这项工作得到了加拿大自然科学与工程委员会对AK的发现资助。图中使用的原理图是用 BioRender.com 创建的。

1.实验准备
<ol><li>用目的基因纯化质粒pUC18T-mini-Tn7T-LAC-Gm9 （插入质粒， 图2A）。 注意:在这里，感兴趣的基因是 adeIJK。最终质粒浓度应为 ≥100 ng/μL。</li>
	<li>纯化辅助质粒（pTNS2）9和切除质粒（pFLP2ab）6（分别为图2B，C），理想情况下最终质粒DNA浓度为≥100ng/μL。</li>
	<li>准备 50 mL 无菌超纯水和 25 mL 无菌 LB （Lennox） 肉汤（参见 材料表）。</li>
	<li>制备至少 10 LB 琼脂平板，每个平板含有以下添加剂:普通（无添加剂）、50 μg/mL 的庆大霉素 （Gm）、200 μg/mL 的羧苄青霉素 （Cb）（ 通过 氨苄青霉素耐药基因进行选择）和 5% 蔗糖（参见 材料表）。</li>
	<li>划出用于插入LB琼脂的菌株，并在37°C下孵育16-18小时。 注意:该协议必须尽可能在无菌环境中通过使用工作台上的本生燃烧器或生物安全柜进行。所有耗材（移液器吸头、接种环、微量离心管等）都必须是无菌的。</li>
</ol>2. 培养准备
<ol><li>使用无菌接种环或无菌木制接种棒，将 鲍曼不动 杆菌 AB258 的单个菌落接种到无菌 13 mL 培养管中的 4 mL LB 肉汤中。 注意:单个 4 mL 培养物用于一个样品和一个对照。根据所需的样品数量增加培养物的数量。</li>
	<li>在37°C振荡（250rpm）下孵育过夜。</li>
	<li>将一瓶无菌蒸馏水（25-50mL）置于4°C过夜，用于步骤3。</li>
</ol>3. 电感受态电池的制备
<ol><li>将所有无菌 1.5 mL 微量离心管（每次培养两个）和无菌电穿孔比色皿（每个培养物两个）放在冰上（参见 材料表）。在整个过程中，尽可能多地将样品放在冰上。</li>
	<li>将储存在4°C的无菌水瓶放在冰上。</li>
	<li>将 1.5 mL 过夜细菌培养物转移到其中一个 1.5 mL 微量离心管中。</li>
	<li>以13,000× g 离心2分钟以沉淀细胞。 注意:理想情况下，离心应在4°C下进行，但环境温度离心是可以接受的，并且不会有害。</li>
	<li>使用 1 mL 移液管，在不干扰细胞沉淀的情况下除去所有上清液。</li>
	<li>将另外 1.5 mL 细菌培养物加入同一微量离心管中。以13,000× g 离心2分钟，然后除去所有上清液。</li>
	<li>用剩余的 1 mL 培养物最后一次重复步骤 3.6。</li>
	<li>向细胞沉淀中加入 1 mL 冰冷无菌水，并用轻柔的移液液管重悬，直到沉淀不再位于微量离心管的底部。</li>
	<li>将重悬细胞以13,000× g 离心2分钟。</li>
	<li>使用 1 mL 移液管小心地除去上清液。不要倒掉上清液，特别是在随后的洗涤步骤中，当细胞倾向于形成不太紧凑的沉淀时。</li>
	<li>用冰冷的无菌水重复此洗涤步骤，步骤3.8至步骤3.10，再重复两次。</li>
	<li>将最终细胞沉淀轻轻重悬于200μL冰冷无菌水中。</li>
	<li>将 100 μL 最终细胞悬液转移到第二个冰冷的 1.5 mL 微量离心管中。第二个等分试样将成为电穿孔的阴性对照。将细胞样品放在冰上。</li>
</ol>4. 电穿孔
<ol><li>预热每个样品的1mLLB肉汤和一个LB + Gm50 琼脂平板（在步骤1.4中制备），并在设置为37°C的静态培养箱中对照。</li>
	<li>在 5 μL 或更小的总体积中，将 100-200 ng pTNS2 辅助质粒和 pUC18T-mini-Tn7T-LAC-Gm-adeIJK 插入质粒加入等分试样的电感受态细胞中。</li>
