京王收集菌株来自国家 BioResource 项目 ( 日本 ) :大肠杆菌。我们感谢德克左翼 (奥斯陆大学生物科学系) 继续提供支持。这项工作由挪威青年研究员补助金 249793 (对杰克. 里奥) 的研究理事会资助。

1. 菌株和质粒
<ol><li>
菌株
	<ol>
<li>使用大肠杆菌菌株 BW251135, JW4179 (BW25113摩:: 菅直人)7, BL21 (DE3)20, BL21ΔABCF21。有关详细信息, 请参阅材料表。</li>
	</ol>
</li>
	<li>
噬菌体
	<ol>
<li>使用噬菌体 P1vir的一般转导。将噬菌体储存在少量氯仿滴下的液体储存中 (见步骤 2.2)。有关详细信息, 请参阅材料表。</li>
	</ol>
</li>
	<li>
质 粒
	<ol>
<li>在本协议中使用以下质粒: pCP2022、pIBA2-YadA23和 pEibD1024。作为控制质粒, 使用 pASK-IBA2 和 pET22b (见材料表)。</li>
	</ol>
</li>
	<li>
生长条件
	<ol>
<li>在含有 pCP20 的 BL21ΔABCF 和菌株的情况下, 以剧烈震动 (180–200 rpm) 在37摄氏度或30摄氏度的溶源性肉汤 (磅) 培养基25中传播细菌。</li>
		<li>进行质粒固化43摄氏度。</li>
		<li>对于固体培养基, 补充 LB 与1% 琼脂 (w/v)。</li>
		<li>对于顶琼脂, 补充 LB 与0.7% 琼脂和10毫米 CaCl2和高压釜的培养基。使用 SOC 介质进行电穿孔26后的恢复。</li>
		<li>使用以下抗生素浓度: 100 微克/毫升为安培和25微克/毫升为菅直人。</li>
	</ol>
</li>
</ol>2. 制备噬菌体裂解液
<ol><li>
供体菌株感染
	<ol>
<li>生长捐赠者应变 JW4197 在5毫升 LB 介质补充与10毫米 CaCl2和可选与菅直人 (25 µg/毫升) 到光学密度在600毫微米 (OD600) ~ 1.0。使用分光光度计测量 OD600值。</li>
		<li>在 LB 介质中做一个现有 P1 噬菌体库存的稀释系列: 推荐的稀释介于 10-3到 10-7之间。</li>
		<li>混合 200 ul 的细菌悬浮和 100 ul 的特定噬菌体稀释在15毫升离心管或同等。准备尽可能多的管作为噬菌体稀释。在37摄氏度处孵育20分钟, 不摇晃。</li>
		<li>添加〜3毫升的熔融顶琼脂 (~ 50 °c) 补充10毫米 CaCl2的管, 混合的内容, 通过涡流的管很快, 并倒入 prewarmed LB 板上的混合物, 使均匀层。</li>
		<li>在37摄氏度一夜之间孵化盘子。</li>
	</ol>
</li>
	<li>
裂解制剂
	<ol>
<li>第二天选择一个有半汇合生长的噬菌体斑块的盘子。在半汇合板上, 大约一半的板块表面是清晰的 (图 3)。</li>
		<li>使用接种回路或类似的工具从这样的盘子刮上琼脂层, 并将顶琼脂放在离心管中。添加一毫升的 LB 和一滴氯仿和漩涡的管大力1分钟. 在通风罩中加入氯仿。</li>
		<li>离心管15分钟, 在 4000 x克或更快的颗粒的琼脂和细菌细胞。</li>
		<li>将上清移至新鲜的离心管, 避免从颗粒中携带任何碎片。加入2滴氯仿, 在4–10°c 储存裂解液。在通风罩中加入氯仿。不要冻结噬菌体裂解物, 因为这将导致显著减少感染微粒的数量。</li>
	</ol>
</li>
	<li>
测定裂解液滴度
	<ol>
<li>生长 BW25113 在 LB 补充与10毫米 CaCl2在37°c, 直到文化达到 OD600的 ~ 1.0。</li>
		<li>准备一个稀释系列的噬菌体裂解液在 LB (例如, 10-6–10-9)。 注意: 小心吸管从裂解液顶部取样, 避免将氯仿转移到稀释。</li>
