Name: determination of the optimal chromosomal location(s) for a dna element in escherichia coli using a novel transposon-mediated approach
Uploaded: 2017-09-11T13:00:00
Description: Watch this Scientific Journal Video about Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach at JoVE.com

作者的资金来自诺德基金会, Lundbeck 基金会, 和丹麦国家研究基金会 (DNRF120) 通过细菌应激反应和持久性中心 (BASP) 的赠款。

1. 转库的集合 注意: 染色体 DARS2 轨迹被克隆到迷你 Tn 10 基转, NKBOR (在 pNKBOR) 21 , 从而导致NKBOR:: DARS2 (pJFM1)。pNKBOR 可以在线获得 22 。pNKBOR 是一个 R6K-based 的自杀载体, 需要启动蛋白和 #960; 对于复制 23 。质粒 pJFM1 因此能够复制在一个 大肠杆菌 菌株 ( 例如, Dh5 和 #945; 和 #955; 红外) ...

<ol>
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这里使用的方法论利用了最先进的技术来回答一个关于遗传元素的最佳基因组位置的难题。随机插入的遗传元素 (介导的转) 使数以千计的克隆快速和容易的收集, 然后可以进行竞争, 以选择的最佳位置的研究遗传元素 (即,最适的克隆)。
在这里 , DARS2入到基于最小 Tn10的转 , NKBOR 。转的选择对于插入点的下游分析非常重要。在转定向插入站点测序 (TraDIS) 实验...

任何基因元素的功能都可能受到其在基因组中的位置的影响。在细菌, 这主要起因于干涉由邻居基因的转录, 地方脱氧核糖核酸拓扑结构和/或者复制相关的基因剂量。特别是, DNA 复制和分离的过程被控制, 至少部分, 由编码染色体区域1, 并且这些区域的适当的作用取决于基因组位置/上下文。在大肠杆菌中, 示例为姐妹染色体解析2所需的家庭综合分析站点;KOPS 序列, 染色体分离所需的3;和数据、 DARS1和DARS2区域, 用于正确的染色体复制控制 (如下所示)4). 我们提出了一种方法, 允许随机迁移, 选择和确定任何给定的遗传元素的最佳遗传上下文, 这里的例子是通过研究的DARS2编码区域。
在大肠杆菌中, DnaA 是负责在单个复制源、oriC和旋 DnaB5、67中进行 DNA 链打开的启动蛋白。DnaA 属于 AAA+ (即atp 与不同活动相关的) 蛋白质, 可以将 ATP 和 ADP 与类似的高亲和力5结合在一起。DnaAatp的级别在初始化8处达到峰值, 其中 DnaAatp在oriC上形成一个触发 DNA 双工打开9的 multimer。启动后, oriC暂时无法用于起爆, 原因是由包含 SeqA 蛋白绑定的机制到 hemimethylated oriC10,11。在隔离期间, DnaAATP的级别至少减少了两种机制: DnaA (达)12、13和数据相关 DnaAatp水解 (DDAH)14 的调节失活 ,15。达和 DDAH 都促进将 DnaAATP转换为 DnaAADP。在新一轮启动之前, DnaAADP在特定的 DnaA 重新激活序列 (DARS) 上恢复 DnaAATP , DARS1 和DARS216,17。染色体数据、DARS1、和 DARS2区域是编码的, 并以类似于监护人的方式来调节 DnaAATP/DnaAADP转换。这些区域位于复制源之外, 使 DnaA 复合体的程序集可以用于失活 (数据;14) 或激活 (DARS1和DARS2;17) DnaA。删除单元格中的DARS2不会改变质量加倍的时间,但会导致异步复制启动15、16、18。然而, DARS2-不足的细胞有一个健身成本相比, 否则基因野生在持续增长的竞争中, 在丰富的培养基或建立殖民在老鼠肠道18。这表明, 即使是微小的变化, 在异步/起源浓度有一个负面影响的细菌健身。在大肠杆菌中, 有一个选择性的压力来维持染色体对称性 (即,两个几乎相等长度的复制臂)19。数据、 DARS1和DARS2区域在所有大肠杆菌菌株中都具有与oriC相同的相对距离, 尽管在染色体大小上有很大的差异.
在这里, 我们使用大肠杆菌的DARS2区域作为示例, 以确定其功能的最佳染色体位置。DARS2入到 NKBOR 转中 , 结果 NKBOR :DARS2转随后随机插入到 MG1655 ΔDARS2的基因组中。因此, 我们生成了一组单元格, 每一个都拥有位于染色体上不同位置的DARS2 。一个体外竞争实验, 其中所有的细胞汇集在一起, 并相互竞争, 在持续增长的 LB 为估计700代, 执行。竞争实验的结果是监测/确定使用南部印迹, 容易的基因行走, 和全基因组测序 (WGS;图 1)。采用流式细胞术对细胞周期参数进行了评价, 以方便基因行走的终点克隆为特征。在流动细胞分析中, 细胞大小, DNA 含量, 和启动同步性可以测量大量的细胞。在流式细胞术中, 单个细胞的流动通过一束适当波长的光束来激发染色的 dna, 然后由倍增同时注册, 收集发射的荧光, 一种 dna 含量的测量, 提供细胞被染色为 DNA。前向散射光是单元格质量20的度量。
我们这里的体外竞争实验是用来解决与染色体位置和基因组背景的重要性有关的问题。该方法无偏且易于使用。