	<li>用指尖轻敲击混合，以确保质粒与电感受态细胞完全混合，而不会引入气泡。</li>
	<li>将等量的无菌蒸馏水加入阴性对照细胞等分试样中，并如上所述轻轻混合。</li>
	<li>将样品在冰上孵育20分钟。</li>
	<li>将整个细胞样品转移到冰冷的电穿孔比色皿中，然后将比色皿放回冰上。对阴性对照细胞样品重复上述步骤。</li>
	<li>电穿孔细胞样品。<ol><li>打开电穿孔仪并将其设置为 2.0 kV （25 μF， 200 Ω）（参见 材料表）。</li>
		<li>用软纸擦拭比色皿表面，以去除任何粘附的冰或水分。</li>
		<li>将比色皿插入电穿孔器并进行电击。</li>
		<li>立即将 0.9 mL 预热的 LB 肉汤加入比色皿中的细胞中，并轻轻上下移液以将细胞与培养基混合。</li>
		<li>将整个细胞悬液转移到新的 1.5 mL 微量离心管（室温）中。</li>
		<li>检查电穿孔仪上的时间常数值;为获得最佳结果，此值应介于 4 和 6 之间。</li>
	</ol></li>
	<li>对阴性对照细胞样品重复电穿孔程序（步骤4.7.1至步骤4.7.6）。</li>
	<li>将电穿孔样品在37°C下以250rpm孵育1小时，以允许细胞回收。</li>
	<li>使用接种撒布器，将每个电穿孔细胞样品的100μL铺在预热的LB + Gm50 琼脂平板上。<ol><li>将剩余样品以13,000× g 离心2分钟以沉淀细胞。</li>
		<li>使用移液管除去所有上清液后，将细胞重悬于100μLLB肉汤中。</li>
		<li>将每个样品铺在单个预热的LB + Gm50 琼脂平板上。 注意:电穿孔效率可能取决于应变。首次对菌株进行电穿孔时，可以板出不同体积的电穿孔样品，甚至稀释度，以确保所得板具有分离的菌落。</li>
	</ol></li>
	<li>将板在37°C孵育16-18小时。</li>
</ol>5. 选择转化菌落进行基于PCR的筛选
<ol><li>检查电穿孔板。阴性对照不应有菌落;样品应具有不同的菌落。 注意:在这一步以及产生带有菌落或贴片的琼脂平板的所有后续步骤中，具有菌落的平板可以在4°C下储存长达3天，然后再继续。</li>
	<li>使用无菌牙签，从LB + Gm50 琼脂平板中取出多达10个单菌落，并将它们贴在新鲜的LB + Gm50 琼脂平板上。</li>
	<li>将板在37°C孵育16-18小时。</li>
</ol>6. 通过菌落PCR验证染色体插入
<ol><li>从培养箱中取出含有贴片菌落的LB + Gm50 琼脂平板。</li>
	<li>使用无菌牙签或无菌移液管吸头，将贴片中的一小部分（约一个大菌落大小）从贴片中取出到0.2 mL PCR管中的20 μL无菌蒸馏水中;搅拌均匀。当细胞从牙签中释放出来时，水样应该变得明显浑浊。准备任意数量的需要筛选的样品，但 6 个就足够了。</li>
	<li>将含有细菌悬浮液的PCR管在100°C下孵育5-10分钟。</li>
	<li>使用微型离心机（参见 材料表），以固定的最大速度旋转样品2分钟以沉淀细胞碎片。</li>
	<li>将上清液转移到新的 0.2 mL PCR 管中，并将其置于冰上。该样品包含用于PCR反应的模板DNA。 注意:在进行PCR之前，该模板DNA可以储存在-20°C。</li>
	<li>制备含有 1x 聚合酶缓冲液、200 μM dNTP、0.18 μM ABglmS2_F_New正向引物、0.18 μM Tn7R 反向引物（表 1）、1 U Taq DNA 聚合酶和 1 μL 制备的模板 DNA 的 PCR 反应混合物，总体积为 25 μL。 制备无模板对照（NTC），包括除模板DNA以外的所有DNA（参见 材料表）。</li>
	<li>将反应条件输入热循环仪:95°C2分钟;95°C30秒，49°C30秒，72°C30秒，共35个循环;72°C10分钟;12°C保持。运行示例。</li>