		<li>混合 200 ul 的细菌悬浮和 100 ul 的特定噬菌体稀释在15毫升离心管或同等。准备尽可能多的管作为噬菌体稀释。在37摄氏度处孵育20分钟, 不摇晃。</li>
		<li>添加〜3毫升的熔融顶琼脂 (~ 50 °c) 补充10毫米 CaCl2的管, 混合的内容, 通过涡流的管很快, 并倒入 prewarmed LB 板上的混合物, 使均匀层。</li>
		<li>在37摄氏度一夜之间孵化盘子。</li>
		<li>第二天计算每个板块的斑块数量 (细菌垫中的清除区域), 并使用以下公式计算裂解液的滴度: <img alt="Equation 1" src="/files/ftp_upload/58267/58267eq1.jpg"> 例子: 在板材与稀释 10-7, 10-8和 10-9, 分别有 231, 27 和2个匾。当 100 ul 每稀释被使用了为传染, 并且, 因为效价被表达作为斑块形成的单位 (pfu)/mL, 电镀因素是10。这些数字输入到公式中: <img alt="Equation 2" src="/files/ftp_upload/58267/58267eq2.jpg"> 因此, 滴度是大约 2 x 1010传染性噬菌体粒子/毫升。</li>
	</ol>
</li>
</ol>3. P1 转导
<ol><li>
准备收件人单元格
	<ol>
<li>增加接受者应变 BL21ΔABCF 在 LB 补充到光学密度在600毫微米 (OD600) 1.0。使用分光光度计测量 OD600值。</li>
		<li>计算所需的噬菌体裂解液的体积, 以达到0.5 的感染多样性 (语言) 值。计算语言, 估计细菌数量的基础上600的文化。假设 OD600值1.0 对应于 ~ 109 cfu/毫升。根据已知的裂解液滴度计算所需的体积。 示例 (基于在步骤2.3.6 后的示例中计算的效度): <img alt="Equation 3" src="/files/ftp_upload/58267/58267eq3.jpg">
</li>
		<li>添加 CaCl2到接受菌株的文化, 以10毫米和混合。采取1毫升的文化进行转导。</li>
	</ol>
</li>
	<li>
执行转导
	<ol>
<li>添加适当数量的噬菌体裂解到1毫升的接受者文化 (包括 CaCl2在10毫米), 计算在步骤3.1.2 和轻轻混合。 注意: 小心把样品从裂解液的顶部移除, 避免将氯仿转移到混合物中。</li>
		<li>静态孵化混合20分钟, 在37摄氏度。</li>
		<li>通过添加柠檬酸钠, pH 值 5.5, 到100毫米停止感染。</li>
		<li>离心的细菌 (5000 x g 2 分钟) 和清除上清, 然后并用重悬他们在1毫升的新鲜磅补充100毫米柠檬酸钠, pH 5.5。</li>
		<li>在步骤3.2.4 中清洗细胞两次, 以确保去除游离噬菌体和钙。</li>
		<li>并用重悬1毫升新鲜磅的细菌补充100毫米柠檬酸钠, pH 5.5。用震动 (> 100 rpm) 在30摄氏度孵化细菌1小时。</li>
		<li>收集细菌的离心 (5000 x g 2 分钟) 和并用重悬他们在 100 ul 的 LB 与100毫米柠檬酸钠, pH 5.5。</li>
		<li>在 LB 板上传播细菌, 辅以25µg/毫升和10毫米柠檬酸钠, pH 5.5, 并将细菌生长在30摄氏度, 直到菌落出现 (~ 24 h)。</li>
	</ol>
</li>
	<li>
选择 transductants
	<ol>
<li>一旦殖民地生长在选择板块上, restreak 他们在 LB + 菅直人为单一的殖民地和增长30摄氏度, 直到单一的殖民地出现。</li>
	</ol>
</li>
</ol>4. 切除菅直人卡带
<ol><li>
重组质粒的转化
	<ol>
