这项工作是由美国国家航空和航天局西弗吉尼亚州的太空格兰特联盟（NASA WVSGC），囊性纤维化基金会（CFF-YU11G0）和美国国立卫生研究院P20RR016477和P20GM103434到西弗吉尼亚理念营销网络的生物医学研究的卓越支持。我们感谢Vonya M.艾辛格对这项工作的技术援助。

1。细菌菌株的制备与双亲共轭<ol><li> E.接种大肠杆菌 SM10/λpir/pFAC在5ml的Luria肉汤（LB）中在37℃下补充有庆大霉素和地方的15微克/毫升在振荡孵化过夜</li><li>接种P.假单胞菌菌株PAO1于5ml的LB，并将其放置在摇床过夜，42°C </li><li>测量外径600过夜培养和混合PAO1和E等量大肠杆菌 PFAC使得最终体积为1-1.4毫升之间。 </li><li>离心混合物以6,000×g离心5分钟。 </li><li>与此同时，干燥的LB平板的结合：离开板的孵化器开放在37℃10-15分钟。 </li><li>删除所有，但50微升上清液从细胞混合物。 </li><li>悬浮在剩余的50微升上清液和吸管的细胞沉淀到干LB平板在一个紧凑的液滴。 </li><li>小心地将板在通风橱中晾干（IMPORTANT注意：不要让细胞分布在盘子里，这将显著降低共轭的效率）。 </li><li>一旦液滴干燥，颠倒LB平板上并在37℃下进行4-6小时。 </li><li>而细胞混合物孵化，准备大（外径150×15毫米），PIA板与庆大霉素的300微克/毫升。在使用之前，通过放置在培养箱中在37℃下15-20分钟除去残留的水分。 </li><li>后的细胞混合物的温育结束后，用无菌接种环收集细胞并在含有1ml LB和涡流，或吸管微量离心管中，以彻底混合。 </li><li>地摊细胞均匀涂于大的PIA板含有300微克/毫升的庆大霉素。 （重要提示：对于此步骤，最好是增加细胞混合物的体积增加来分隔板（ 如板1：10微升，板2：50微升，板3：100微升，标牌4：500微升， 等 ） 。 </li><li>孵育过夜，在37°C。 </li></ol> 2。检测和粘液殖民地的隔离<ol><li>从孵化器中取出板并检查粘液菌落。 </li><li>分离出单个菌落粘液和条纹隔离在中小板（100外径×10毫米），PIA和庆大霉素的300微克/毫升。 </li><li>孵育过夜，在37°C。 </li><li>上一过夜培养重复步骤2.2。 </li><li>重复步骤2.2-2.4两次，以确定粘液分离的稳定性。 </li><li>经过最后的分离步骤，接种4.5毫升LB与单个粘液菌落孵育摇床中过夜，在37°C。 </li><li>第二天，标签3的离心管中的每个粘液突变体。 </li><li>准备过夜培养两个1.25毫升分装成2管基因组DNA的提取和鉴定转座子插入位点（见协议3）。 </li><li>此外，吹打1毫升过夜培养成一个适用情况选择归档的每个粘液突变含10％脱脂牛奶等量伊利标记的离心管。贮存于-86°C。 </li></ol> 3。基因组DNA酶切和结扎<ol><li>使用任何惯用的方法（ 例如 ，酚-氯仿，旋转柱等 ）的粘液突变体中提取基因组DNA。 </li><li>确定基因组DNA的浓度，消化共2微克与1微升的限制性内切酶SalI位，0.5微升的牛血清白蛋白，5微升SalI位酶缓冲液（100mM的NaCl，50mM的Tris-盐酸，10mM的MgCl 2的，1mM的二硫苏糖醇（DTT），pH值为7.9，在室温下）和DH 2 O的适当体积达到50μl的总体积。 </li><li>过夜消化的DNA在37°C （要点：根据不同的限制性内切酶的效率，它可能需要额外的1微升加至过夜消化和孵育在37℃下1-2小时，这是除了过夜消化。） </li><li>净化DIGESTED的DNA使用任何首选方法（ 如琼脂糖凝胶纯化，离心柱）和洗脱与25微升缓冲。 </li><li>通过将11微升纯化的DNA用的〜10-20纳克/微升的优选​​浓度，1.5微升连接缓冲液中（330毫摩尔Tris-乙酸盐pH为7.5，660 mM的醋酸钾，100mM的醋酸镁，5mM的结扎消化的DNA DTT），1.5微升100毫摩尔ATP和1μlDNA连接酶。 </li><li>为15分钟，在室温下温育该混合物，然后通过在70℃下温育15分钟，使酶失活（注：在室温下温育该混合物不再可能增加连接的成功）。 </li></ol> 4。反向PCR（反向PCR）及序列分析<ol><li>使用下列的正向和反向引物和热循环条件对连接产物进行反向PCR： - 正向引物（Gm3OUT）：GGGCATACGGGAAGAAGTGA - 反向引物（Gm5OUT）：GACTGCCCTGCTGCGTAACA 热循环康迪系统蒸发散： 1。 94℃1分钟; 2。 94℃1分钟; 3。 58℃持续2分钟。 4。 72℃2分钟。 5。 72°C为8分钟。 6。 10°C保温。 重复步骤2-4为34个周期。 （注：扩增反向PCR产品可以保存在4°C） </li><li>进行琼脂糖凝胶电泳，以确认成功的反向PCR扩增。 </li><li>对反向PCR产物使用Gm3OUT和Gm5OUT引物进行测序。插入位点和himar1的取向被映射出来。 </li></ol>