	<li>将DNA上样染料加入至终浓度为1x后，将10μL每个PCR反应上样于2%琼脂糖凝胶上，并在80V下运行40分钟。该引物对的预期扩增子大小为 382 bp。NTC 不应有条带。</li>
	<li>确定产生预期PCR产物的样品。从任何PCR阳性贴片在LB + Gm50 琼脂平板上制备条纹板;在37°C孵育16-18小时。目标是生成具有单个菌落的板，用于制备甘油储备液以保存该标记菌株，并作为创建未标记菌株的起点（如下所述）。</li>
</ol>7. 使用 pFLP2ab 去除 Gm R 标记物
<ol><li>按照步骤 2:培养物制备中提到的步骤进行操作。用于制备过夜培养物的样品是来自LB + Gm50 琼脂平板的单个菌落，在步骤6.9中制备以产生离散菌落。</li>
	<li>按照步骤3中提到的步骤:电感受态电池的制备。从过夜培养物中制备细胞进行电穿孔。</li>
	<li>按照步骤4:电穿孔中提到的步骤进行操作。在这里，引入细胞的质粒是pFLP2ab （100-200ng），它将去除庆大霉素抗性基因，位于染色体插入的感兴趣基因的侧翼。</li>
	<li>细胞恢复1小时（步骤4.9）后，将100μL电穿孔细胞样品铺在预热的LB + Cb200 琼脂平板上（在步骤1.4中制备）。这种选择性压力确保只有携带 pFLP2ab 切除质粒的细胞才能生长。</li>
	<li>将板在37°C孵育16-18小时。</li>
	<li>检查平板是否有菌落。阴性对照板不应有菌落;LB + Cb200 琼脂样品板应具有不同的菌落。</li>
	<li>使用无菌牙签，将多达 20 个分离的菌落交叉贴在 LB + Cb200 琼脂平板和 LB + Gm50 琼脂平板上。</li>
	<li>将板在37°C下孵育16-18小时。</li>
	<li>检查板是否有补丁。在 LB + Cb200 琼脂平板上生长但不在 LB + Gm50 琼脂平板上生长的克隆已从原始插入片段中去除庆大霉素抗性基因。</li>
	<li>选择对羧苄苄青霉素耐药且庆大霉素易在补充有 5% （w/v） 蔗糖的单个 LB 琼脂平板上划线的贴片，这迫使 pFLP2ab 质粒从细菌中排出。最多选择 10 个菌落。</li>
	<li>将板在37°C下孵育16-18小时。</li>
	<li>检查平板是否有菌落。</li>
	<li>作为pFLP2ab 质粒丢失的最终确认，将5%蔗糖平板中的4-6个分离菌落交叉贴片到LB + Cb200 琼脂平板和LB琼脂平板上。</li>
	<li>将板在37°C下孵育16-18小时。</li>
	<li>选择在LB琼脂平板上生长的克隆，该克隆对制备甘油原液对羧苄青霉素敏感，以保存这种未标记的菌株。<ol><li>使用靶向庆大霉素抗性基因的引物，可以通过集落PCR（步骤6:通过集落PCR验证染色体插入）对未标记菌株进行基因验证。</li>
		<li>制备含有 1x 聚合酶缓冲液、200 μM dNTP、0.18 μM Gm_F正向引物、0.18 μM Gm_R反向引物（表 1）、1 U Taq DNA 聚合酶和 1 μL 制备的模板 DNA 的 PCR 反应混合物，总体积为 25 μL。 制备一个无模板对照（NTC），包括除模板DNA以外的所有模板， 以及使用来自标记菌株的 DNA 的阳性对照。</li>
		<li>将反应条件输入热循环仪:95°C2分钟;95°C30秒，50°C30秒，72°C40秒，共35次循环;72°C10分钟;12°C保持。运行示例。</li>
		<li>在加入DNA上样染料至终浓度为1x后，将10μL每个PCR反应上样于1%琼脂糖凝胶上，并在80V下运行40分钟。该引物对的预期扩增子大小为 525 bp。阳性对照应具有单个条带;样品和NTC不应有条带。</li>
	</ol></li>
</ol>