<li>使抗 BL21ΔABCF 应变 electrocompetent。<ol>
<li>生长菌株从一个单一的蚁群在新鲜磅 (5 毫升) 补充与菅直人 (25 µg/毫升) 在30°c, 直到 OD600是 ~ 0.5–0.7。</li>
			<li>从这里, 执行以下步骤在4°c 或在冰上。将细菌 (5000 x g 10 分钟) 离心, 取出上清液。</li>
			<li>重复先前的离心步骤, 并在100µL 的冰冷10% 甘油中并用重悬细胞颗粒, 用冰冷的蒸馏水冲洗两次细胞颗粒。</li>
		</ol>
</li>
		<li>冷却1毫米电穿孔小试管在冰上。</li>
		<li>在细胞悬浮液中加入 1 pg 质粒 DNA (pCP20), 轻轻混合悬浮液, 将其转移到冷电穿孔试管。</li>
		<li>将 electroporator 设置为1.8 伏, 并 electroporate 单元格。</li>
		<li>通过添加1毫升的 SOC 培养基, 并在30摄氏度的1小时内生长, 来抢救转化后的细胞。</li>
		<li>板100µL 的细胞在 LB + 安培板和增长的细胞在30°c 过夜。</li>
	</ol>
</li>
	<li>
重组的诱导
	<ol>
<li>从 lb + 安培板中挑选单一的菌落, 接种新鲜的 lb, 省略所有的抗生素。</li>
		<li>在不允许的温度下 (43 摄氏度) 一夜之间生长细胞, 以诱导 FLP recombinase 的表达。</li>
	</ol>
</li>
	<li>
选择重组
	<ol>
<li>使系列稀释和板材50µL 105-106稀释在非选择性板材和增长它隔夜在30°c。</li>
	</ol>
</li>
</ol>5. 基因删除的验证
<ol><li>
质粒丧失与成功重组的验证
	<ol>
<li>在这一顺序中, 4.3.1 在 lb、lb、磅和无抗生素的 lb 板上进行的平板上的单菌落。要帮助裸奔, 请使用殖民地网格 (参见辅助文件 1)。将盘子生长在30摄氏度, 直到菌落出现 (~ 24 小时)。</li>
		<li>选择在非选择性板上生长的10–20克隆, 但在选择性介质上无法生长以进行进一步的验证。</li>
	</ol>
</li>
	<li>
用菌落 PCR 法进行附加验证
	<ol>
<li>执行一个群体 PCR 与引物侧翼的多重摩编码序列 (见材料表)。</li>
		<li>准备一个主 PCR 组合。所需的混合量取决于要测试的菌落数 (参见表 1中的示例)。将试剂混合在冰上, 最后加入聚合酶。</li>
		<li>将 pcr 大师组合彻底混合, 然后将20µL 放入 pcr 条的管中。选择一个小数量的每个殖民地使用不育的吸管提示, 并添加到管。请记住包含原始的收件人应变以进行比较。另外, 还包括捐助者的应变。</li>
		<li>运行 PCR 反应 (见表 2所用的程序)。</li>
		<li>准备1% 琼脂糖凝胶。<ol>
<li>50毫升凝胶, 测量0.5 克琼脂糖和添加50毫升的泰缓冲器 (40 毫米三, 20 毫米醋酸, 1 毫米 EDTA, pH 值 8.0)。在微波炉中加热混合物, 直到所有琼脂糖都溶解。</li>
			<li>一旦琼脂糖溶解, 冷却溶液约50摄氏度, 然后添加 5 ul 的 DNA 染色染料。把溶液搅拌好, 倒入浇注室的凝胶盘中。插入一个或多个行的好梳子, 以便有足够的水井为所有样品, 以及分子尺寸标记。允许凝胶设置为30分钟。</li>
		</ol>
</li>
		<li>一旦 PCR 运行完成, 添加4µL 的 6x DNA 负载染料的每个样本。将凝胶放在电泳室中, 在井盖前加泰缓冲器。</li>
		<li>应用10–15µL 从每个 PCR 反应到一个良好的凝胶。还添加5µL 的分子尺寸标记。然后, 在 75 v 上运行30分钟的凝胶。</li>
		<li>一旦运行完成, 图像的凝胶使用蓝光成像仪。</li>
	</ol>