<ol>
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笔者宏伟D.羽的首席科学官兼联合创始人Progenesis科技有限责任公司。

要注意，这种方法可以在其他假单胞菌属与这些改变使用是很重要的：孵化P.荧光假单胞菌和P.恶臭在30°C和P。施氏假单胞菌在42℃; P。 stuzeri应培养于补充有150微克/毫升庆大霉素的LB平板上，在步骤1.11，P。斯氏假单胞菌细胞应被转移到500微升的LB代替1毫升。此外，还有对本协议的两个关键步骤。首先，受体菌株应在适当的温度下进行培养。例如，PAO1，应?...

机会主义，革兰氏阴性病原体绿脓杆菌的生产过剩海藻酸钠的能力是其建立了生物膜能力的主要因素。藻酸盐的过量产生是一个表型通常被称为mucoidy。粘液状菌落从患有囊性纤维化（CF）个人痰隔离指示一种慢性感染，并直接与病人的健康1整体下降有关。目前，应当理解的是藻酸盐中P的调节和生产假单胞菌主要发生在两个操纵子。首先是海藻酸钠生物合成操纵子，其中包含12个基因（ALGD - alg8 - alg44 - algK - 代数 - algG - algX - algL - ALGI - algJ - algF - 藻类 ），负责海藻酸盐聚合物的合成及出口对面在周质的细胞外环境为NT 2-5。第二个操纵子是基因的集群开头的替代σ因子algU /吨 ，与mucA，mucB和MUCD继续。 AlgU / T是正的调节器，而mucAB-D被划分为藻酸盐生产6-8的负调节因子。此外，转录调节子，如ALGB，AlgQ，ALGR和RpoN，以及转录后和翻译后修饰通过降解物阻遏控制，激酶活性（KinB）和膜内蛋白水解作用，也被证明是参与海藻酸钠调控9-14。 被称为PFAC迷你himar1转座子载体最初是在Mekalanos博士的实验室在哈佛医学院15创建的。该PFAC质粒组成由27个基点2反向重复序列和庆大霉素抗性盒在中间（aacC1：534 bp）的两侧转座元件，编码hyperact的基因的ive 水手转座16，和一个基因编码的β-内酰胺酶（BLA）（ 图1）。可在GenBank登录号为PFAC的转座子序列信息：DQ366300 13。在PFAC，有一个多克隆位点（MCS），其用于使用反向PCR的染色体插入位点的鉴定aacC1基因的后面。使用迷你himar1转座子的一个主要优点是所需的换位（诱变）没有具体的宿主因素。此外，还有高丰度的整个P的基因组中发现了TA二核苷酸插入位点铜绿假单胞菌 。例如，TA二核苷酸插入位点发生94404和100229倍，在PAO1（6264404基点，GenBank登录号NC_002516.2）和PA14（6537648基点; GenBank登记接入号码NC_008463）基因组中，分别为。因为TA二核苷酸的基因组中，微型himar1丰度的</em&gt;转座子可导致高密度和随机诱变，这是特别适合的毒力基因的分析，并且是高度调控的基因。理论上，迷你himar1座子可以插入到P的基因组中的任何非必需基因铜绿假单胞菌 。这提供了每在PAO1基因组开放阅读框约18 TA的插入位点。 在这里，我们描述了一个协议，使用mini-himar1转座子介导的诱变识别mucoidy在体育新监管铜绿假单胞菌 。更具体地说，我们biparentally缀含有微型himar1座子来自大肠杆菌的PFAC矢量大肠杆菌 SM10/λpir到nonmucoid原养型菌株PAO1。后转座子整合在基因组中，受体菌株进行培养的假单胞菌分离琼脂（PIA）的含有三氯生，其抑制大肠杆菌的生长大肠杆菌 。因此，突变体的文库<em&gt; P。假单胞菌可以通过生长在PIA板补充有庆大霉素和粘液表型的存在进行选择。请注意，一个真正的转座子介导与整合的突变会产生耐庆大霉素和羧苄青霉素敏感的表型。在这项研究中，PAO1约80,000插入突变体通过四个独立的变体的分离。然后，我们筛选粘液株，并确定插入的酶切，连接和反向PCR的网站。我们使用庆大霉素抗性盒作为探针看每个基因组插入的数目进行Southern印迹分析。我们确定了90％以上使用该协议每缀合得到的突变体有himar1只有单拷贝基因组中并显示在庆大霉素抗性和羧苄青霉素敏感的表型。共有32粘液株进行了鉴定，其中22映射到不同的位点P的铜绿假单胞菌 PAO1染色体。插入的这个比率提供足够的覆盖范围，以确定海藻酸钠生产过剩几种新型稳压器。

<table border="0" cellpadding="0" cellspacing="0" width="780">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:267px;">Luria Broth</td>
			<td style="width:187px;">Difco</td>