<table width="819">
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	<tbody>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">Autoclaved Mili-Q water</td>
			<td style="width:164px;"></td>
			<td style="width:119px;">None</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">Electroporation Cuvettes, 0.1 cm</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">P41050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">Bio-Rad MicroPulser Electroporation System</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:119px;">165-2100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">LB Broth</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12780029&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">LB Agar, powder (Lennox L agar)</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">22700025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">Glycerol</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">17904</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Fisherbrand&#160;Plastic Petri Dishes</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:119px;">S33580A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Falcon 50mL Conical Centrifuge Tubes</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Falcon 15mL Conical Centrifuge Tubes</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:119px;">14-959-53A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Nunc&#160;CryoTubes</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">V7634&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">Phusion High-Fidelity DNA Polymerase (2 U/&#181;L)</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">F530S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">dATP, [&#945;-32P]- 3000Ci/mmol 10mCi/ml, 250 &#181;Ci</td>
			<td style="width:164px;">PerkinElmer</td>
			<td style="width:119px;">BLU012H250UC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">DECAprime II DNA Labeling Kit</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM1455</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="width:369px;height:20px;">Spectrophotometer&#160; SF/MBV/03.32</td>
			<td style="width:164px;">Pharmacia</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Hermle Centrifuge&#160;&#160;&#160; SF/MBV/03.46</td>
			<td style="width:164px;">Hermle</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Ole Dich Centrifuge&#160; SF/MBV/03.29</td>
			<td style="width:164px;">Ole Dich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Eppendorftubes 1.5 mL</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">T9661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Eppendorftubes 2.0 mL</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">T2795</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Merck</td>
			<td>6404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">96% Ethanol</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">16368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Trizma HCl</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">T-3253</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Phenol Ultra Pure</td>
			<td>BRL</td>
			<td>5509UA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Chloroform</td>
			<td>Merck</td>
			<td>2445</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Ribonuclease A type II A</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td>R5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Sodiumdodecylsulphate (SDS)</td>
			<td>Merck</td>
			<td>13760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Lysozyme</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td>L 6876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Isopropanol</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td>405-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">0.5M Na-EDTA pH 8.0</td>
			<td>BRL</td>
			<td>5575 UA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Kanamycin sulfate</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">10106801001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">PvuI (10 U/&#181;L)</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">ER0621</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">UltraPure Agarose</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">16500500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:369px;height:42px;">DNA Gel Loading Dye (6X)</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">R0611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Tris-Borate-EDTA buffer</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Ethidium bromide</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">E7637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Hydrochloric acid</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">433160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Sodium Hydroxide</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">71687</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Whatman&#160;3MM papers</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">WHA3030931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">SSC Buffer 20&#215; Concentrate</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">S6639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Amersham Hybond-N+</td>
			<td style="width:164px;">GE Healthcare</td>
			<td style="width:119px;">RPN119B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Ficoll&#160;400</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">F8016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Polyvinylpyrrolidone</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">PVP40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Bovine Serum Albumin - Fraction V</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">85040C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Deoxyribonucleic acid sodium salt from salmon testes</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">D1626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Carestream Kodak &#160;BioMax &#160;light film</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z373494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">GenElute &#160;Gel Extraction Kit</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">NA1111&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">GenElute &#160;PCR Clean-Up Kit</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">NA1020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">T100&#160; Thermal Cycler</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">SmartSpec Plus Spectrophotometer</td>
			<td style="width:164px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Rifampicin</td>
			<td style="width:164px;">Serva</td>
			<td style="width:119px;">34514.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Cephalexin</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">C4895</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Mithramycin</td>
			<td style="width:164px;">Serva</td>
			<td>29803.02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Magnesium chloride hexahydrate</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:119px;">246964</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:369px;height:21px;">Apogee A10 instrument</td>
			<td style="width:164px;">Apogee</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of the optimal chromosomal location(s) for a dna element in escherichia coli using a novel transposon-mediated approach