</li>
</ol>6. 其他技术
<ol><li>
蛋白表达 注: 测试蛋白 (亚 EibD) 的表达在其他地方23,24被详细描述。这是对主要步骤的简要总结。<ol>
<li>将所需的质粒 (pIBA2-YadA 和 pEibD10 及相应的控制质粒) 转换成 BL21ΔABCF Δ多摩 w, 并选择在 LB + Amp 上转化。</li>
		<li>对于蛋白表达, 接种100毫升的 LB 中 + 安培与1毫升的一夜文化转化细菌和增长这些30°c, 直到中期记录阶段, 在这段时间, 诱导蛋白质生产与任何乳糖 (在 100 ng/毫升) 或异丙基-β-d (0.5 毫米)。</li>
		<li>在 2 h 以后归纳在30°c, 测量文化的浊度和收集对应于50毫升的细胞在 OD600 = 1.0 通过离心文化为15分钟在 4000 x g。用10毫米 HEPES, pH 7.4, 然后将球团 1x, 然后将其存储在-20 摄氏度或进一步处理, 如步骤6.2。</li>
	</ol>
</li>
	<li>
外膜萃取 注: 外膜萃取是由 Leo等详细描述的。27. 下面概述了主要步骤。<ol>
<li>并用重悬细胞颗粒在1毫升10毫米 HEPES, pH 7.4, 补充10毫米氯化镁2和 MnCl2, 溶菌酶 (0.1 毫克/毫升), 和一捏 DNase i。</li>
		<li>溶解细胞 (例如, 使用一个珠子搅拌器)。</li>
		<li>将细胞尽快离心 (15600 x g2 分钟) 去除细胞碎片, 将上清移至新鲜管。</li>
		<li>离心细胞为30分钟在 1.6万 x g, 在之后, 并用重悬颗粒在 400 ul 1% n-月桂肌氨酸在10毫米 HEPES, pH 7.4。</li>
		<li>在室温下, 用搅拌30分钟孵育细胞, 然后将其离心30分钟以上。</li>
		<li>清洗半透明颗粒1x 与 200 ul 10 毫米 HEPES, pH 7.4, 然后并用重悬它在 30 ul 的10毫米 HEPES 和添加 10 ul 4x SDS 页样本缓冲区。</li>
	</ol>
</li>
	<li>
活动化验 注: 执行 SDS 页和活动化验为亚多和 EibD 如28所述。主要步骤概述如下。</li>
	<li>
SDS 页
	<ol>
<li>将样品加热50摄氏度5分钟, 然后再将其装入聚丙烯酰胺凝胶上, 以避免蛋白质变性。</li>
		<li>在 SDS 页分离后, 将蛋白质转移到聚偏聚氟 (PVDF) 膜上。</li>
		<li>转移后, 在 PBS 中用2% 脱脂奶粉堵住膜。</li>
	</ol>
</li>
	<li>
远西胶原蛋白印迹
	<ol>
<li>阻断后, 加入牛胶原 i 型稀释在阻塞缓冲液中浓度为10微克/毫升, 并孵育膜1小时。</li>
		<li>用 pbs + 0.05% Tween20 (pbs t) 冲洗膜2x。</li>
		<li>将原抗体 (单克隆抗胶原 COL-1) 添加到膜中, 稀释 1:2, 000 在阻塞缓冲液中。</li>
		<li>孵化后1小时的膜, 洗 2x, 如步骤6.3.2.2 中所述, 然后添加二次抗体 [山羊抗鼠 IgG-辣根过氧化物酶 (HRP) 共轭], 稀释1:10,000 在阻塞缓冲。</li>
		<li>将膜孵化1小时, 然后用 PBS 清洗2x。根据制造商的指示, 在膜上添加增强的化学发光基板, 并使用 CCD 摄像机检测波段。</li>
	</ol>
</li>
	<li>
EibD IgG 结合试验
	<ol>
<li>阻断后, 添加二次抗体 (山羊抗兔 HRP), 稀释 1:2, 000 在阻塞缓冲。</li>
		<li>将膜孵化1小时, 然后用 PBS 冲洗2x。执行步骤6.3.2.5 中提到的化学发光检测。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