用于表征多药物外排系统的单拷贝基因表达

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counterselectable+markers:+untapped+tools+for+bacterial+genetics+and+pathogenesis.">Counterselectable markers: untapped tools for bacterial genetics and pathogenesis.</a> Infect Immun. 66 (9), 4011-4017 (1998).</li><li>Gay, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+selection+procedure+for+entrapment+of+insertion+sequence+elements+in+gram-negative+bacteria.">Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria.</a> J Bacteriol. 164, 918-921 (1985).</li><li>Kyriakidis, I., Vasileiou, E., Pana, Z. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tragiannidis,+Acinetobacter+baumannii+antibiotic+resistance+mechanisms.">Tragiannidis, Acinetobacter baumannii antibiotic resistance mechanisms.</a> Pathogens. 10 (3), 373 (2021).</li><li>Clinical and Laboratory Standards Institute. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+standards+for+antimicrobial+susceptibility+testing.">Performance standards for antimicrobial susceptibility testing.</a> CLSI Supplement M100., 31st edn. , (2021).</li><li>Dijkshoorn, L., Nemec, A., Seifert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+increasing+threat+in+hospitals:+multidrug-resistant+Acinetobacter+baumannii.">An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii.</a> Nature Reviews Microbiology. 5, 939-951 (2007).</li><li>Yildirim, S., Thompson, M. G., Jacobs, A. C., Zurawski, D. V., Kirkup, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+parameters+for+high+efficiency+transformation+of+Acinetobacter+baumannii.">Evaluation of parameters for high efficiency transformation of Acinetobacter baumannii.</a> Sci Rep. 25 (6), 22110 (2016).</li><li>Thompson, M. G., Yildirim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+Acinetobacter+baumannii:+electroporation.">Transformation of Acinetobacter baumannii: electroporation.</a> Methods Mol Biol. 1946, 69-74 (2019).</li><li>Choi, K. H., DeShazer, D., Schweizer, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-Tn7+insertion+in+bacteria+with+multiple+glmS-linked+attTn7+sites:+example+Burkolderia+mallei+ATCC+2334.">Mini-Tn7 insertion in bacteria with multiple glmS-linked attTn7 sites: example Burkolderia mallei ATCC 2334.</a> Nat Protoc. 1 (1), 162-169 (2006).</li><li>Jittawuttipoka, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-Tn7+vectors+as+genetic+tools+for+gene+cloning+at+a+single+copy+number+in+an+industrially+important+and+phytopathogenic+bacteria,+Xanthomonas+spp.">Mini-Tn7 vectors as genetic tools for gene cloning at a single copy number in an industrially important and phytopathogenic bacteria, Xanthomonas spp.</a> FEMS Microbiol Lett. 298 (1), 111-117 (2009).</li><li>P&#233;rez-Varela, M., Tierney, A. R. P., Kim, J. S., V&#225;zquez-Torres, A., Rather, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+RelA+in+Acinetobacter+baumannii.">Characterization of RelA in Acinetobacter baumannii.</a> J Bacteriol. 202 (12), e00045 (2020).</li><li>Williams, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Acinetobacter+baumannii+copper+resistance+reveals+a+role+in+virulence.">Characterization of Acinetobacter baumannii copper resistance reveals a role in virulence.</a> Front Microbiol. 11, 16 (2020).</li></ol>

作者没有什么可透露的。

尽管在 鲍曼不动杆 菌中插入可诱导的单拷贝基因表达系统的染色体插入过程在技术上很简单且不费力，但有几个重要步骤需要强调。首先，感受态细胞的制备需要尽可能在冰上进行，因为在用冰冷水替换培养基的过程中细胞会变得脆弱。理想情况下，离心步骤在4°C下进行，但在室温下离心是可以接受的。鉴于水洗过程中细胞的脆弱性越来越强，轻柔的移液也很重要。其次，电穿孔对离子?...

