5 R6K&#947;ori/KAN-2 Tnp Transposome Kit</td>
			<td style="width:145px;">Epicentre (Illumina)</td>
			<td style="width:119px;">TSM08KR</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">EC100D pir+ Electrocompetent E. coli</td>
			<td style="width:145px;">Epicentre (Illumina)</td>
			<td style="width:119px;">ECP09500</td>
			<td style="width:227px;">Capable of replicating plasmids with an R6K&#947; replication origin at a low copy number</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">EC100D pir-116 Electrocompetent E. coli</td>
			<td style="width:145px;">Epicentre (Illumina)</td>
			<td style="width:119px;">EC6P095H</td>
			<td style="width:227px;">Capable of replicating plasmids with an R6K&#947; replication origin at a high copy number</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5X M-9 Salts</td>
			<td style="width:145px;">Thermo Fisher</td>
			<td style="width:119px;">DF048517</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lennox LB Broth</td>
			<td style="width:145px;">Thermo Fisher</td>
			<td style="width:119px;">BP1427-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Agar</td>
			<td style="width:145px;">Amresco, Inc.</td>
			<td style="width:119px;">J637-1KG</td>
			<td style="width:227px;">Solid media contained 1.6% (w/v) agar</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Kanamycin Sulfate&#160;</td>
			<td style="width:145px;">Amresco, Inc.</td>
			<td style="width:119px;">0408-25G</td>
			<td style="width:227px;">When required, media contained 50 &#956;g/ml kanamycin sulfate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">D-Glucose Monohydrate</td>
			<td style="width:145px;">Amresco, Inc.</td>
			<td style="width:119px;">0643-1KG</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yeast Extract</td>
			<td style="width:145px;">Amresco, Inc.</td>
			<td style="width:119px;">J850-500G</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tryptone</td>
			<td style="width:145px;">Amresco, Inc.</td>
			<td style="width:119px;">J859-500G</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4504-500G</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:145px;">Amresco, Inc.</td>
			<td style="width:119px;">X190-1KG</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">MgCl2</td>
			<td style="width:145px;">Fisher Scientific</td>
			<td style="width:119px;">BP214-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">MgSO2</td>
			<td style="width:145px;">Fisher Scientific</td>
			<td style="width:119px;">BP213-1</td>
			<td></td>
		</tr>
		<tr height="92">
			<td height="92" style="height:92px;width:216px;">Super Optimal Broth with Catabolite Repression (SOC) medium</td>
			<td style="width:145px;"></td>
			<td style="width:119px;"></td>
			<td style="width:227px;">0.5% (w/v) yeast extract, 2% (w/v) tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM MgSO4 and 20 mM Glucose</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BfuC I</td>
			<td style="width:145px;">NEB</td>
			<td style="width:119px;">R0636S</td>
			<td>Partial digestion of genomic DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xho I</td>
			<td style="width:145px;">NEB</td>
			<td style="width:119px;">R0146S</td>
			<td>Plasmid digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T4 DNA Ligase</td>
			<td style="width:145px;">NEB</td>
			<td style="width:119px;">M0202S</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Nuclease Free Water</td>
			<td style="width:145px;">Amresco, Inc.</td>
			<td style="width:119px;">E476-1L</td>
			<td style="width:227px;">For dissolving precipitated DNA and restriction endonuclease reactions</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GenomeLab DTCS - Quick Start Kit</td>
			<td style="width:145px;">Beckman Coulter</td>
			<td style="width:119px;">608120</td>
			<td>DNA sequencing</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wizard Genomic DNA Purification Kit</td>
			<td style="width:145px;">Promega</td>
			<td style="width:119px;">A1120A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wizard Plus SV Minipreps DNA Purification System</td>
			<td style="width:145px;">Promega</td>
			<td style="width:119px;">A1460</td>
			<td>Plasmid purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Replica Plating Block</td>
			<td style="width:145px;">Thermo Fisher</td>
			<td style="width:119px;">09-718-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Velveteen Squares</td>
			<td style="width:145px;">Thermo Fisher</td>
			<td style="width:119px;">09-718-2</td>
			<td>Replica plating</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PetriStickers</td>
			<td style="width:145px;">Diversified Biotech</td>
			<td style="width:119px;">PSTK-1100</td>
			<td>Grid for replica plating</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri Dishes</td>
			<td style="width:145px;">Thermo Fisher</td>
			<td style="width:119px;">FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gene Pulser II</td>
			<td style="width:145px;">BioRad</td>
			<td style="width:119px;"></td>
			<td>Electroporation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Electroporation Cuvettes &#8211; 2 mm</td>
			<td style="width:145px;">BioExpress</td>
			<td style="width:119px;">E-5010-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CentriVap DNA Vacuum Concentrator</td>
			<td style="width:145px;">Labconco</td>
			<td style="width:119px;">7970010</td>
			<td>Drying DNA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CEQ 2000XL DNA Analysis System</td>
			<td style="width:145px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td>DNA sequencing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vector NTI Advance 11.5.0</td>
			<td style="width:145px;">Life Technologies</td>
			<td style="width:119px;">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

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

科研

生物学

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

自制的网站定向诱变整个质粒

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

改造使用含RNA寡核苷酸基因组DNA的RNA / DNA杂交一代

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

蠕虫病毒实验室介绍:从培养蠕虫突变

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

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.

导演蛋白质的演化突变和功能选择协议 E。大肠杆菌</em

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

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

单卵母细胞的亚硫酸氢钠诱变

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

从血液高效的iPS细胞生成使用附加体和HDAC抑制剂

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...