P1 转导是一种快速、稳健、可靠的方法, 可在大肠杆菌中产生基因缺失。这在这里展示了传感一个摩中的删除突变从一个王庆会捐献者的应变 BL21-derived 接受。转导过程的主要阶段是传感裂解液的产生、转导本身、菅直人抗性盒的切除以及 PCR 的验证。总的来说, 这个过程需要大约1周的时间, 不需要使用分子生物学方法, 除了最终的 PCR 进行验证。因此, P1 转导可以在花费的努力和时间?...

研究基因功能的一个共同的第一种方法是执行敲出诱变和观察结果表型。这也被称为反向遗传学。细菌大肠杆菌一直是分子生物学的主力, 在过去的70年左右, 由于它的培养和它的顺从的遗传操作1。在大肠杆菌中产生基因缺失的方法有好几种, 包括标记交换诱变2、3和最近的重组工程使用λ红色或 Rac ET 系统4,5,6。
在广泛使用的系统中, 单个基因的编码序列被一种抗生素耐药性盒取代, 以后可以从5、7号染色体中切除。编码序列被替换, 例如由卡那霉素 (菅直人) 抵抗卡带, 由 flippase (FLP) 识别目标 (首次登记) 站点在两边旁边。首次登记税的地点是由 recombinase FLP 认可的, 该处介导在首次登记税地点之间, 导致删除菅直人卡带的地点特定的重组。这样, 可以实现对给定基因编码序列的完全删除, 只留下大约100基对 (bp) 的最小疤痕序列 (图 1)。
就在十年前, 所谓的京王收藏被开发出来了。这是一个基于标准实验室大肠杆菌K12 菌株的细菌库, 几乎所有非必需基因都被λ红色重组7,8单独删除。此集合中的克隆每个都有一个编码序列, 替换为可抽税的坎电阻盒。在许多应用中, 京王系列已被证明是一个有用的工具9。其中一个应用是在其他大肠杆菌菌株中产生删除突变体。从一个给定的删除克隆的菅直人可以动员一般传感噬菌体, 如 P110,11,12,13,14。从这种菌株中制备的噬菌体可以用来感染受体大肠杆菌, 在低但可靠的频率下, 可以通过同源重组将菅直人盒包含的区域纳入受体基因组。(图 2)。Transductants 可以选择在含菅直人的培养基上生长。在此之后, 如果需要去除抗生素耐药性盒, FLP recombinase 可以提供给 transductant 菌株的反式。对含有氨苄西林 (amp) 抗性标记物的 FLP 质粒进行了筛选, 对其进行了筛查, 并对野型编码序列和菅直人盒的正确切除进行了菌落 PCR 验证。
在这里, 提出了一个详细的协议, 描述了根据上述战略生产出的大肠杆菌菌株的每一个步骤。作为一个例子, 一个删除的摩的基因是证明。多摩编码的外层膜β-桶蛋白是运输和组装模块 (TAM) 的一部分, 涉及某些 autotransporter 蛋白的合成和毛菌15,16,17。然后用这个敲出的菌株来考察多摩的删除对两个三联 autotransporter adhesins (TAAs)、合成耶尔森氏杆菌和大肠杆菌免疫球蛋白 (Ig) 结合黏附 TAA的影响。18,19。

<table><tbody><tr><td>&lt;strong&gt;Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BW25113</td><td>NIG</td><td>ME6092</td><td>Wild-type strain of Keio collection</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL21(DE3)</td><td>Merck</td><td>69450-3</td><td>Expression strain</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL21DABCF</td><td>Addgene</td><td>102270</td><td>Derived from BL21(DE3)</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; JW4179</td><td>NIG</td><td>JW4179-KC</td><td>&lt;em&gt;tamA&lt;/em&gt; deletion mutant</td></tr><tr><td>P1 &lt;em&gt;vir&lt;/em&gt;</td><td>NIG</td><td>HR16</td><td>Generally transducing bacteriophage</td></tr><tr><td>&lt;strong&gt;Plasmids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pCP20</td><td>CGSC</td><td>14177</td><td>conditionally replicating plasmid with FLP</td></tr><tr><td>pASK-IBA2</td><td>IBA GmbH</td><td>2-1301-000</td><td>expression vector</td></tr><tr><td>pEibD10</td><td>N/A</td><td>N/A</td><td>for production of EibD; plasmid available on request</td></tr><tr><td>pET22b+</td><td>Merck</td><td>69744-3</td><td>expression vector</td></tr><tr><td>pIBA2-YadA</td><td>N/A</td><td>N/A</td><td>for production of YadA; plasmid available on request</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetic acid</td><td>ThermoFisher</td><td>33209</td><td></td></tr><tr><td>Agar</td><td>BD Bacto</td><td>214010</td><td></td></tr><tr><td>Agarose</td><td>Lonza</td><td>50004</td><td></td></tr><tr><td>Ampicillin</td><td>Applichem</td><td>A0839</td><td></td></tr><tr><td>Anhydrotetracycline</td><td>Abcam</td><td>ab145350</td><td></td></tr><tr><td>anti-collagen type I antibody COL-1</td><td>Sigma</td><td>C2456</td><td></td></tr><tr><td>Bovine collagen type I</td><td>Sigma</td><td>C9791</td><td></td></tr><tr><td>Calcium chloride</td><td>Merck</td><td>102382</td><td></td></tr><tr><td>Chloroform</td><td>Merck</td><td>102445</td><td></td></tr><tr><td>Di-sodium hydrogen phosphate</td><td>VWR</td><td>28029</td><td></td></tr><tr><td>DNA dye</td><td>Thermo</td><td>S33102</td><td></td></tr><tr><td>DNA molecular size marker</td><td>New England BioLabs</td><td>N3232S</td><td></td></tr><tr><td>DNase I</td><td>Sigma</td><td>DN25</td><td></td></tr><tr><td>dNTP mix</td><td>New England Biolabs</td><td>N0447</td><td></td></tr><tr><td>ECL HRP substrate</td><td>Advansta</td><td>K-12045</td><td></td></tr><tr><td>EDTA</td><td>Applichem</td><td>A2937</td><td></td></tr><tr><td>Glycerol</td><td>VWR</td><td>24388</td><td></td></tr><tr><td>goat anti-mouse IgG-HRP</td><td>Santa Cruz</td><td>sc-2005</td><td></td></tr><tr><td>goat anti-rabbit IgG-HRP</td><td>Agrisera</td><td>AS10668</td><td></td></tr><tr><td>HEPES</td><td>VWR</td><td>30487</td><td></td></tr><tr><td>Isopropyl thiogalactoside</td><td>VWR</td><td>43714</td><td></td></tr><tr><td>Kanamycin</td><td>Applichem</td><td>A1493</td><td></td></tr><tr><td>Lysozyme</td><td>Applichem</td><td>A4972</td><td></td></tr><tr><td>Magnesium chloride</td><td>VWR</td><td>25108</td><td></td></tr><tr><td>Manganese chloride</td><td>Sigma</td><td>221279</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-lauroyl sarcosine</td><td>Sigma</td><td>L9150</td><td></td></tr><tr><td>Skim milk powder</td><td>Sigma</td><td>70166</td><td></td></tr><tr><td>Sodium chloride</td><td>VWR</td><td>27808</td><td></td></tr><tr><td>&lt;em&gt;tamA&lt;/em&gt; forward primer</td><td>Invitrogen</td><td>N/A</td><td>Sequence 5&#039;-GAAAAAAGG&lt;br /&gt;ATATTCAGGAGAAAATGTG-3&#039;</td></tr><tr><td>&lt;em&gt;tamA&lt;/em&gt; reverse primer</td><td>Invitrogen</td><td>N/A</td><td>Sequence 5&#039;-TCATAATT&lt;br /&gt;CTGGCCCCAGACC-3&#039;</td></tr><tr><td>Taq DNA polymerase</td><td>New England Biolabs</td><td>M0267</td><td></td></tr><tr><td>Tri-sodium citrate</td><td>Merck</td><td>106448</td><td></td></tr><tr><td>Tryptone</td><td>VWR</td><td>84610</td><td></td></tr><tr><td>Tween20</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>Yeast extract</td><td>Merck</td><td>103753</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agarose gel electrophoresis chamber</td><td>Hoefer</td><td>SUB13</td><td></td></tr><tr><td>Bead beater</td><td>Thermo</td><td>FP120A-115</td><td></td></tr><tr><td>CCD camera</td><td>Kodak</td><td>4000R</td><td></td></tr><tr><td>Electroporation cuvettes</td><td>Bio-Rad</td><td>165-2089</td><td></td></tr><tr><td>Electroporation unit</td><td>Bio-Rad</td><td>1652100</td><td></td></tr><tr><td>Gel imager</td><td>Nippon Genetics</td><td>GP-03LED</td><td></td></tr><tr><td>Incubating shaker</td><td>Infors HT</td><td>Minitron</td><td></td></tr><tr><td>Incubator</td><td>VWR</td><td>390-0482</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5415D</td><td></td></tr><tr><td>Microwave oven</td><td>Samsung</td><td>CM1099A</td><td></td></tr><tr><td>PCR machine</td><td>Biometra</td><td>Tpersonal</td><td></td></tr><tr><td>PCR strips</td><td>Axygen</td><td>PCR-0208-CP-C</td><td></td></tr><tr><td>pH meter</td><td>Hanna Instruments</td><td>HI2211-01</td><td></td></tr><tr><td>PVDF membrane</td><td>ThermoFisher</td><td>88518</td><td></td></tr><tr><td>SDS-PAG electrophoresis chamber</td><td>ThermoFisher</td><td>A25977</td><td></td></tr><tr><td>Tabletop centrifuge</td><td>Beckman Coulter</td><td>B06322</td><td></td></tr><tr><td>Vortex mixer</td><td>Scientific Industries</td><td>SI-0236</td><td></td></tr><tr><td>Water bath</td><td>GFL</td><td>D3006</td><td></td></tr><tr><td>Wet transfer unit</td><td>Hoefer</td><td>TE22</td><td></td></tr></tbody></table>