鲍曼不动杆 菌是世界卫生组织的头等大事原体，因为它对所有类别的抗生素都具有耐药性1。它是一种机会性病原体，主要影响住院、受伤或免疫功能低下的人。 鲍曼不动杆 菌主要 通过 外排泵逃避抗生素，最相关的是抗结瘤分裂 （RND） 出口商家族 2。了解这些外排泵的机械工作原理将使人们能够开发有针对性的治疗方案。
可以特异性区分细胞过程的一种常见方法是通过基因操作。然而，可用于鲍曼不动杆菌遗传研究的工具有限，并且进一步混淆了实验设计，临床分离株通常对遗传操作中常规用于选择的抗生素具有耐药性3。在专门研究外排泵时遇到的第二个障碍是，它们受到严格监管（通常受到未知因素的影响），因此很难准确地将功能隔离并归因于单个泵4。鉴于需要扩展研究工具箱，我们开发了一种基于 Mini-Tn7 的单拷贝插入诱导型表达系统，该系统包含 Flp 重组酶靶标 （FRT） 盒，允许去除选择标记 5,6,7（图 1）。这种优雅的克隆和表达系统最初是为假单胞菌 8,9,10 创建的，用于将单拷贝外排泵补体生成到我们生成的鲍曼不动杆菌 （ATCC 17978::ΔadeIJK，Δ adeFGH，Δ adeAB:以下简称鲍曼不动杆菌 AB258） 的 RND 外排泵缺陷菌株中11.能够一次研究一个外排泵，并且不会以高拷贝表达淹没细菌细胞（如通常在基于质粒的表达系统中看到的那样），人们可以更好地了解每个外排泵的关键生理方面，同时将干扰降至最低，并减少并发症。
本文介绍了如何使用 mini-Tn7 系统通过在 9 天内执行的一系列简单步骤，将缺失的目的基因 RND 外排泵 adeIJK 补合成鲍曼不动杆菌 AB258 的染色体7。第一组步骤在保守的 glmS 基因下游的单个 attTn7 插入位点（图 3A）重新引入克隆到基于 mini-Tn7 的插入质粒（图 2A）中的缺失的外排泵基因。该过程由编码 Tn7 驱动插入所需的转座酶基因的非复制辅助质粒 （图 2B） 促进。第二组步骤使用切除质粒（图2C）进行Flp重组酶介导的庆大霉素基因的去除，两侧是FRT位点（图3B），以产生未标记的菌株。虽然该系统用于阐明RND外排泵在抗生素耐药性方面的基本作用和可能的抑制剂，但它可用于研究任何感兴趣的基因。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 mL PCR tube</td>
			<td>VWR</td>
			<td>20170-012</td>
			<td>For colony boil preparations and PCR reactions</td>
		</tr>
		<tr>
			<td>1.5 mL microfuge tubes</td>
			<td>Sarstedt</td>
			<td>72-690-301</td>
			<td>General use</td>
		</tr>
		<tr>
			<td>13-mL culture tubes, Pyrex</td>
			<td>Fisher</td>
			<td>14-957K</td>
			<td>Liquid culture vessels</td>
		</tr>
		<tr>
			<td>6x DNA loading buffer</td>
			<td>Froggabio</td>
			<td>LD010</td>
			<td>Agarose gel electrophoresis sample loading dye</td>
		</tr>
		<tr>
			<td>Acetic acid, glacial</td>
			<td>Fisher</td>
			<td>351271-212</td>
			<td>Agarose gel running buffer component</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Bioshop</td>
			<td>AGR003</td>
			<td>Solid growth media</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>BioBasic</td>
			<td>D0012</td>
			<td>Electrophoretic separation of PCR reaction products; used at a concentration of 0.8&#8211;2%</td>
		</tr>
		<tr>
			<td>Agarose gel electrophoresis unit</td>
			<td>Fisher</td>
			<td>29-237-54</td>
			<td>Agarose gel electrophoresis; separation of PCR reaction products</td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>Fisher</td>
			<td>50841231</td>
			<td>Selective media</td>
		</tr>
		<tr>
			<td>Culture tube closures</td>
			<td>Fisher</td>
			<td>13-684-138</td>
			<td>Stainless steel closure for 13-mL culture tubes</td>
		</tr>
		<tr>
			<td>Deoxynucleotide triphosphate (dNTP) set</td>
			<td>Biobasic</td>
			<td>DD0058</td>
			<td>PCR reaction component; supplied as 100 mM each dATP, dCTP, dGTP, dTTP; mixed and diluted for 10 mM each dNTP</td>
		</tr>
		<tr>
			<td>Dry bath/block heater</td>
			<td>Fisher</td>
			<td>88860023</td>
			<td>Isotemp digital dry bath for boil preparations</td>
		</tr>
		<tr>
			<td>Electroporation cuvettes</td>
			<td>VWR</td>
			<td>89047-208</td>
			<td>2 mm electroporation cuvettes with round cap</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Cole Parmer</td>
			<td>940000009</td>
			<td>110 VAC, 60 Hz electroporator</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Fisher</td>
			<td>BP102-1</td>
			<td>Visualization of PCR reaction products and DNA marker in agarose gel</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>VWR</td>
			<td>CA-EM4050</td>
