Name: testing the role of multicopy plasmids in the evolution of antibiotic resistance
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Description: Watch this Scientific Journal Video about Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance at JoVE.com

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)。
<ol><li>
在λ噬菌体集成站点 (attB)20的 MG1655 染色体中插入耐药性基因。
	<ol>
<li>通过 PCR 方法放大染色体attB站点两侧的500个 bp 长区域。使用寡核苷酸 YbhC (5 '-CCTGTACCGTACAGAGTAAT-3 ') 和 attB (5 '-GCCCGCCACCCTCCGGGCCGGTATAAAAAAGCAGGCTTCA-3 ') 为左同源区和 attB F (5 '-AGCGCCCTAGCGCCCGCTCCTTATACTAACTTGAGCGAAA-3 ') 和 YbhB/R (5 '-TGGCGATAATATTTCACCGC-3 ') 为正确的一个。使用引物 Tem1-pBAD-F (5 '-TGAAGCCTGCTTTTTTATACCGGCCCGGAGGGTGGCGGGC-3 ') 和 Tem1-pBAD-R (5 '-TTTCGCTCAAGTTAGTATAAGGAGCGGGCGCTAGGGCGCT-3 ') 放大血乳酸TEM-1抗性基因。设计底漆与大约 20 bp (在 5 ' 末端) 序列显示互补到将被熔化的片断。</li>
		<li>将同源区与抗生素耐药性基因 PCR 产品 (包括其启动子区域) 结合使用等温组件21, 在50摄氏度为30分钟。</li>
		<li>Electroporate MG1655 细胞含有质粒 pKOBEG。 注: pKOBEG 是一种热敏载体, 含有λ红色机械, 促进染色体和 PCR 产品之间的同源性等位基因交换22。<ol>
<li>使用1µL 的产品从步骤 1.1.223到40µL electrocompetent 细胞在2毫米小试管在4°c 和2.5 伏。并用重悬细胞在1毫升 LB 汤 + 0.2% 阿拉伯糖, 以保持λ红色机械的表达。</li>
			<li>将总容积转移到离心管, 在30摄氏度 (允许温度为 pKOBEG) 上孵化2小时, 并进行剧烈晃动 (在一个轨道振动筛中 200 rpm), 以便在染色体中插入电阻标记的表型表达式。</li>
			<li>在 LB 琼脂上板上含有适当抗生素的细胞, 用于选择抗生素耐药性基因 (氨苄西林或羧苄青霉素100毫克/升用于血乳酸 TEM-1). </li>
		</ol>
</li>
		<li>板100µL 在一个培养皿, 并旋转下来的其余的体积, 并用重悬它在100µL 新鲜的 LB 汤, 并在另一培养皿盘子。孵化过夜在42摄氏度。 注: 此温度不允许复制 pKOBEG。能够在这些条件下生长的菌落将失去 pKOBEG, 并将抵抗基因集成到attB站点中 (为了简单起见, 这一菌株将被称为MG1655:瑞莎从此处起)。</li>
		<li>通过用氯霉素 (pKOBEG 含有抗性标记的抗生素) 复制板上的兴趣菌落来测试 pKOBEG 的损失。 注: 在30ºC 的许可温度下, 氯霉素板缺乏生长, 表明质粒缺乏。</li>
		<li>为了验证克隆, PCR 放大结构使用外部引物 YbhC (5 '-TTTGTGACCAGAAGACCGCA-3 ') 和 YbhB-外部 (5 '-CTCATCAGTAACGATCTGCG-3 '), 通过凝胶电泳验证扩增子的正确大小和序列的纯化的产品, 以确保插入基因的顺序是正确的。</li>
	</ol>
</li>
	<li>
在多拷贝产质粒中插入耐药性基因。 注: 由于具有极高拷贝数的质粒倾向于大量减少细菌宿主的适用性, 我们建议使用具有自然复制起源的多拷贝产质粒, 如 p3655 (pSU18T-pBAD gfp2, ColE1-type 起源于复制24)。这些质粒通常为每个细胞提供大约15-20 个拷贝的适度拷贝数。<ol>
<li>pcr 放大选择的抗生素耐药性基因 (包括启动子区), phosphorylate pcr 产物, 并将其结扎到 pcr 扩增的质粒:<ol>
<li>PCR 放大抗生素耐药性基因;对于乳酸TEM-1使用底漆 Tem1-pBAD-F (5 '-TGAAGCCTGCTTTTTTATACCGGCCCGGAGGGTGGCGGGC-3 ') 和 Tem1-pBAD-R (5 '-TTTCGCTCAAGTTAGTATAAGGAGCGGGCGCTAGGGCGCT-3 ')。</li>
			<li>Phosphorylate 的纯化产品使用 T4 核苷酸激酶, 按照制造商的指示。</li>
			<li>应用高保真聚合酶和寡核苷酸 pBAD (5 '-CGTTGATCGGCACGTAAGAG-3 ') 和 pBAD R (5 '-AAACGACGGCCAGTGCCAAG-3 '), PCR 扩增质粒骨干。</li>
			<li>结扎两个 PCR 片段使用 T4-ligase 以下制造商的指导方针。</li>
		</ol>
</li>
		<li>Electroporate, 如前所述, 40 µL 的 electrocompetent大肠杆菌DH5-α细胞最多1µL 结扎产品, 并选择适当的抗生素耐药性基因 (氨苄西林或羧苄青霉素在100毫克/升用于血乳酸TEM-1) 和质粒主干。 注: 补充磅与阿拉伯糖这里是不必要的。</li>
