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

<ol>
	<li>Prioritization of pathogens to guide research, discovery, research and development of new antibiotics for drug-resistant bacterial infections, including tuberculosis. World Health Organization Available from: <a target="_blank" href="https://www.who.int/publications/i/item/WHO-EMP-IAU-2017.12">https://www.who.int/publications/i/item/WHO-EMP-IAU-2017.12</a> (2017)</li><li>Kornelsen, V., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+multidrug+resistance+efflux+pumps+in+Acinetobacter+spp.">Update on multidrug resistance efflux pumps in Acinetobacter spp.</a> Antimicrob Agents Chemother. 65 (7), e00514-00521 (2021).</li><li>Sykes, E. M., Deo, S., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+genetic+tools+for+Acinetobacter+baumannii.">Recent advances in genetic tools for Acinetobacter baumannii.</a> Front Genet. 11, 601380 (2020).</li><li>Kumar, A., Chua, K. L., 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=Method+for+regulated+expression+of+single-copy+efflux+pump+genes+in+a+surrogate+Pseudomonas+aeruginosa+strain:+identification+of+the+BpeEF-OprC+chloramphenicol+and+trimethoprim+efflux+pump+of+Burkholderia+pseudomallei+1026b.">Method for regulated expression of single-copy efflux pump genes in a surrogate Pseudomonas aeruginosa strain: identification of the BpeEF-OprC chloramphenicol and trimethoprim efflux pump of Burkholderia pseudomallei 1026b.</a> Antimicrob Agents Chemother. 50 (10), 3460-3463 (2006).</li><li>Kumar, A., Dalton, C., Cortez-Cordova, J., 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+vectors+as+genetic+tools+for+single+copy+gene+cloning+in+Acinetobacter+baumannii.">Mini-Tn7 vectors as genetic tools for single copy gene cloning in Acinetobacter baumannii.</a> J Microbiol Methods. 82 (3), 296-300 (2010).</li><li>Ducas-Mowchun, K., 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=Next+generation+of+Tn7-based+single-copy+insertion+elements+for+use+in+multi-+and+pan-drug+resistant+strains+of+Acinetobacter+baumannii.">Next generation of Tn7-based single-copy insertion elements for use in multi- and pan-drug resistant strains of Acinetobacter baumannii.</a> Appl Environ Microbiol. 85 (11), e00066-00119 (2019).</li><li>Ducas-Mowchun, K., De Silva, P. M., Patidar, R., Schweizer, H. P., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tn7-based+single-copy+insertion+vectors+for+Acinetobacter+baumannii.">Tn7-based single-copy insertion vectors for Acinetobacter baumannii.</a> Methods Mol Biol. 1946, 135-150 (2019).</li><li>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=Applications+of+the+Saccharomyces+cerevisiae+Flp-FRT+system+in+bacterial+genetics.">Applications of the Saccharomyces cerevisiae Flp-FRT system in bacterial genetics.</a> J Mol Microbiol Biotechnol. 5 (2), 67-77 (2003).</li><li>Choi, K. H., 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=A+Tn7-based+broad-range+bacterial+cloning+and+expression+system.">A Tn7-based broad-range bacterial cloning and expression system.</a> Nat Methods. 2 (6), 443-448 (2005).</li><li>Choi, K. H., 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+single+attTn7+sites:+example+Pseudomonas+aeruginosa.">Mini-Tn7 insertion in bacteria with single attTn7 sites: example Pseudomonas aeruginosa.</a> Nat Protoc. 1 (1), 153-161 (2006).</li><li>Kornelsen, V., Unger, M., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atorvastatin+does+not+display+an+antimicrobial+activity+on+its+own+nor+potentiates+the+activity+of+other+antibiotics+against+Acinetobacter+baumannii+ATCC+17978+or+A.+baumannii+AB030.">Atorvastatin does not display an antimicrobial activity on its own nor potentiates the activity of other antibiotics against Acinetobacter baumannii ATCC 17978 or A. baumannii AB030.</a> Access Microbiol. 3, 000288 (2021).</li><li>Reyrat, J. 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><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&amp;ndash;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&amp;nbsp;</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 &amp;mu;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 pFLP2&lt;sup&gt;ab&lt;/sup&gt; 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

Universidad de Cartagena UDEC

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

479 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

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 ...

科研

遗传学

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

基于荧光的感染组织细菌基因调控方法

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

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

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

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

实验进化方法中疟原虫介导抗生素耐药性的定量化

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

生物化学

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 &#215; 105-1.2 &#215; 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of &#8805;0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae,&#160;and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (&#946;-lactams), aadA, aac (6&#39;)-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, &#946;-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the&#160;chloramphenicol acetyltransferase gene catB10, and&#160;the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Here, we present a detailed protocol for the isolation and identification of antibiotic-resistant bacteria from water and the molecular characterization of their antibiotic resistance genes (ARGs). The use of culture-based and non-culture-based (metagenomic analysis) techniques provides complete information about the total bacterial diversity and the total pool of different ARGs present in freshwaters from Mumbai, India.

水性抗生素耐药细菌的分离和鉴定及其抗生素耐药基因的分子表征

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the ...