			<td>Agarose gel running buffer component</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Biobasic</td>
			<td>GB0217</td>
			<td>For the preparation of selective media</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher</td>
			<td>G33</td>
			<td>Preparation of bacterial stocks for long-term storage in an ultra-low freezer</td>
		</tr>
		<tr>
			<td>Incubator (shaking)</td>
			<td>New Brunswick Scientific</td>
			<td>M1352-0000</td>
			<td>Excella E24 Incubator Shaker for liquid culture growth</td>
		</tr>
		<tr>
			<td>Incubator (static)</td>
			<td>Fisher</td>
			<td>11-690-550D</td>
			<td>Isotemp Incubator Oven Model 550D for solid (LB agar) culture growth</td>
		</tr>
		<tr>
			<td>Inoculation loop</td>
			<td>Sarstedt</td>
			<td>86.1562.050</td>
			<td>Streaking colonies onto agar plates</td>
		</tr>
		<tr>
			<td>Inoculation spreader</td>
			<td>Sarstedt</td>
			<td>86.1569.005</td>
			<td>Spreading of culture onto agar plates</td>
		</tr>
		<tr>
			<td>Lysogeny broth (LB) broth, Lennox</td>
			<td>Fisher</td>
			<td>BP1427</td>
			<td>Liquid growth media (20 g/L: 5 g/L sodium chloride, 10 g/L tryptone, 5 g/L yeast extract)</td>
		</tr>
		<tr>
			<td>Microfuge</td>
			<td>Fisher</td>
			<td>75002431</td>
			<td>Sorvall Legend Micro 17 for centrifugation of samples</td>
		</tr>
		<tr>
			<td>Mini-centrifuge</td>
			<td>Fisher</td>
			<td>S67601B</td>
			<td>Centrifugation of 0.2 mL PCR tubes</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>SPL Life Sciences</td>
			<td>10090</td>
			<td>For solid growth media (agar plates): 90 x 15 mm</td>
		</tr>
		<tr>
			<td>Pipettes&#160;</td>
			<td>Mandel</td>
			<td>Various</td>
			<td>Gilson single channel pipettes (P10, P20, P200, P1000)</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>Biorad</td>
			<td>1645050</td>
			<td>PowerPac Basic power supply for electrophoresis</td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>IDT</td>
			<td>NA</td>
			<td>PCR reaction component; specific to gene of interest; prepared at 100 &#956;M as directed on the product specification sheet</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>BioBasic</td>
			<td>SB0498</td>
			<td>For the preparation of counterselective media for removal of the pFLP2ab plasmid from transformed A. baumannii</td>
		</tr>
		<tr>
			<td>Taq DNA polymerase</td>
			<td>FroggaBio</td>
			<td>T-500</td>
			<td>PCR reaction component; polymerase supplied with a 10x buffer</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>Biorad</td>
			<td>1861096</td>
			<td>Model T100 for PCR</td>
		</tr>
		<tr>
			<td>Toothpicks</td>
			<td>Fisher</td>
			<td>S24559</td>
			<td>For patching colonies onto agar plates</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T1503</td>
			<td>Agarose gel running buffer component</td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>Millipore Sigma</td>
			<td>ZLXLSD51040</td>
			<td>MilliQ water purification system: ultra pure water for media and solution preparation, and cell washing</td>
		</tr>
		<tr>
			<td>Wide range DNA marker</td>
			<td>Biobasic</td>
			<td>M103R-2</td>
			<td>Size determination of PCR products on an agarose gel</td>
		</tr>
		<tr>
			<td>Wooden inoculating sticks</td>
			<td>Fisher</td>
			<td>29-801-02</td>
			<td>Inoculating cultures with colonies from agar plates</td>
		</tr>
	</tbody>
</table>

