Name: testing the role of multicopy plasmids in the evolution of antibiotic resistance
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这项工作得到了干杯研究所的支持 (计划 Estatal de i + 2013-2016): 赠款 CP15-00012、PI16-00860 和 CIBER (CB06/02/0053), 由欧洲发展区域基金共同出资 "实现欧洲的一种方式" (ERDF)。talento (2016-T1/生物-1105) 和西班牙 Ministerio Economía、工业 y Competitividad (BIO2017-85056-P) 的 Excelencia, 得到了马德里地区政府的 Atracción de ( ) 计划的支持。ASM 是由由欧洲社会基金 "投资于你的未来" (Servet) 和 ERDF 共同出资的干杯 (MS15/00012) 的一支米格尔的奖学金。

1. 抗生素耐药性基因实验系统的构建
注: 以大肠杆菌MG1655 作为质粒或染色体编码抗生素耐药性基因的受体菌株。抗生素耐药性基因编码在染色体或多拷贝产质粒中, 否则等基因系应变(图 1)。
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在λ噬菌体集成站点 (attB)20的 MG1655 染色体中插入耐药性基因。
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<li>通过 PCR 方法放大染色体attB站点两...

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我们提出了一个新的协议, 结合分子生物学, 实验进化和深 DNA 测序, 旨在研究多拷贝产质粒在细菌耐药性进化中的作用。虽然该协议结合了不同领域的技术, 但开发它所需的所有方法都很简单, 可以在常规微生物学实验室中进行。该协议中最关键的步骤可能是建立模型系统菌株和重建实验后观察到的突变 (使用完全相同的方法进行)。然而, 等温装配系统21, 大大简化了此协议, 因此?...

细菌耐药性是一个主要的健康问题1。在基本水平上, 抗生素耐药性在致病细菌中的传播是自然选择的一个简单的例子, 这是自然选择2,3。简而言之, 抗生素的使用会产生抗性菌株的选择。因此, 进化生物学的一个关键问题是了解影响细菌种群对抗生素的抗药性的能力的因素。选择实验已经成为一个非常强大的工具来调查细菌的进化生物学, 这个领域已经产生了令人难以置信的洞察到广泛的进化问题4,5,6。在实验性进化过程中, 由单亲菌株引发的细菌种群在定义和严格控制的条件下依次传代。在这些文化的生长过程中发生的一些突变增加了细菌的适应性, 这些变化通过自然选择的文化传播。在试验过程中, 种群的样本定期低温保存, 以创建一个非进化的冰冻化石记录。大量的方法可以用来表征不断变化的细菌种群, 但最常见的两种方法是体能测定, 即测量进化细菌与远方祖先竞争的能力, 以及整个基因组测序, 即用于识别驱动适应的遗传变化。经过理查德 Lenski 和同事的开创性工作7,8, 实验进化的标准方法是挑战相对较少数量的复制人口 (通常 < 10), 适应新的环境挑战, 如新的碳源, 温度, 或捕食性的噬菌体。
抗生素耐药性细菌引起的感染成为一个很大的问题, 当抗药性足够高, 不能增加抗生素浓度, 以致命的水平在病人组织。因此, 临床医生对允许细菌进化为高剂量抗生素的耐药性有兴趣, 这是高于此阈值的抗生素浓度, 临床断点。如何进行实验研究？如果少量的细菌种群受到高剂量抗生素的挑战, 就像在 Lenski 的实验中那样, 最可能的结果是抗生素会驱使所有的人群灭绝。同时, 如果使用的抗生素剂量低, 低于最低限度的抑制浓度 (MIC) 的父母菌株, 那么它是不可能的细菌群体将发展临床相关水平的抗药性, 特别是如果阻力承载着很大的成本。这两种情况的一个折中方案是使用 "进化营救" 实验9,10,11。在这种方法中, 大量的文化 (通常 > 40) 受到挑战, 随着时间的推移, 抗生素的剂量增加, 通常是通过加倍抗生素浓度每天12。这个实验的特点是, 任何没有进化出抗药性的种群都将被迫灭绝。以这种方式受到挑战的大多数人口将被驱赶绝种, 但少数族裔将坚持不断发展的高水平的抵抗。本文介绍了该实验设计如何能够用于研究多拷贝产质粒对抗性进化的贡献。
细菌通过两条主要途径、染色体突变和获取移动遗传元素, 主要是质粒13获得对抗生素的抗药性。质粒在抗生素耐药性的演变过程中起着关键作用, 因为它们能够通过共轭14,15在细菌间转移抗性基因。质粒可根据其大小和生物学分为两组: "小", 每个细菌细胞的拷贝数和 "大", 低拷贝数16,17。大型质粒在抗生素耐药性演变中的作用已被广泛记录在案, 因为它们包括共轭质粒, 这是细菌传播抗药性和多种抗药性的关键驱动因素15。在细菌17、18中, 小多拷贝产质粒也非常常见, 它们经常编码抗生素耐药性基因19。然而, 小多拷贝产质粒在抗生素耐药性演变中的作用在较小程度上被研究。
在最近的一项工作中, 我们建议多拷贝产质粒可以通过增加基因突变率来加速它们携带的基因的进化, 因为每个细胞的基因复制数都是12。使用具有大肠杆菌菌株 MG1655 和β-酶基因血乳酸TEM-1的实验模型, 表明多拷贝产质粒加速了 TEM-1 突变的出现率, 赋予了第三代抗性头孢他啶。结果表明, 多拷贝产质粒在抗生素耐药性的演变过程中可能起着重要的作用。
在这里, 我们提供了一个详细的描述, 我们已经开发的方法来研究多拷贝产质粒介导的抗生素耐药性的演变。该方法有三种不同的步骤: 一是在多拷贝产质粒或宿主细菌的染色体中插入研究中的基因。第二, 利用实验进化 (进化拯救) 来评估不同菌株的潜能, 以适应选择性压力。第三, 用 DNA 测序法确定质粒介导的进化的分子基础, 并在父母基因型中单独重建疑似突变。
最后, 虽然这里描述的协议是为了调查抗生素耐药性的演变而设计的, 但人们可以争辩说, 这种方法对于分析任何多拷贝产突变所获得的创新的进化是很有用的。质粒编码基因。