producing gene deletions in escherichia coli by p1 transduction with excisable antibiotic resistance cassettes

第一种研究未知基因在细菌中的作用的方法是创建一个基因的敲除。在这里, 我们描述了一个健壮和快速的协议, 将基因缺失突变从一个大肠杆菌的菌株转移到另一个, 利用广义转导与噬菌体 P1。这种方法要求突变是可选择的 (例如,基于基因中断使用抗生素盒插入)。这种抗生素盒可以从捐献者的应变中调动出来, 并引入一个感兴趣的受体菌株, 迅速而容易地产生基因删除突变体。该抗生素盒可以设计为包括 flippase 识别网站, 允许通过特定地点的 recombinase 切除卡带, 以产生一个干净的敲出, 只有一个〜100基对长的疤痕序列在基因组。我们通过敲除 autotransporter 合成中涉及的一个装配因子的多摩基因来证明该协议, 并测试这一击出对两三联 autotransporter adhesins 合成和功能的影响。虽然 P1 转导基因的缺失有其局限性, 但其实施的简便性和速度使其成为其他基因缺失的一种有吸引力的替代方法。

多摩BL21ΔABCF 的产生:
上面概述的战略以前曾被用来生产 BL21 (DE3) 的衍生物菌株, 这是用于蛋白质生产的标准实验室菌株, 它被优化用于外膜蛋白生产, 并被称为 BL21ΔABCF21。这种菌株缺乏四基因编码的丰富的外膜蛋白, 因此, 能够产生更多的 heterologously 表达外膜蛋白比野生型菌株。为了测试 TAM 是否参与了 TA...

Watch this Scientific Journal Video about Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes at JoVE.com

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

可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失

在这里, 我们提出了使用预先存在的抗生素耐药性-盒式删除结构作为一个基础, 使删除突变体在其他大肠杆菌菌株。这种删除突变可以调动和插入到相应的受体菌株的位置使用 P1 噬菌体转导。

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 ...

producing-gene-deletions-escherichia-coli-p1-transduction-with

Research

JoVE Journal

Genetics

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

16.9K 次观看.  University of Oslo. 在这里, 我们提出了使用预先存在的抗生素耐药性-盒式删除结构作为一个基础, 使删除突变体在其他大肠杆菌菌株。这种删除突变可以调动和插入到相应的受体菌株的位置使用 P1 噬菌体转导。

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

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 ...

科研

生物工程

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

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

Here we present an experimental method to test 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

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

遗传学

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

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

全基因组基因缺失<em&gt;链球菌血统</em通过高通量PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas aeruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative to E. coli. However, P. aeruginosa is an opportunistic human pathogen. Here, we detail a procedure that can be used to generate nonpathogenic strains of P. aeruginosa through sequential genomic deletions using the pEX100T-NotI plasmid. The main advantage of this method is to produce a marker-free strain. This method may be used to generate highly attenuated P. aeruginosa strains for the production of commercial products, or to design strains for other specific uses. We also describe a simple and reproducible mouse model of bacterial systemic infection via intraperitoneal injection of validated test strains to test the attenuation of the genetically engineered strain in comparison to the FDA-approved BL21 strain of E. coli.