在给定的 DNA 元素的最佳染色体位置 (s) 是由转介导的随机插入后, 适应选择。在细菌中, 遗传环境对遗传因素的影响可能难以评估。一些机制, 包括拓扑效应, 邻近基因的转录干扰, 和/或复制相关的基因剂量, 可能会影响一个特定的遗传因素的功能。在这里, 我们描述一种方法, 允许随机集成的 DNA 元素到染色体的大肠杆菌和选择最有利的位置, 使用简单的生长竞争实验。该方法利用了描述 transposon-based 系统的随机插入, 再加上选择的最合适的克隆 (s) 的增长优势, 一个过程, 可以很容易地调整到实验需要。最适合的克隆 (s) 的性质可以由全基因组测序对复杂的克隆种群或容易的基因走, 以快速识别选定的克隆。在这里, 编码 DNA 区域DARS2, 它控制了在大肠杆菌中染色体复制的起始, 被用作一个例子. 已知的DARS2的功能受复制相关基因剂量的影响;DARS2越接近 DNA 复制的起源, 它就越活跃。DARS2被随机插入到DARS2删除的应变的染色体中。包含单个插入的结果克隆被汇集在一起, 并相互竞争数百代。最后, 对最合适的克隆进行了描述, 并发现它包含了与原始DARS2位置紧邻的DARS2 。

为了验证DARS2是否在转库 (t = 0) 的整个染色体中随机分布, 并且最适宜的克隆将随着时间的推移而持续存在。南部的印迹是从最初的转池 (t = 0) 提取的 DNA 进行的, 每700代的竞争中估计有100个 (图 3)。这里, 从每个时点的总细胞 DNA 被消化与PvuI 限制酵素, 已知切开转 NKBOR::DARS2一次只在没有被探针覆盖的区域。用放射性标记的 DNA 片段?...

Watch this Scientific Journal Video about Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach at JoVE.com

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

在这里, 转介导的随机插入编码 DNA 元素的力量被用来解决它的最佳染色体位置。

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 ...

Research

JoVE Journal

Genetics

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

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

使用了大气颗粒物引起的染色体畸变分析<em&gt;体外</em&gt;系统

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

科研

遗传学

Detection of Copy Number Alterations Using Single Cell Sequencing

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

通过DNA含量测定的哺乳动物复制时序的全基因组的测定

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

一种新的饱和诱变方法: 利用 Phosphorothioates 对 RNA 调控蛋白结合位点的单步表征

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to ...

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

利用体外和细胞内形状研究小分子诱导的预 mrna 结构变化

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

remtoe 控制系统在内源基因表达调控中的应用

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

基于质谱的蛋白质组学分析, 利用 OpenProt 数据库揭开从非规范开放阅读框架翻译的新蛋白质

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

在彗星分析中使用冷冻组织评估DNA损伤

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...