<table border="0" cellpadding="0" cellspacing="0" style="width:668px;" width="668">
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		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thermocycler</td>
			<td style="width:167px;">BioRad</td>
			<td style="width:119px;">C1000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electroporator</td>
			<td style="width:167px;">BiorRad</td>
			<td style="width:119px;">1652660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electroporation cuvettes</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z706078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NanoDrop 2000/2000c</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">ND-2000</td>
			<td>Determine DNA quality measuring the ratios of absorbance 260nm/280nm and 260nm/230nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubator</td>
			<td style="width:167px;">Memmert</td>
			<td style="width:119px;">UF1060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubator (shaker)</td>
			<td style="width:167px;">Cole-Parmer Ltd</td>
			<td style="width:119px;">SI500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrophoresis power supply</td>
			<td style="width:167px;">BioRad</td>
			<td style="width:119px;">1645070</td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrophoresis chamber</td>
			<td style="width:167px;">BioRad</td>
			<td style="width:119px;">1704405</td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pippettes</td>
			<td style="width:167px;">Biohit</td>
			<td style="width:119px;">725020, 725050, 725060, 725070</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Multi-channel pippetes</td>
			<td style="width:167px;">Biohit</td>
			<td style="width:119px;">728220, 728230, 
			728240</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plate reader Synergy HTX</td>
			<td style="width:167px;">BioTek</td>
			<td style="width:119px;">BTS1LF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Inoculating loops</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">I8388</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well plates</td>
			<td style="width:167px;">Falcon</td>
			<td style="width:119px;">351172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB</td>
			<td style="width:167px;">BD Difco</td>
			<td style="width:119px;">DF0446-17-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LB agar</td>
			<td style="width:167px;">Fisher scientific</td>
			<td style="width:119px;">BP1425-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phusion Polymerase</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">F533S</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:216px;">Gibson Assembly</td>
			<td style="width:167px;">New England Biolabs</td>
			<td style="width:119px;">E2611S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Resctriction enzymes</td>
			<td style="width:167px;">Fermentas FastDigest</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:216px;">Antibiotics</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:167px;">Qiagen</td>
			<td style="width:119px;">27104</td>
			<td>Plasmid extraction kit</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:216px;">Wizard Genomic DNA Purification Kit</td>
			<td style="width:167px;">Promega</td>
			<td style="width:119px;">A1120</td>
			<td>gDNA extraction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNeasy Blood &#38; Tissue Kits</td>
			<td style="width:167px;">Qiagen</td>
			<td style="width:119px;">69506</td>
			<td>gDNA extraction kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electroporation cuvettes</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z706078</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dishes</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cryotubes</td>
			<td style="width:167px;">ClearLine</td>
			<td style="width:119px;">390701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well plates (-80&#186;C storage)</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">249945</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QuantiFluor dsDNA System</td>
			<td style="width:167px;">Promega</td>
			<td style="width:119px;">E2670</td>
			<td>Quantification of DNA concentartion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:167px;">BioRad</td>
			<td style="width:119px;">1613100</td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50x TAE buffer</td>
			<td style="width:167px;">BioRad</td>
			<td style="width:119px;">1610743</td>
			<td>Agarose gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T4 Polynucleotide Kinase</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">EK0031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T4 DNA Ligase</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">EL0014</td>
			<td></td>
		</tr>
	</tbody>
</table>