		<li>使用读者选择的商业迷你准备套件提取质粒:<ol>
<li>在室温下, 通过离心在 1.2万 x g 上收获5毫升的隔夜文化。并用重悬250µL 泥沙溶液中的细胞, 混合均匀。加入250µL 的溶解液, 混合均匀。添加350µL 中和, 混合良好。</li>
			<li>将上清液转移到所提供的 DNA 结合柱, 离心机1分钟, 并丢弃流经。洗两次柱与500µL 的洗涤液, 离心一分钟, 并丢弃 flowthrough 在每次洗涤。离心空列1余分钟, 以除去残留洗涤缓冲器。</li>
			<li>把柱子放在干净的管子里, 将50µL 的超纯净水添加到膜中, 洗脱纯化的 DNA。离心机为1-2 分钟, 收集含有纯化质粒的流动。利用插入序列外的引物对质粒中感兴趣的基因进行序列化, 以确认序列是正确的, 在进化实验之前, 抗性基因中没有突变存在。</li>
		</ol>
</li>
		<li>一旦序列被证实, electroporate 的质粒在大肠杆菌MG1655 菌株, 如上文所述。 注意: 这会产生应变MG1655/pRESA.
</li>
	</ol>
</li>
</ol>2. 进化拯救方法以实验方式发展抗生素耐药性 (图 1)
<ol><li>对 LB 板研究中的菌株进行条纹: MG1655::瑞莎和 MG1655/pRESA 加上敏感的父母应变, MG1655。 注: 质粒 pRESA 应稳定在 MG1655, 所以没有必要添加抗生素的 LB 板。在大肠杆菌中的突变率足够低, 因此一旦验证了构造, 就不必在每一步验证质粒序列。</li>
	<li>在每个井中准备96井板和200µL 磅, 并接种独立井每株48个独立的菌落 (每株一张)。在37摄氏度和200转每晚孵化板块。对这些创始人种群保持冻结的库存。 注: 为防止和控制96井板中的文化交叉污染, 在整个96井板上使用无菌培养基的锂接种井进行棋盘板设计。在整个实验过程中使用这个板块设计。</li>
	<li>启动进化救援实验, 接种2µL 从板块的每个井与创建者人口在新的96井板材与198µL 磅在每个井与抗生素的亚抑制浓度在研究中。为进化抢救方法开始与¼或1/8 最小的抑制浓度 (麦克风, 确定以前的25) 的抗生素在 LB 为每三株和双倍的浓度的抗生素每日。使用此方法可以最大限度地提高种群获取电阻突变的几率26。孵化的板块在37摄氏度和 200 rpm 20 小时。 注: 测量创始人群体的隔夜 OD。如果菌株之间的 OD 有显著的差异, 正确的初始接种, 以相同数量的细胞每井开始实验。</li>
	<li>每天, 衡量每一个人口的 OD, 在隔夜文化和执行转移的文化, 如2.3 所述, 到新的96井板与双倍的抗生素浓度比前一天。在37摄氏度和200转每分钟孵化20小时的板材。 注: 1:100 稀释因子每天产生大约6-7 代。</li>
	<li>同时, 在2.3 和2.4 所述的相同条件下, 传播每个菌株的控制种群, 但缺乏抗生素。 注意: 控制人群将有助于区分由于抗生素的存在和一般突变帮助细菌适应实验条件而产生的突变。在缺乏抗生素的情况下发生的平行突变可能有助于细菌适应实验条件, 而与抗生素耐药性无关。</li>
	<li>通过用平板阅读器测量600毫微米 (OD) 的吸收量, 每天跟踪幸存人口的数量。 注: 光密度值低于0.1 表示种群的灭绝。有关生存曲线的示例, 请参见图 2 。</li>
	<li>定期 (每3-5 天) 保存所有人口的冰冻库存。</li>
	<li>使用日志秩测试 [RStudio (版本 0.99.486) 中的 "包" 生存期], 以确定在抗生素浓度不断增加的情况下, 不同菌株的种群存活率的统计差异12。 注: 本实验将确定多拷贝产质粒是否事实对特定抗生素和基因研究的抗生素耐药性的演变。</li>
</ol>3. 抗生素耐药性演变的分子基础 (图 1)
<ol><li>执行总 DNA 提取从代表性数量的人口和克隆跨菌株和治疗。包括父母菌株 (MG1655、MG1655:瑞莎和 MG1655/pRESA), 以便能够检测在实验过程中积累的突变。 注: 有关 DNA 提取套件的示例, 请参阅材料表。每个套件都有不同的协议;遵循制造商的指示。</li>
	<li>量化 DNA 质量和浓度。有不同的方法来确定 DNA 的质量和浓度。测定吸收率 260 nm/280 nm 和 260 nm/230 nm 的质量。使用荧光, DNA 结合染料测量 dna 浓度后, 制造商的指示, 和琼脂糖凝胶电泳, 以确认没有 DNA 降解或 RNA 污染。</li>
	<li>利用来自全种群和个体克隆样本的深 DNA 测序, 研究抗生素耐药性的遗传基础。 注: 我们在英国牛津大学人类遗传学信托中心进行了所有的测序。有关详细信息, 请参阅 San 文澜et 。201612。</li>
	<li>使用 breseq 0.26.1 管线27、28检测突变, 使用多态性模式估计种群中突变的频率。将不同的进化基因组与父母基因组进行比较, 以检测在实验中积累的突变。</li>
	<li>比较在对照人群中积累的突变与在抗生素浓度不断增加的人群中的变化, 以区分帮助细菌适应一般实验条件的突变 (在控制人群中发现的那些) 和那些涉及抗生素耐药性的人 (那些仅在受到抗生素压力的人群中发现)。 注意: 平行突变的数量通常是低的, 所以治疗之间的不同的平行突变可以很容易地评估。除了研究中的抗生素耐药性基因突变外, 它很可能在染色体中发现与抗生素耐药性相关的其他突变, 例如孔蛋白或排便系统中的这种突变 12.</li>
	<li>采用与本议定书1节相同的方法, 重建父母菌株中抗生素耐药性基因的突变。按照《议定书》1.1 和1.2 的规定, 在父母背景中引入新的变异 (使用进化后的基因作为 PCR 模板)。分析这些新结构的耐药性表型, 以确定突变的作用。</li>
</ol>

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Approved standard M100-S19. , (2009).</li><li>Lindsey, H. A., Gallie, J., Taylor, S., Kerr, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+rescue+from+extinction+is+contingent+on+a+lower+rate+of+environmental+change.">Evolutionary rescue from extinction is contingent on a lower rate of environmental change.</a> Nature. 494 (7438), 463-467 (2013).</li><li>Deatherage, D. E., Barrick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+mutations+in+laboratory-evolved+microbes+from+next-generation+sequencing+data+using+breseq.">Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq.</a> Methods Mol Biol. 1151, 165-188 (2014).</li><li>Barrick, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+structural+variation+in+haploid+microbial+genomes+from+short-read+resequencing+data+using+breseq.">Identifying structural variation in haploid microbial genomes from short-read resequencing data using breseq.</a> BMC Genomics. 15, 1039 (2014).</li><li>Salverda, M. L., De Visser, J. A., Barlow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+evolution+of+TEM-1+&#946;-lactamase:+experimental+reconstruction+and+clinical+relevance.">Natural evolution of TEM-1 &#946;-lactamase: experimental reconstruction and clinical relevance.</a> FEMS Microbiol Rev. 34 (6), 1015-1036 (2010).</li><li>Ramirez, M. S., Tolmasky, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aminoglycoside+modifying+enzymes.">Aminoglycoside modifying enzymes.</a> Drug Resist Updat. 13 (6), 151-171 (2010).</li><li>Jerison, E. R., Desai, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+investigations+of+evolutionary+dynamics+and+epistasis+in+microbial+evolution+experiments.">Genomic investigations of evolutionary dynamics and epistasis in microbial evolution experiments.</a> Current Opinion in Genetics &amp; Development. 35, 33-39 (2015).</li><li>Toll-Riera, M., San Millan, A., Wagner, A., MacLean, R. 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作者没有什么可透露的。

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

测试多拷贝质粒在抗生素耐药性进化中的作用|遗传学 |视频

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 ...

testing-role-multicopy-plasmids-evolution-antibiotic

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SCIENTISTS IN ACTION

11.5K 次观看.  Universidad Complutense de Madrid. 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。

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

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.

This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs.

食品筛查为1类整合和基因盒

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major ...

科研

环境科学

High-Resolution Comparison of Bacterial Conjugation Frequencies

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

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

细菌结合频率的高分辨率比较

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

遗传学

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

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial populations. This is especially the case under rapidly changing environments such as fluctuating antibiotics exposure. We recently showed that plasmids maintain antibiotic resistance genes in Escherichia coli without positive selection for the plasmid presence. Here we describe an experimental system that allows following both the plasmid genotype and phenotype in long-term evolution experiments. We use molecular techniques to design a model plasmid that is subsequently introduced to an experimental evolution batch system approach in an E. coli host. We follow the plasmid frequency over time by applying replica plating of the E. coli populations while quantifying the antibiotic resistance persistence. In addition, we monitor the conformation of plasmids in host cells by analyzing the extent of plasmid multimer formation by plasmid nicking and agarose gel electrophoresis. Such an approach allows us to visualize not only the genome size of evolving plasmids but also their topological conformation-a factor highly important for plasmid inheritance. Our system combines molecular strategies with traditional microbiology approaches and provides a set-up to follow plasmids in bacterial populations over a long time. The presented approach can be applied to study a wide range of mobile genetic elements in the future.

Our experimental approach provides a strategy to follow plasmid abundance and antibiotic resistance over time in bacterial populations.

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 ...

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the study of the mobilization of naturally occurring conjugative plasmids, using two naturally-occurring plasmids as an example. These protocols rely on the differential presence of selectable markers in donor, recipient, and conjugative plasmid. Specifically, the methods described include 1) the identification of natural conjugative plasmids, 2) the quantification of conjugation rates in solid culture, and 3) the diagnostic detection of the antibiotic resistance genes and plasmid replicon types in transconjugant recipients by polymerase chain reaction (PCR). The protocols described here have been developed in the context of studying the evolutionary ecology of horizontal gene transfer, to screen for the presence of conjugative plasmids carrying antibiotic-resistance genes in bacteria found in the environment. The efficient transfer of conjugative plasmids observed in these experiments in culture highlights the biological relevance of conjugation as a mechanism promoting horizontal gene transfer in general and the spread of antibiotic resistance in particular.

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation mediates horizontal gene transfer by mobilizing plasmid DNA across two different cells, facilitating the spread of beneficial genes. This work describes a widely-used method for the efficient detection of conjugative plasmid transfer, based on the use of differential markers in the conjugative plasmid, donor, and recipient to detect transconjugation.

大肠杆菌中天然偶联质粒介导的水平基因转移检测

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the ...

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 ...

免疫与感染

Antibiotic Dereplication Using the Antibiotic Resistance Platform

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this challenge, dereplication, which is the process of identifying known compounds, is performed on samples of interest. Methods for dereplication such as analytical separation followed by mass spectrometry are time-consuming and resource-intensive. To improve the dereplication process, we have developed the antibiotic resistance platform (ARP). The ARP is a library of approximately 100 antibiotic resistance genes that have been individually cloned into Escherichia coli. This strain collection has many applications, including a cost-effective and facile method for antibiotic dereplication. The process involves the fermentation of antibiotic-producing microbes on the surface of rectangular Petri dishes containing solid medium, thereby allowing for the secretion and diffusion of secondary metabolites through the medium. After a 6 day fermentation period, the microbial biomass is removed, and a thin agar-overlay is added to the Petri dish to create a smooth surface and enable the growth of the E. coli indicator strains. Our collection of ARP strains is then pinned onto the surface of the antibiotic-containing Petri dish. The plate is next incubated overnight to allow for E. coli growth on the surface of the overlay. Only strains containing resistance to a specific antibiotic (or class) grow on this surface enabling rapid identification of the produced compound. This method has been successfully used for the identification of producers of known antibiotics and as a means to identify those producing novel compounds.

We describe a platform that utilizes a library of isogenic antibiotic resistant Escherichia coli for the dereplication of antibiotics. The identity of an antibiotic produced by bacteria or fungi can be deduced by the growth of E. coli expressing its respective resistance gene. This platform is economically effective and time-efficient.

使用抗生素耐药性平台进行抗生素脱除

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this ...

用于表征多药物外排系统的单拷贝基因表达

Characterizing Multidrug Efflux Systems in Acinetobacter baumannii Using an Efflux&#45;Deficient Bacterial Strain and a Single&#45;Copy Gene Expression System

Acinetobacter baumannii is recognized as a challenging Gram-negative pathogen due to its widespread resistance to antibiotics. It is crucial to comprehend the mechanisms behind this resistance to design new and effective therapeutic options. Unfortunately, our ability to investigate these mechanisms in A. baumannii is hindered by the paucity of suitable genetic manipulation tools. Here, we describe methods for utilizing a chromosomal mini-Tn7-based system to achieve single-copy gene expression in an A. baumannii strain that lacks functional RND-type efflux mechanisms. Single-copy insertion and inducible efflux pump expression are quite advantageous, as the presence of RND efflux operons on high-copy number plasmids is often poorly tolerated by bacterial cells. Moreover, incorporating recombinant mini-Tn7 expression vectors into the chromosome of a surrogate A. baumannii host with increased efflux sensitivity helps circumvent interference from other efflux pumps. This system is valuable not only for investigating uncharacterized bacterial efflux pumps but also for assessing the effectiveness of potential inhibitors targeting these pumps.

作者聚焦:使用外排缺陷型细菌菌株和单拷贝基因表达系统推进抗生素耐药性研究

We describe a facile procedure for the single-copy chromosomal complementation of an efflux pump gene using a mini-Tn7-based expression system into an engineered efflux-deficient strain of Acinetobacter baumannii. This precise genetic tool allows for controlled gene expression, which is key for the characterization of efflux pumps in multidrug resistant pathogens.

使用外排缺陷细菌菌株和单拷贝基因表达系统表征 鲍曼不动杆 菌的多药外排系统

Acinetobacter baumannii is recognized as a challenging Gram-negative pathogen due to its widespread resistance to antibiotics. It is crucial to comprehend ...

Bacterial Transformation

<h4>Background</h4>

In early 20th century, pneumonia was accountable for a large portion of infectious disease deaths1. In order to develop an effective vaccine against pneumonia, Frederick Griffith set out to study two different strains of the Streptococcus pneumoniae: a non-virulent strain with a rough appearance (R-strain) and a virulent strain with a smooth appearance (S-strain) due to an outer polysaccharide capsule2. This outer layer of the S-strain bacteria enabled them to withstand the host immune system, eventually leading to a life-threatening illness. When Griffith separately injected mice with heat-killed bacteria of either strain, the mice lived. However, when he injected mice with a combination of the heat-killed S-strain with live R-strain, the mice died2. When he analyzed the samples obtained from dead mice injected with the combination, he observed live S-strain bacteria to be present. In 1928, Griffith noted that a &#8220;transformation&#8221; process must have occurred to change the nonvirulent bacteria into the virulent strain. As the first known discovery of bacterial transformation, his discovery paved the way for the development of an essential tool in genetic engineering - transformation3.

Transformation is the genetic change in a cell due to the intake of DNA from the environment. In Griffith&#8217;s experiment, the DNA that encodes the protective polysaccharide coating of the S-strain bacteria did not break down from the heat shock and was introduced into the R-strain, allowing the latter to bypass the immune system of the mouse. While this process constantly occurs in the wild between organisms and even different species, scientists transform bacteria in laboratory settings for research purposes4.

<h4>Bacterial Transformation using Plasmids</h4>

Bacteria are the ideal organisms for transformation as they can easily take in exogenous genetic material into their genome and quickly amplify it3,5. They have one circular chromosome and multiple small circular pieces of double-stranded DNA called plasmids within the cytoplasm. These plasmids can replicate independently from the chromosomal DNA and generally provide certain functional benefits, such as resistance to antibiotics6,7. In their natural environment, bacteria undergo &#8220;bacterial transformation&#8221; by taking in plasmids from other bacteria in a process called conjugation8. Moreover, when they multiply, each of their progeny receives a copy of the new plasmid.

Plasmids that are used for experimental purposes are called plasmid vectors. In a laboratory setting, scientists can artificially create &#8220;recombinant plasmids&#8221; that are approximately 5,000-10,000 base pairs in length by inserting DNA fragments into a plasmid vector. These recombinant plasmids usually have certain components: an origin of replication (ORI), an antibiotic resistance gene, a multiple cloning site, a promoter, a selection marker, and the gene of interest. The origin of replication is where replication begins. The antibiotic resistance gene allows the bacteria that take in the plasmid to survive on plates in the presence of a certain antibiotic drug. Although plasmids are relatively small pieces of DNA, scientists need to treat the host cells to enable the plasmid penetration through the cell membrane. Hence, the efficiency of the transformation is directly related to porosity of the host membrane. One common approach is to heat-shock the bacteria that have been treated with a calcium chloride solution9. The bacteria that do not incorporate the plasmid will not transform, and therefore have no resistance to survive on the plate and be visible. The multiple cloning sites aid in DNA insertion by containing sites for restriction enzymes to cut the plasmid where the gene of interest can be inserted and ligated. The promoter drives transcription of the gene of interest. It is tagged by a marker, usually a fluorescent protein such as a green fluorescent protein (GFP), or can be an additional antibiotic resistance gene. The antibiotic resistance gene and the other selection markers help us determine whether the collected bacteria contain the plasmid of interest.

<h4>Applications</h4>

Effective transformation methods enabled scientists to isolate and profile genes and gene products and led to many advancements in life sciences and medicine, such as development of effective drugs, generation of genetically-modified crops, and advanced diagnostic tools10. In addition, with technological advancements, new methods of transformation have emerged. For instance, Gateway Cloning allows insertion of multiple DNA fragments into different vectors as well as transfer of DNA sequences between plasmids11. Furthermore, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 is a gene-editing technique that directly modifies nucleotides in the genome and does not require the use of plasmids12. After transformation, researchers often isolate and profile the gene of interest and its products. Subsequently, the whole process of genetic cloning has opened up a new field of genetic manipulation. Thanks to genetic cloning, researchers can manipulate bacteria to produce large amounts of specific human proteins, such as insulin to treat diabetes patients10. Cloning also has served a major importance in modern day agriculture. Genetically modified organisms (GMO) are a direct result of genetic cloning and bacterial transformation10. For instance, scientists are working to generate genetically modified crops with nitrogen-fixing genes incorporated into their genome to boost food production and reduce fertilizer use, thus, diminishing the economic and environmental impact of fertilizers13. In sum, bacterial transformation is the first step of modern-day biotechnology and the foundation of future research discoveries.