环境科学

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated anti-cancer drugs or antibiotics, is a current serious problem in public health. This multidrug resistance is largely due to energy-dependent drug efflux pumps. The pumps expel anti-cancer drugs or antibiotics into the external medium, lowering their intracellular concentration below a toxic threshold. We are studying multidrug resistance in Pseudomonas aeruginosa, an opportunistic bacterial pathogen that causes infections in patients with many types of injuries or illness, for example, burns or cystic fibrosis, and also in immuno-compromised cancer, dialysis, and transplantation patients. The major MDR efflux pumps in P. aeruginosa are tripartite complexes comprised of an inner membrane proton-drug antiporter (RND), an outer membrane channel (OMF), and a periplasmic linker protein (MFP) 1-8. The RND and OMF proteins are transmembrane proteins. Transmembrane proteins make up more than 30% of all proteins and are 65% of current drug targets. The hydrophobic transmembrane domains make the proteins insoluble in aqueous buffer. Before a transmembrane protein can be purified, it is necessary to find buffer conditions containing a mild detergent that enable the protein to be solubilized as a protein detergent complex (PDC) 9-11. In this example, we use an RND protein, the P. aeruginosa MexB transmembrane transporter, to demonstrate how to express a recombinant form of a transmembrane protein, solubilize it using detergents, and then purify the protein detergent complexes. This general method can be applied to the expression, purification, and solubilization of many other recombinantly expressed membrane proteins. The protein detergent complexes can later be used for biochemical or biophysical characterization including X-ray crystal structure determination or crosslinking studies.

In this protocol we demonstrate the expression, solubilization, and purification of a recombinantly expressed membrane protein, MexB, as a soluble protein detergent complex. MexB is a multidrug resistance membrane transporter from the opportunistic bacterial pathogen Pseudomonas aeruginosa.

表达，洗涤剂溶解，和一个膜转运，MexB多药耐药蛋白的纯化

Multidrug resistance (MDR), the ability of a cancer cell or pathogen to be resistant to a wide range of structurally and functionally unrelated ...

生物学

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 ...

免疫与感染

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram-negative bacteria Pseudomonas aeruginosa in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport, and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution, and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient.

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

We present a protocol for the in vitro investigation of efflux pumps from Pseudomonas aeruginosa. This protocol allows for the generation of a robust, reversible, and tunable proton gradient within the liposome membrane and hence should be adaptable to any membrane protein energized by the protomotive force.

在 MexAB外排泵从铜绿假单胞菌的体外研究

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution ...

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans constitute a strain-specific barcode. The associated glycans show higher diversity in sugar composition and structure than those of eukaryotes and are important in bacterial-host recognition processes and interaction with the environment. In pathogenic bacteria, glycoproteins have been involved in different stages of the infectious process, and glycan modifications can interfere with specific functions of glycoproteins. However, despite the advances made in the understanding of glycan composition, structure, and biosynthesis pathways, understanding of the role of glycoproteins in pathogenicity or interaction with the environment remains very limited. Furthermore, in some bacteria, the enzymes required for protein glycosylation are shared with other polysaccharide biosynthetic pathways, such as lipopolysaccharide and capsule biosynthetic pathways. The functional importance of glycosylation has been elucidated in several bacteria through mutation of specific genes thought to be involved in the glycosylation process and the study of its impact on the expression of the target glycoprotein and the modifying glycan. Mesophilic Aeromonas have a single and O-glycosylated polar flagellum. Flagellar glycans show diversity in carbohydrate composition and chain length between Aeromonas strains. However, all strains analyzed to date show a pseudaminic acid derivative as the linking sugar that modifies serine or threonine residues. The pseudaminic acid derivative is required for polar flagella assembly, and its loss has an impact on adhesion, biofilm formation, and colonization. The protocol detailed in this article describes how the construction of null mutants can be used to understand the involvement of genes or genome regions containing putative glycosyltransferases in the biosynthesis of a flagellar glycan. This includes the potential to understand the function of the glycosyltransferases involved and the role of the glycan. This will be achieved by comparing the glycan deficient mutant to the wild-type strain.

Here, we describe the construction of null mutants of Aeromonas in specific glycosyltransferases or regions containing glycosyltransferases, the motility assays, and flagella purification performed to establish the involvement and function of their encoded enzymes in the biosynthesis of a glycan, as well as the role of this glycan in bacterial pathogenesis.

产生空突变体以阐明细菌糖基转移酶在细菌运动中的作用

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans ...

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 ...

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

副本

摘要

探索更多视频

此视频中的章节

多拷贝产质粒在抗生素耐药性演变中的作用试验

基于荧光的感染组织细菌基因调控方法

实验进化方法中疟原虫介导抗生素耐药性的定量化

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

水性抗生素耐药细菌的分离和鉴定及其抗生素耐药基因的分子表征

表达，洗涤剂溶解，和一个膜转运，MexB多药耐药蛋白的纯化

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

在 MexAB外排泵从铜绿假单胞菌的体外研究

产生空突变体以阐明细菌糖基转移酶在细菌运动中的作用

的产生

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

副本

摘要

探索更多视频

此视频中的章节

多拷贝产质粒在抗生素耐药性演变中的作用试验

基于荧光的感染组织细菌基因调控方法

实验进化方法中疟原虫介导抗生素耐药性的定量化

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

水性抗生素耐药细菌的分离和鉴定及其抗生素耐药基因的分子表征

表达，洗涤剂溶解，和一个膜转运，MexB多药耐药蛋白的纯化

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

在 MexAB外排泵从铜绿假单胞菌的体外研究

产生空突变体以阐明细菌糖基转移酶在细菌运动中的作用

的产生

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