characterizing multidrug efflux systems in acinetobacter baumannii using an efflux&#45;deficient bacterial strain and a single&#45;copy gene expression system

鲍曼不动杆 菌因其对抗生素的广泛耐药性而被公认为一种具有挑战性的革兰氏阴性病原体。了解这种耐药性背后的机制对于设计新的有效治疗方案至关重要。不幸的是，由于缺乏合适的基因操作工具，我们在 鲍曼不动 杆菌中研究这些机制的能力受到阻碍。在这里，我们描述了利用基于染色体 mini-Tn7 的系统在缺乏功能性 RND 型外排机制的 鲍曼不动杆 菌菌株中实现单拷贝基因表达的方法。单拷贝插入和诱导型外排泵表达非常有利，因为细菌细胞通常对高拷贝数质粒上存在 RND 外排操纵子的耐受性较差。此外，将重组 mini-Tn7 表达载体掺入替代鲍 曼不动 杆菌宿主的染色体中，具有更高的外排灵敏度，有助于规避来自其他外排泵的干扰。该系统不仅对于研究未表征的细菌外排泵很有价值，而且对于评估靶向这些泵的潜在抑制剂的有效性也很有价值。

染色体插入过程在3天内总共只需要2小时，即可看到在选择性琼脂平板上生长的结果菌落（图1A-C）。转化板上的预期菌落数量取决于菌株:人们可能会看到 20-30 甚至数百个菌落，因为在 attTn7 位点插入 Tn7 是特异性和有效的9。将转化板菌落贴到选择性培养基上（图4A）可保留转化的菌株，并为菌落PCR筛选...

Watch this Scientific Journal Video about Author Spotlight: Advancing Antibiotic Resistance Research Using an Efflux-Deficient Bacterial Strain and a Single-Copy Gene Expression System at JoVE.com

作者聚焦:使用外排缺陷型细菌菌株和单拷贝基因表达系统推进抗生素耐药性研究

我们描述了一种简单的程序，使用基于 mini-Tn7 的表达系统将外排泵基因进行单拷贝染色体互补，并将其转化为鲍 曼不动杆菌的工程外排缺陷菌株。这种精确的遗传工具允许受控的基因表达，这是表征多重耐药病原体中外排泵的关键。