<h4>References</h4>

<ol class="references">
	<li>GL, Armstrong, LA, Conn and RW, Pinner. Trends in Infectious Disease Mortality in the United States During the 20th Century. JAMA. 1999, Vol. 281, 1 (61-66).</li>
	<li>F, Griffith. The Significance of Pneumococcal Types. J Hyg (Lond). . 1928 , 2 (113-59).</li>
	<li>Lorenz, MG and Wackernagel, F. Bacterial gene transfer by natural genetic transformation in the environment. Microbiol Rev. . 1994 Sep;, Vol. 58, 3: (563-602).</li>
	<li>S, Domingues, et al. Natural Transformation Facilitates Transfer of Transposons, Integrons and Gene Cassettes between Bacterial Species. PLOS Pathogens. 2012, 8(8): e1002837.</li>
	<li>Dubnau, D. DNA uptake in bacteria. Annu Rev Microbiol. . 1999, 53: (217-44).</li>
	<li>Solar, G del, et al. Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev. . 1998 , Vol. 62, 2: (434-64).</li>
	<li>Bennett, PM. Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria. Br J Pharmacol. . 2008 , Vol. 153, S1: (S347-S357).</li>
	<li>MLlosa, et al. Bacterial conjugation: a two-step mechanism for DNA transport. Mol Microbiol. 2002 Jul;, Vol. 45, 1: (1-8).</li>
	<li>Roychoudhury, A, Basu, S and Sengupta, DN. Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors. Indian J Biochem Biophys. . 2009 , Vol. 46, 5: (395-400).</li>
	<li>Khan, S, et al. Role of Recombinant DNA Technology to Improve Life. I. nt J Genomics. . 2016, 2016:2405954.</li>
	<li>Marsischky, G and LaBaer, J. Many Paths to Many Clones: A Comparative Look at High-Throughput Cloning Methods. Genome Res. 2004, Vol. 14, 2020-28.</li>
	<li>Doudna, JA and Charpentie, E. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014, Vol. 346, 6213-1258096.</li>
	<li>Good, A. Toward nitrogen-fixing plants. Science. 2018, Vol. 359, 6378: 869-70.</li>
</ol>

Bacterial Transformation using Plasmids - Concept

细菌转化

Background

In early 20th century, pneumonia was accountable for a large portion of infectious disease deaths1. In order to develop an effective vaccine ...

多拷贝产质粒在抗生素耐药性演变中的作用试验

摘要

探索更多视频

此视频中的章节

食品筛查为1类整合和基因盒

细菌结合频率的高分辨率比较

实验进化方法中疟原虫介导抗生素耐药性的定量化

大肠杆菌中天然偶联质粒介导的水平基因转移检测

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

使用抗生素耐药性平台进行抗生素脱除

使用外排缺陷细菌菌株和单拷贝基因表达系统表征鲍曼不动杆菌的多药外排系统

使用外排缺陷细菌菌株和单拷贝基因表达系统表征鲍曼不动杆菌的多药外排系统

使用外排缺陷细菌菌株和单拷贝基因表达系统表征鲍曼不动杆菌的多药外排系统

细菌转化

多拷贝产质粒在抗生素耐药性演变中的作用试验

摘要

探索更多视频

此视频中的章节

食品筛查为1类整合和基因盒

细菌结合频率的高分辨率比较

实验进化方法中疟原虫介导抗生素耐药性的定量化

大肠杆菌中天然偶联质粒介导的水平基因转移检测

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

使用抗生素耐药性平台进行抗生素脱除

使用外排缺陷细菌菌株和单拷贝基因表达系统表征 鲍曼不动杆 菌的多药外排系统

使用外排缺陷细菌菌株和单拷贝基因表达系统表征 鲍曼不动杆 菌的多药外排系统

使用外排缺陷细菌菌株和单拷贝基因表达系统表征 鲍曼不动杆 菌的多药外排系统

细菌转化

使用外排缺陷细菌菌株和单拷贝基因表达系统表征鲍曼不动杆菌的多药外排系统

使用外排缺陷细菌菌株和单拷贝基因表达系统表征鲍曼不动杆菌的多药外排系统

使用外排缺陷细菌菌株和单拷贝基因表达系统表征鲍曼不动杆菌的多药外排系统