testing the role of multicopy plasmids in the evolution of antibiotic resistance

多拷贝产质粒在原核生物中非常丰富, 但它们在细菌进化中的作用仍不甚了解。我们最近发现, 多拷贝产质粒提供的每个细胞基因拷贝数的增加, 都可以加速粒编码基因的进化。在这项工作中, 我们提出了一个实验系统, 以测试多拷贝产质粒的能力, 促进基因进化。使用简单的分子生物学方法, 我们构建了一个模型系统, 在这种模式下, 抗生素耐药性基因可以插入到大肠杆菌 MG1655, 无论是在染色体或多拷贝产质粒. 我们使用一种实验进化方法在越来越多的抗生素浓度下传播不同的菌株, 我们测量细菌种群的存活时间。抗生素分子和抗性基因的选择是为了使基因只能通过获取突变来获得抵抗。这种 "进化拯救" 方法提供了一种简单的方法来检测多拷贝产质粒的潜能, 以促进抗生素耐药性的获取。在实验系统的下一步, 对抗生素耐药性的分子基础进行了表征。为了找出负责获取抗生素耐药性的突变, 我们使用从整个种群和克隆获得的样本的深 DNA 测序。最后, 为了确认基因突变在研究中的作用, 我们在父母的背景下重建它们, 并测试结果菌株的抗性表型。

在我们以前的研究中, 研究了β-酶基因乳酸TEM-1对第三代头孢霉素12抗性的进化。这个基因之所以被选择是因为, 虽然 TEM-1 并没有对头孢他啶的抗性, 但在血乳酸TEM-1中的突变可以扩大 TEM-1 的活性范围, 以水解以甲霉素29为孢菌素抗生素耐药性酶的突变, 如β-lactamases 或氨基糖苷类改变酶导致其活动范围的变化?...

Watch this Scientific Journal Video about Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance at JoVE.com

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

多拷贝产质粒在抗生素耐药性演变中的作用试验

在此, 我们提出了一个实验方法来检验多拷贝产质粒在抗生素耐药性进化中的作用。

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

Research

Education

JoVE Journal

JoVE Core

Biology

Genetics

Evolution

Natural Selection

SCIENTISTS IN ACTION

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

设计与低成本的使用，自动Morbidostat细菌的适应性进化在抗生素药物的选择

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

科研

遗传学

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

程序的微生物实验室自适应演化的恒化

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

Tools to Study the Role of Architectural Protein HMGB1 in the Processing of Helix Distorting, Site-specific DNA Interstrand Crosslinks

工具来研究蛋白质建筑HMGB1的作用，螺旋窜改，站点特定的DNA间交联处理

High mobility group box 1 (HMGB1) protein is a non-histone architectural protein that is involved in regulating many important functions in the genome, ...

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 ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

利用 niper-cas9 在不损失目标活动的情况下通过定向进化, 最大限度地减少 crispr-cas9 的非目标效应

The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the ...

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

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 ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

CRISPR/Cas9核糖核酸蛋白在急性骨髓性白血病细胞中的RUNX1电子消光器的转录作用研究

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

从果蝇雄性附件腺的二级细胞中分离RNA的FACS协议

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of ...

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

实验进化方法中疟原虫介导抗生素耐药性的定量化

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial ...