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) 含有的染色体副本的 红外 基因。然而, 当 pJFM1 转化为缺乏红外野生 MG1655, pJFM1 不能复制, 导致选择的卡那霉素抗性克隆产生的随机插入 NKBOR:: DARS2 进入细菌染色体。为了简单起见, 这些被称为 DARS2 插入。有关方法的示意图演示, 请参见 图 1 . 
<ol> <li> 准备 electrocompetent MG1655 和 #916; DARS2 从积极生长的细胞性肉汤 (LB) 生长在37和 #176; C 24 . </li>
	<li> Electroporate pJFM1 到 electrocompetent MG1655 和 #916; DARS2 , 根据冈萨雷斯 et al. 24 <ol> <li> 添加1和 #181; g pJFM1 (1 和 #181; l) 到40和 #181; electrocompetent 大肠杆菌 MG1655 和 #916; DARS2 , 并将此混合物转换为 pre-chilled、无菌0.2 厘米间隙试管。将试管插入电穿孔室。Electroporate 在18伏, 500 和 #937;, 和25和 #181; F;时间常数应为 ~ 5.0 毫秒, 不应发生电弧. </li>
		<li> 通过溶液1毫升的 5ml LB 肉汤快速恢复细菌悬浮液, 并将其转移到15毫升的试管中. </li>
		<li> 通过在37和 #176 的充气生长条件下孵化, 使细胞恢复; C 为30分钟, 没有抗生素选择。将细菌放在 LB 琼脂板上, 辅以50和 #181; 卡那霉素和孵育在37和 #176; C 过夜. </li>
	</ol> </li> <li> 从电穿孔中计数菌落: 一个菌落被认为等于一个染色体转插入. 
	注意: 菌落可以用眼睛或菌落计数。菌落数与每个克隆的染色体上的转的平均距离成反比. </li>
	<li> 在每个盘子上加入1毫升的 lb 肉汤, 洗去所有的菌落; 1 毫升的 lb 肉汤应该足够洗掉10万菌落。再用同样的1毫升的 LB 肉汤来增加细菌的浓度。池所有的殖民地在同一50毫升管. </li>
	<li> 涡流管并冻结起始材料 (t = 0)。为了冷冻它, 在冰上混合1毫升的细胞悬浮液和1毫升的50% 甘油。把管子转到干冰上10分钟。一旦文化被冻结, 把它们转移到-80 和 #176; C 冷冻. 
	注: 在这个步骤中, 细胞浓度至少为 5万 CFU/毫升. </li>
</ol> 2。竞争实验在 LB <ol> <li> 从-80 和 #176 解冻转库; 冰上的 C由移混合. </li>
	<li> 传输100和 #181; 转库的 l 在 15 ml 测试管中为10毫升的 LB. </li>
	<li> 通过连续晃动 (250 rpm) 来增加细胞的生长, 8 小时为37和 #176; C 到固定相位 ( 即 OD 600 = ~ 4.0)。根据需要将这些参数调整为不同的生长条件. </li>
	<li> 每10代将细菌种群连续转移到新鲜的 prewarmed 培养基中传播。通过转移10和 #181 的方法来完成这一步. 以前固定相培养的 L 到10毫升的新鲜 LB 和成长为另 8 h 到固定阶段 ( 即, OD 600 = ~ 4.0). 
	注: 由于起始和结束光学密度相同, 1000 倍的稀释相当于 ~ 10 代的生长 (2 10 = 1024)。细菌种群可以直接传播到新鲜的 prewarmed 培养基中, 或保存在0-4 和 #176; C (冰上), 第二天传播。LB 在这里使用, 以确保尽可能短的时间尽可能多的细胞 doublings (一倍的时间接近22分钟). </li>
	<li> 在每100代竞争之后, 在-80 和 #176 中保存五 1 mL 样本 (如步骤3.3 所述). </li>
	<li> 如果包含单个转插入的单元格之间的适应性差异预计较小, 则保持竞争持续时间长; 在这里, 700 代 (t = 700) 被使用. </li>
</ol> 3。南方印迹分析监测竞争的实验随着时间的推移 <ol> <li> 通过使用特定引物进行聚合酶链式反应 (PCR) 的方法, 为南部印迹 (NKBOR 的 1 kb 部分) 准备探针: NKBOR_Probe_FW: gatgttggacgagtcggaat 和 NKBOR_Probe_RV: cgttacatccctggcttgtt. 
	<ol> <li> 孵育在98和 #176; C 用于三十年代初始变性。然后, 执行35周期在98和 #176; c 为十年代, 60 和 #176; c 为三十年代, 和72和 #176; c 为四十五年代。对于最后的扩展, 使用72和 #176; C 为 10 min. 
		注: PCR 反应的模板是纯化的 pNKBOR 质粒 21 . </li>
		<li> 使用随机引物系统将合成的 PCR 片段标记为 [和 #945;-32 p] dATP, 根据 Smith 25 . </li>
	</ol> </li> <li> 根据 L 和 #248 准备总细胞 DNA; bner 奥尔森和冯 Freiesleben 26 为 t = 0 和选择的时间点直到 t = 700 估计的世代竞争. </li>
	<li> 消化总细胞 dna 与 Pvu i, 它减少包含区域利益的 NKBOR 一次仅在没有被探针包括的区域; 1 单位 Pvu 我可以用来完全消化1和 #181; g 的基质 dna. 
	注意: 探针将识别包含部分转的片段, 以及不同长度的染色体 DNA, 这取决于插入点. </li>
	<li> 根据 L 和 #248 使用0.7% 琼脂糖凝胶执行南部印迹; bner-奥尔森和冯 Freiesleben 26 . </li>
</ol> 4。最适克隆的标识 <ol> <li> 使用简单的基因遍历, 如哈里森 et al. 所述 27 , 用于从单个克隆 (在 LB 板上隔离) 中确定 DARS2 插入点 (在700估计的几代竞争之后);南部印迹上的波段越多, 就需要更多的隔离物来覆盖所有的转插入. 
	<ol> <li> 将基因组 DNA 与 PCR 模板分离。将细菌散布在含有50和 #181 的 LB 琼脂板上; 在37和 #176; C/毫升卡那霉素和孵育;在37和 #176 的 LB 肉汤中, 一夜之间生长单一的菌落; C, 然后转移20和 #181; l 进入200和 #181; l 蒸压蒸馏水 (或使用 DNA 纯化, 如步骤 3.2)。
		要混合的 <ol> <li> 旋涡。在100和 #176 加热混合; c 为10分钟和离心机在最大速度为 5 min. 将细胞裂解液保存在-20 和 #176; c, 以备将来使用. </li>
		</ol> </li> <li> 设计三嵌套底漆以在选择的转中退火 (请参见 图 2 以显示 PCR 策略的图形表示). 
		注意: 设计嵌套的底漆应确保嵌套的底漆3最靠近已知的5和 #39; 转 DNA 的末端, 后跟嵌套的引物2和1。嵌套入门3和5和 #39 之间的间距; 已知 t 的结尾ransposon dna 应该是足够的序列通读从已知的 dna 到未知的 dna (找到特定的染色体转插入点)。对于 NKBOR, 使用了以下嵌套底漆: pNKBOR_Nested3_RV: gcagggctttattgattcca、pNKBOR_Nested2_RV: tcagcaacaccttcttcacg 和 pNKBOR_Nested1_RV: actttctggctggatgatgg。还使用了含有已知限制性酶的识别点的随机引物。为了确保一个结果, 你应该至少使用两个不同的随机引物。限制酶的限制点 ( 如, 秀 3AI 和 显 dIII) 应位于3和 #39; 末端和前面十随机基地, 使5和 #39;-NNNNNNNNNNGATC-3 和 #39; 和5和 #39;-NNNNNNNNNNAAGCTT-3 和 #39; 是底漆分别包含 秀 3AI 站点 (GATC) 和 显 dIII 站点 (AAGCTT), 其中 N = a、T、G 或 C. </li>
		<li> 在20和 #181 中使用 200 ng 基因组 DNA, PCR 反应。孵育于98和 #176; C 用于三十年代的初始变性。在98和 #176 执行25次循环; c 为三十年代, 50 和 #176; c 为十五年代, 72 和 #176; c 为 4 min。对于最后的扩展, 使用72和 #176; C 为 10 min. 使用由嵌套底漆1和随机引物之一组成的底漆对. </li>
		<li> 使用1和 #181 的二次放大中的嵌套底漆2和相同的随机底漆的底漆对. </li>
		<li> 重复步骤4.1.4 与底漆对组成的嵌套底漆3和相同的随机底漆. </li>
		<li> 从1.5% 琼脂糖凝胶和溴溴化物的最终 PCR 反应中运行产品。根据制造商和 #39 的方向, 在 100-800 bp 的范围内切割出一个波段, 并使用任何商业 dna 凝胶提取试剂盒来分离 dna。重15和 #181 中的 DNA; 水的 L. 
		注意: 小心。溴溴化物有毒, 必须小心处理. </li>
		<li> 将上一步中隔离的频带序列 (步骤 4.1.6) 与嵌套的入门3作为排序入门, 使用在商业提供商的桑格排序. </li>