使用外排缺陷细菌菌株和单拷贝基因表达系统表征 鲍曼不动杆 菌的多药外排系统

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

我们研究了革兰氏阴性菌的多重耐药性外排系统。我们有兴趣了解这些泵的天然功能以及这些泵如何用作潜在的药物靶点。多重耐药性外排系统的表达受到严格调控，因此难以研究其功能。因此，绕过调节系统的表达系统在表征这些泵时非常有用。该协议允许在自然环境中通常看到的水平上表达基因。因此，基因产物的活性模仿了自然界中观察到的。

characterizing-multidrug-efflux-systems-acinetobacter-baumannii-using

Research

JoVE Journal

Immunology and Infection

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

462 次观看.  University of Manitoba. 我们研究了革兰氏阴性菌的多重耐药性外排系统。我们有兴趣了解这些泵的天然功能以及这些泵如何用作潜在的药物靶点。多重耐药性外排系统的表达受到严格调控，因此难以研究其功能。因此，绕过调节系统的表达系统在表征这些泵时非常有用。该协议允许在自然环境中通常看到的水平上表达基因。因此，基因产物的活性模仿了自然界中观察到的。

视频: 作者聚焦:使用外排缺陷型细菌菌株和单拷贝基因表达系统推进抗生素耐药性研究

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

科研

免疫与感染

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

包装艾滋病毒或FIV的基于Lentivector表达结构与VSV-G假型病毒颗粒转导

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac转座子初级人类T细胞系统改造

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene transcripts from infected tissues. Pathogen RNA constitutes a tiny portion of the total RNA isolated from infected tissue samples. Both microarray and RNAseq technologies have difficulties in generating reliable reads for weakly expressed pathogen genes. Mutant pathogen strains with reduced in vivo proliferation pose an even bigger challenge. Here we describe an in vivo gene expression profiling protocol that is very fast, extremely sensitive and highly reproducible. We developed this protocol during our investigation of the fungal pathogen Candida albicans in a murine model of hematogenously disseminated candidiasis. Using this protocol, we have documented time courses of dynamically regulated C. albicans gene expression during kidney infection, and discovered unexpected features of gene expression responses to antifungal drug treatment in vivo.

We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

侵染微生物利用数字条码平台的基因表达谱

For most mammalian pathogens, gene expression profiling studies have been limited by technical difficulties to accurately quantify pathogen gene ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

在抗菌蓝光治疗的体内研究的多药耐药鲍曼不动杆菌感染烧伤利用生物发光成像

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system&#8217;s reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

在浮动水培系统中监测阿拉伯菌的殖民化与维护

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

外迁移系统中淋巴细胞迁移的评估

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

腹部主动脉瘤小鼠模型中脉冲传播速度、失散性和应变的测量

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to assess the purity of the bilayers. The OM has an increased overall density compared to the inner membrane, largely due to the presence of lipooligosaccharides (LOS) and lipopolysaccharides (LPS) within the outer leaflet. LOS and LPS molecules are amphipathic glycolipids that have a similar structure, which consists of a lipid-A disaccharolipid and core-oligosaccharide substituent. However, only LPS molecules are decorated with a third subunit known as the O-polysaccharide, or O-antigen. The type and amount of glycolipids present will impact an organism&#8217;s OM density. Therefore, we tested whether the membranes of bacteria with varied glycolipid content could be similarly isolated using our technique. For the LPS-producing organisms, Salmonella enterica serovar Typhimurium and Escherichia coli, the membranes were easily isolated and the LPS O-antigen moiety did not impact bilayer partitioning. Acinetobacter baumannii produces LOS molecules, which have a similar mass to O-antigen deficient LPS molecules; however, the membranes of these microbes could not initially be separated. We reasoned that the OM of A. baumannii was less dense than that of Enterobacteriaceae, so the sucrose gradient was adjusted and the membranes were isolated. The technique can therefore be adapted and modified for use with other organisms.

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

Gram-negative bacteria produce two spatially segregated membranes. The outer membrane is partitioned from the inner membrane by a periplasm and a peptidoglycan layer. The ability to isolate the dual bilayers of these microbes has been critical for understanding their physiology and pathogenesis.

将葛兰阴性细菌的细胞包络分离到内膜和外膜分数，对阿辛托巴基的培养素进行技术调整

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to ...

使用外排缺陷细菌菌株和单拷贝基因表达系统表征 鲍曼不动杆 菌的多药外排系统

使用外排缺陷细菌菌株和单拷贝基因表达系统表征鲍曼不动杆菌的多药外排系统