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

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

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

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

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

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

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

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

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

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

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

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

Universidad San Sebastian

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

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

接合交配测定在接合介入转移蛋白的序列特异性分析

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

科研

遗传学

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

网站特定的细菌染色体工程：ΦC​​31整合介导的盒式交换（IMCE）

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

生物工程

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 ...

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 ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

使用细菌共轭的 Enterobacterial 菌株 (如大肠杆菌或志贺菌福氏) 创建稠密的转插入库

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

免疫与感染

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

测绘细菌功能的网络和途径<em&gt;大肠杆菌</em使用合成基因阵列

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

生物学

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

的产生<em&gt;肠杆菌</em&gt; SP。 YSU营养缺陷型使用转座子突变

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure. Antimicrobial resistance is defined as bacterial growth in the presence of otherwise lethal antibiotics. The &#8220;last-line&#8221; antimicrobial colistin is used to treat Gram-negative bacterial infections. However, several Gram-negative pathogens, including Acinetobacter baumannii can develop colistin resistance through a range of molecular mechanisms, some of which were characterized using Tn-seq. Furthermore, signal transduction pathways that regulate colistin resistance vary within Gram-negative bacteria. Here we propose an efficient method of transposon mutagenesis in A. baumannii that streamlines generation of a saturating transposon insertion library and amplicon library construction by eliminating the need for restriction enzymes, adapter ligation, and gel purification. The methods described herein will enable in-depth analysis of molecular determinants that contribute to A. baumannii fitness when challenged with colistin. The protocol is also applicable to other Gram-negative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.

We describe a method to generate saturating transposon mutant libraries in Gram-negative bacteria and subsequent preparation of DNA amplicon libraries for high-throughput sequencing. As an example, we focus on the ESKAPE pathogen, Acinetobacter baumannii, but this protocol is amenable to a wide range of Gram-negative organisms.

在革兰氏负性细菌中生成跨波子插入库，用于高通量测序

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways ...

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological processes. Advances in molecular microbiology have seen the emergence of diverse mutagenesis techniques for the manipulation of genes. Among them, transposon-insertion sequencing (Tn-seq) is a valuable tool to simultaneously assess the functionality of many candidate genes in an untargeted way. The technique has been key to identify&#160;molecular mechanisms for the colonization of eukaryotic hosts in several pathogenic microbes and a few beneficial symbionts.
Here, Tn-seq is established as a method to identify colonization factors in a mutualistic Burkholderia gladioli symbiont of the beetle Lagria villosa. By conjugation, Tn5 transposon-mediated insertion of an antibiotic-resistance cassette is&#160;carried out at random genomic locations in B. gladioli. To identify the effect of gene disruptions on the ability of the bacteria to colonize the beetle host, the generated B. gladioli transposon-mutant library is inoculated on the beetle eggs, while a control is grown in vitro in a liquid culture medium. After allowing sufficient time for colonization, DNA is extracted from the in vivo and in vitro grown libraries. Following a DNA library preparation protocol, the DNA samples are prepared for transposon-insertion sequencing. DNA fragments that contain the transposon-insert edge and flanking bacterial DNA are selected, and the mutation sites are determined by sequencing away from the transposon-insert edge. Finally, by analyzing and comparing the frequencies of each mutant between the in vivo and in vitro libraries, the importance of specific symbiont genes during beetle colonization can be predicted.

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

This is an adapted method for identifying candidate insect colonization factors in a Burkholderia beneficial symbiont. The beetle host is infected with a random mutant library generated via transposon mutagenesis, and library complexity after colonization is compared to a control grown in vitro.

跨波森插入测序作为工具，以阐明细菌殖民因素在拉克霍尔德里亚角膜甲虫

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological ...

Real-Time Quantification of the Effects of IS200/IS605 Family-Associated TnpB on Transposon Activity

Here, a protocol is outlined to perform live, real-time imaging of transposable element activity in live bacterial cells using a suite of fluorescent reporters coupled to transposition. In particular, it demonstrates how real-time imaging can be used to assess the effects of the accessory protein TnpB on the activity of the transposable element IS608, a member of the IS200/IS605 family of transposable elements. The IS200/IS605 family of transposable elements are abundant mobile elements connected with one of the most innumerable genes found in nature, tnpB. Sequence homologies propose that the TnpB protein may be an evolutionary precursor to CRISPR/Cas9 systems. Additionally, TnpB has received renewed interest, having been shown to act as a Cas-like RNA-guided DNA endonuclease. The effects of TnpB on the transposition rates of IS608 are quantified, and it is demonstrated that the expression of TnpB of IS608 results in ~5x increased transposon activity compared to cells lacking TnpB expression.

A protocol is outlined to perform live real-time imaging to quantify how the accessory protein TnpB affects the dynamics of transposition in individual live Escherichia coli cells.

实时定量IS200/IS605家族相关TnpB对转座子活性的影响

Here, a protocol is outlined to perform live, real-time imaging of transposable element activity in live bacterial cells using a suite of fluorescent ...

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)

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接合交配测定在接合介入转移蛋白的序列特异性分析

网站特定的细菌染色体工程：ΦC31整合介导的盒式交换（IMCE）

细菌结合频率的高分辨率比较

可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失

使用细菌共轭的 Enterobacterial 菌株 (如大肠杆菌或志贺菌福氏) 创建稠密的转插入库

测绘细菌功能的网络和途径

的产生

在革兰氏负性细菌中生成跨波子插入库，用于高通量测序

跨波森插入测序作为工具，以阐明细菌殖民因素在拉克霍尔德里亚角膜甲虫

实时定量IS200/IS605家族相关TnpB对转座子活性的影响

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)

摘要

探索更多视频

此视频中的章节

接合交配测定在接合介入转移蛋白的序列特异性分析

网站特定的细菌染色体工程：ΦC​​31整合介导的盒式交换（IMCE）

细菌结合频率的高分辨率比较

可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失

使用细菌共轭的 Enterobacterial 菌株 (如大肠杆菌或志贺菌福氏) 创建稠密的转插入库

测绘细菌功能的网络和途径

的产生

在革兰氏负性细菌中生成跨波子插入库，用于高通量测序

跨波森插入测序作为工具，以阐明细菌殖民因素在拉克霍尔德里亚角膜甲虫

实时定量IS200/IS605家族相关TnpB对转座子活性的影响

网站特定的细菌染色体工程：ΦC31整合介导的盒式交换（IMCE）