	</ol> </li> <li> 执行 WGS. 
	<ol> <li> 对在竞争实验中收集的选定样本中提取的总 DNA 执行 WGS, 如 Frimodt-M & #248 所述; 缪勒 et al 4 . </li>
	</ol> </li> <li> 执行排序分析。
	<ol> <li> 将配对端读取对齐到 150 Ns 连续到 NKBOR、AF310136.1 1904 和 #8230; 2204 使用 Bowtie2 28 , 并在种子子字符串 (S、11. 15) 和最大数目的不明确字符之间进行间隔 (L, 00. 9). </li>
		<li> 选择对齐的读取, 然后调整 MG1655 ref |NC_000913.3 和 NKBOR gb |AF310136 使用 blastN 29 </li> <li> 在连接 MG1655-NKBOR 处分配换位插入位置。
	</ol> </li> </ol> 5。流式细胞仪 <ol> <li> 收集流式细胞术的样本. 
	<ol> <li> 通过在指数增长阶段保持至少10代, 来平衡每个区域性。检查 OD 600 并确保它永不超过 0.3. </li>
		<li> 收集两种类型的流样本。
		<ol> <li> 对于 "1 ml", 将培养物转移到15毫升的管中, 包含30和 #181; (300 和 #181; 利福平和36和 #181; g/毫升苄溶解于12.5 毫米氢氧化钠)。把它留在37和 #176; C 至少4小时的震动. 
			注意: 利福平间接阻断了染色体复制的启动, 同时允许连续的复制继续终止。苄防止细胞分裂, 这会导致复制的跳动 (跳动) 和细胞的积累与完全复制的染色体。完全复制染色体的数量等于在使用利福平和苄时的起始数量 20 . </li>
			<li> 对于 EXP 样本, 将1毫升的指数增长的文化转移到1.5 毫升的冰上. </li>
		</ol> </li> </ol> </li> <li> 按如下方式修复单元格。以 1.5万 x g 为5分钟的离心法, 在4和 #176 中获得细胞; c. 重100和 #181 中的颗粒; ice-cold 10 mM 的 pH 值 7.5, 添加1毫升 ice-cold 77% 乙醇。将样品存放在4和 #176; C 直到使用. </li>
	<li> 将单元格染色如下。100-300 和 #181; 通过离心法在 1.5万 x g 处固定细胞 L 15 分钟. 在130和 #181 中除去上清和重; #34;D na 染色液和 #34; (90 和 #181; g/毫升 mithramycin, 20 和 #181; g/毫升溴溴, 10 mM 氯化镁 2 和10毫米三 pH 7.5)。把样品放在冰上和黑暗中;样品为流式细胞仪分析10分钟后即可完成. 
	注: 其他 DNA 染色比溴溴化和 mithramycin 也可以使用 30 , 31 。
	<li> 通过流式细胞仪分析确定每个细胞的来源、相对细胞质量和相对 DNA 含量. 
	<ol> <li> 执行流式细胞术, 如前所述 32 。使用流式细胞仪制造商和 #39 的说明 . 对于 mithramycin 和溴溴化染色细胞, 使用励磁波长395和 440 nm, 并收集在 565 nm 以上的荧光。
		<ol> <li> 要确定相对的细胞质量和相对的 DNA 含量, 运行 EXP 样本以获得参数前光散射与细胞数直方图和荧光强度与细胞数直方图的最小3万事件对应的细胞信号. </li>
			<li> 使用前向散射光参数分布来测量平均相对细胞质量. 
			注意: 前向散射光是平均单元格质量 20 的度量值. </li>
			<li> 使用荧光强度参数分布来测量平均 DNA 含量 20 . </li>
			<li> 获取 dna 浓度作为平均 dna 含量与平均细胞质量的比值 20 . </li>
		</ol> </li> <li> 确定每个单元格的起始数。
		<ol> <li> 运行参数的样本以获得与细胞信号对应的至少3万事件的荧光强度与细胞数直方图. </li>
			<li> 使用荧光强度参数分布来确定每个单元格中的染色体数量 20 . </li>
		</ol> </li> </ol> </li> <li> 计算异步索引 (Ai). 
	<ol> <li> 计算 Ai, 如 L 和 #248 所述; bner 奥尔森 et al. 32 , 使用 f3 的荧光强度单参数分布的样本和公式 Ai = (f5 + f6 + f7)/(f2 + f4 + f8), 其中 fx 是分数细胞 x 数的完全复制的染色体。考虑 #62 时的异步提升; 0.1. </li>
	</ol>
	</li>
</ol>

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G., von Meyenburg, K., Boye, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DnaA+protein+determines+the+initiation+mass+of+Escherichia+coli+K-12.">The DnaA protein determines the initiation mass of Escherichia coli K-12.</a> Cell. 57 (5), 881-889 (1989).</li><li>Carver, T., Thomson, N., Bleasby, A., Berriman, M., Parkhill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNAPlotter:+circular+and+linear+interactive+genome+visualization.">DNAPlotter: circular and linear interactive genome visualization.</a> Bioinformatics. 25 (1), 119-120 (2009).</li><li>Eckert, S. 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作者没有竞争的财务利益。

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) 实验中使用了几种不同的转, 如微型 Tn5Km234和更流行的选项, Himar I水手转35, 36 (有关此的最新查看, 请参阅 van Opijnen 和 Camilli36)。我们的目标是7万初始随机插入DARS2, 它在 MG1655 基因组中提供了大约一个DARS2插入埃弗特 65 bp。这很容易通过减少或增加最初收集的菌落池来调整。
在这里, 我们选择在 LB 批培养中进行连续传输, 但竞争实验也可以在不同的介质或不同的条件下执行, 包括使用恒37。此外, 该程序可以修改, 以适应任何条件的选择, 包括各种压力, 如氧化, 渗透, 或抗生素的压力。为了验证存在的持久性克隆随着时间的推移, 你可以做至少三件事: WGS;转测序 (Tn-Seq);或者, 就像这里, 一个南方的污点。最后, 简单的基因走可以做一些克隆, 进一步的优势, 转的插入点被识别在单一的克隆, 这样的效果, 具体插入网站可以型分析。
选择表型测定来评价竞争实验的结果取决于所研究的区域。我们使用流式细胞仪, 这是一个强大的方法来测量细胞周期参数的细菌。在这里, 流式细胞仪显示, 选定的染色体位置 (s) 的DARS2, (即 DARS2插入接近野生DARS2位置) 导致了正确的复制启动规则 (即同步和 DNA 浓度)。选择了DARS2的最佳染色体位置, 部分原因是由于适当的复制相关基因剂量, 部分原因是由于对DARS2函数4非常重要的本地基因组环境有利。
在这里, 一个编码的 DNA 元素被调查, 但这可以扩展到任何编码区域的选择。最近的一项研究发现, 基因表达的水平因其在染色体上的位置而变化300倍, 与复制相关的基因剂量38没有明显的相关性。这里所描述的方法可以对特定基因的染色体定位、转录活性和相关的健身优势之间的关系有重要的了解。这在理论上可以帮助选择基因组的位置, 导致更强的表达一个特定的, 这反过来可能是感兴趣的工程新的, 改良菌株的重组蛋白生产。
由于大肠杆菌包含有限的基因区域39, 因此在大多数情况下, 转插入将破坏基因。这可能偶尔会产生误报, 因为最合适的克隆 (s) 不是由于遗传因素的最佳染色体位置而选择的, 而是因为一个基因被破坏了--在其他方面, 这在竞争实验4。
这种方法利用了一个易于使用的转系统, 其中任何选择区域都可以在随机位置进行集成。设计的竞争实验可以修改, 包括任何数量的选择力以外的纯增长, 如这里所见。设置也可以修改为纯粹基于 Tn-Seq, 它产生的插入点的排序分辨率比 WGS, 这里使用的更大。这种不偏倚的方法应该用来阐明 uncharacterized 生物的令人兴奋的新特性, 这可能表明真染色体组织中存在着共同的趋势。

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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		<col />
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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 片段对 NKBOR 的部分进行了探讨。从图 3中可看出, 初始DARS2池 (t = 0) 缺少不同的带区, 这表明DARS2是在整个染色体中随机插入的。随着时间的推移, 出现了一个模式, 即最初的大池DARS2克隆只发展成一个或几个持久的DARS2克隆 (图 3; t = 0 到 t = 700)。
在所示的示例中, 使用 WGS 和简单的基因行走来识别竞争实验中的DARS2插入点。在这里, WGS 被用来从起始池 (t = 0) 和300、400和700代的竞争中识别插入站点。请注意, 目前深度测序中的覆盖率不足以完成在 t = 0 处插入点的完整映射;但是, 它给出了插入总数的一个具有代表性的子集。WGS 证实了南部的印迹结果 (即选择最合适的DARS2克隆), 结束时约98% 的所有DARS2插入接近野生DARS2染色体位置 (DARS2克隆 IR 和克隆rppH), 其余的2% 在染色体上的其他位置 (图 4)。这强烈表明野生位置对于DARS2函数是最佳的。在 t = 400 处, 在相反的复制臂上找到一个插入, 它与oriC的距离几乎相同, 为野生DARS2位置 (图 4), 但在700代之后未恢复此插入。因此, 复制相关的基因剂量不能成为最佳位置的唯一决定因素。
简单的基因行走被用来确定在700估计数代竞争后孤立的单个克隆中的DARS2插入点。在此处, 标识了上述两个DARS2插入站点 (DARS2克隆红外和克隆rppH)。简单的基因行走只在20克隆上完成, 这解释了为什么所有在 WGS 中映射的DARS2插入站点都未被识别。DARS2-缺陷单元格以前显示为在异步中启动复制, 并且相对于野生单元格的起始浓度降低4、16、17、 18. 因此, 我们使用流式细胞术来解决两个选定菌株 (DARS2克隆红外和克隆rppH) 在启动 DNA 复制和细胞来源含量时的同步性, 在这两种情况下, 它们都还原为野生级别 (图 5)。在终端中有一个具有DARS2单一副本的应变的典型示例 (图 5E)。在这里, 总站中的DARS2元素的存在不会将同步性或单元格原点内容还原到野生级别, 而选定的DARS2克隆 IR 和rppH则不恢复。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55946/55946fig1.jpg"> 
图 1: 方法概述.方法的示意图介绍。染色体DARS2区域被克隆到 pNKBOR 上的迷你Tn10 中, 创建 pJFM1。pJFM1 被转换成大肠杆菌MG1655 δ DARS2, 它将随机插入DARS2链接到大肠杆菌MG1655 δDARS2的染色体上。大约7万克隆, 每个包含一个不同的染色体DARS2插入, 被汇集和竞争直接地互相反对在 LB 肉汤在37° c。在 LB 肉汤中进行的直接竞争试验是在大约700代人的情况下进行的, 在每100代的直接竞争中, 有一个样本被隔离。从每个孤立样本提取的总 DNA, 并用于南部印迹和识别DARS2插入由 WGS。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55946/55946fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55946/55946fig2.jpg"> 
图 2: 易于基因行走的图形演示。这一数字说明了一个未知的 dna 序列的基因组 dna 模板相邻的已知序列与启动点的随机底漆和嵌套引物 1, 2, 和3。使用三设计的嵌套底漆进行的三连续放大的结果如下图所示。最后的产品 (从3轮) 是使用嵌套的底漆3排序。这个数字是改编自哈里森et al.27.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55946/55946fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55946/55946fig3.jpg"> 
图 3: 探测 NKBOR 的南部印迹.南印迹分析的DARS2插入到一个DARS2缺陷应变。从每100代的直接竞争中提取的基因组 DNA, 从 t = 0 开始, 以700代结束, 用PvuI 和凝胶分馏来消化。杂交与 NKBOR 探针 (参见协议)。t 表示竞争的世代数。此图是从 Frimodt-迈克尔·默勒et al.改编的4. 亚基和 lmw-gs 分别为高分子重量和低分子量 DNA。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55946/55946fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55946/55946fig4.jpg"> 
图 4: 决心的图形表示形式ed 转在ΔDARS2中插入站点。指示oriC、数据、DARS1、DARS2、和terC的位置。DARS2在ΔDARS2中插入站点的位置为 t = 0 (黑色条形)、t = 400 (浅蓝色条形)、t = 500 (红色条) 和 t = 700 (绿色条形), 由 full-genome 测序解决。此图是使用 DNAPlotter33进行的, 并且是从 Frimodt-迈克尔·默勒et al.改编的。4.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55946/55946fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55946/55946fig5.jpg"> 
图 5: 具有代表性的流式细胞仪直方图, 由简单的基因在 t = 700 处找到转站点.细胞生长在 AB 最低培养基补充0.2% 葡萄糖, 10 µg/毫升硫胺素, 和 0.5% casamino 酸在37° c。野生和ΔDARS2分别显示在 a 和 B 中。野生应变的衍生物 MG1655 在原始轨迹上没有DARS2 , 而是在已解析的转站点rrpH、 IR 和终点 (terC) 中的DARS2的副本显示在 C、D 和 E 中,分别.此图是从 Frimodt-迈克尔·默勒et al.改编的4显示一个DARS2位置, 它会导致单元格周期异常 (terC) 或恢复野生表型(rrpH, IR)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55946/55946fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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 ...