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Here, we describe a simple and reproducible protocol of mouse model of infection to evaluate the attenuation of the genetically modified strains of Pseudomonas aeruginosa in comparison to the United States Food and Drug Administration (FDA)-approved Escherichia coli for commercial applications.

在伪单体中生成帧内基因删除突变体，并在简单小鼠感染模型中测试病毒衰减

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of ...

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in elucidating the mechanisms that C. trachomatis use to create and maintain a privileged intracellular niche has been limited due to a lack of genetic tools. Fortunately, there have recently been several new advances in genetic manipulation techniques. Among these is the development of fluorescence-reported allelic exchange mutagenesis (FRAEM). This method allows targeted gene deletion coupled with insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Reliance on this strategy can be complicated when targeting genes within polycistronic operons due to the potential of polar effects on downstream genes. Floxed cassette allelic exchange mutagenesis (FLAEM), the protocol for which is described here, was developed to alleviate cassette-induced polar effects. FLAEM utilizes Cre-loxP genome editing to remove the selection cassette after targeted deletion by allelic exchange. The resulting strains contain markerless gene deletions of one or more coding sequences. This technique facilitates direct assessment of gene function and expands the repertoire of tools for genetic manipulation in C. trachomatis.

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Described here is a method for targeted, markerless gene deletion in Chlamydia trachomatis using floxed cassette allelic exchange mutagenesis, FLAEM.

无标记基因删除由絮凝血盒等位基因交换突变在衣原体沙眼

Chlamydia trachomatis is an obligate intracellular pathogen that has been historically difficult to genetically manipulate. Definitive progress in ...

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 ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.

针对性斯尔纳的噬菌体介导的交付构造击倒基因表达<em&gt;电子。大肠杆菌</em

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be ...

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation of cyanobacteria, especially model organisms such as Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002, is a key tool for both basic and applied research. Generation of unmarked mutants, whereby chromosomal alterations are introduced into a strain via insertion of an antibiotic resistance cassette (a manipulatable fragment of DNA containing one or more genes), followed by subsequent removal of this cassette using a negative selectable marker, is a particularly powerful technique. Unmarked mutants can be repeatedly genetically manipulated, allowing as many alterations to be introduced into a strain as desired. In addition, the absence of genes encoding antibiotic resistance proteins in the mutated strain is desirable, as it avoids the possibility of 'escape' of antibiotic resistant organisms into the environment. However, detailed methods for repeated rounds of genetic manipulation of cyanobacteria are not well described in the scientific literature. Here we provide a comprehensive description of this technique, which we have successfully used to generate mutants with multiple deletions, single point mutations within a gene of interest and insertion of novel gene cassettes.

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp. PCC7002.

在型号蓝藻种类标记和无标记突变体的产生

Cyanobacteria are ecologically important organisms and potential platforms for production of biofuels and useful industrial products. Genetic manipulation ...

可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失

摘要

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此视频中的章节

网站特定的细菌染色体工程：ΦC31整合介导的盒式交换（IMCE）

多拷贝产质粒在抗生素耐药性演变中的作用试验

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

全基因组基因缺失

在伪单体中生成帧内基因删除突变体，并在简单小鼠感染模型中测试病毒衰减

无标记基因删除由絮凝血盒等位基因交换突变在衣原体沙眼

的产生

针对性斯尔纳的噬菌体介导的交付构造击倒基因表达

在型号蓝藻种类标记和无标记突变体的产生

可抽税抗生素耐药盒 P1 转导制备大肠杆菌中的基因缺失

摘要

探索更多视频

此视频中的章节

网站特定的细菌染色体工程：ΦC​​31整合介导的盒式交换（IMCE）

多拷贝产质粒在抗生素耐药性演变中的作用试验

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

全基因组基因缺失

在伪单体中生成帧内基因删除突变体，并在简单小鼠感染模型中测试病毒衰减

无标记基因删除由絮凝血盒等位基因交换突变在衣原体沙眼

的产生

针对性斯尔纳的噬菌体介导的交付构造击倒基因表达

在型号蓝藻种类标记和无标记突变体的产生

网站特定的细菌染色体工程：ΦC31整合介导的盒式交换（IMCE）