determination-optimal-chromosomal-location-s-for-dna-element

Nanyang Technological University

Research

JoVE Journal

Genetics

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

7.5K 次观看.  University of Copenhagen. 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。

视频: Determination of the Optimal Chromosomal Locations 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

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

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

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

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

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

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

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 region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

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

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 number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

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

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to control gene expression. These regulatory proteins control gene expression either at the level of DNA (transcription) or at the level of RNA (pre-mRNA splicing, polyadenylation, mRNA transport, decay, and translation). Identification of regulatory sequences helps understand not only how a gene is switched on or off, but also which downstream genes are regulated by a particular regulatory protein. Here, we describe a one-step approach that allows saturation mutagenesis of a protein binding site in RNA. It involves doping DNA template with non-wild-type nucleotides within the binding site, synthesis of separate RNAs with each phosphorothioate nucleotide, and isolation of the bound fraction following incubation with protein. Interference from non-wild-type nucleotides results in their preferential exclusion from the protein-bound fraction. This is monitored by gel electrophoresis following selective chemical cleavage with iodine of phosphodiester bonds containing phosphorothioates (phosphorothioate mutagenesis or PTM). This single-step saturation mutagenesis approach is applicable to the characterization of any protein binding site in RNA.

Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA.

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

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 for transferring gene deletion mutations from one Escherichia coli strain to another by using generalized transduction with the bacteriophage P1. This method requires that the mutation be selectable (e.g., based on gene disruptions using antibiotic cassette insertions). Such antibiotic cassettes can be mobilized from a donor strain and introduced into a recipient strain of interest to quickly and easily generate a gene deletion mutant. The antibiotic cassette can be designed to include flippase recognition sites that allow the excision of the cassette by a site-specific recombinase to produce a clean knock-out with only a ~100-base-pair-long scar sequence in the genome. We demonstrate the protocol by knocking out the tamA gene encoding an assembly factor involved in autotransporter biogenesis and test the effect of this knock-out on the biogenesis and function of two trimeric autotransporter adhesins. Though gene deletion by P1 transduction has its limitations, the ease and speed of its implementation make it an attractive alternative to other methods of gene deletion.

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

Here we present a protocol for the use of pre-existing antibiotic resistance-cassette deletion constructs as a basis for making deletion mutants in other E. coli strains. Such deletion mutations can be mobilized and inserted into the corresponding locus of a recipient strain using P1 bacteriophage transduction.

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

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

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

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

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

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

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

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 environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

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 ...