			<td style="width:125px;">240230</td>
			<td style="width:203px;">via Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pseudomonas isolation agar</td>
			<td>Difco</td>
			<td>292710</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small Plates (100 O.D. x 10 mm)</td>
			<td>Fisher Scientific</td>
			<td>08-757-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Large Plates (150 O.D. x 15 mm)</td>
			<td>Fisher Scientific</td>
			<td>08-757-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benchtop Shaking Incubator</td>
			<td>New Brunswick Scientific</td>
			<td>Innova 4080</td>
			<td>shake at 200 rpm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cabinet Incubator</td>
			<td>VWR</td>
			<td>1540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benchtop Microcentrifuge</td>
			<td>Sorvall</td>
			<td>75-003-287</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SmartSpec Plus Spectrophotometer</td>
			<td>Bio-Rad</td>
			<td>170-2525</td>
			<td>or preferred method/vendor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diposable Inoculation Loops</td>
			<td>Fisher Scientific</td>
			<td>22-363-597</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL Microcentrifuge Tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.0 mL Cryogenic Vials</td>
			<td>Corning</td>
			<td>430659</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL Tubes</td>
			<td>Fisher Scientific</td>
			<td>05-539-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Skim Milk</td>
			<td>Difco</td>
			<td>DF0032-17-3</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNeasy Blood and Tissue (250)&#160;</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td>or preferred method/vendor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick PCR Purification Kit (250)</td>
			<td>Qiagen</td>
			<td>28106</td>
			<td>or preferred method/vendor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAprep Spin Miniprep Kit (250)&#160;</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>or preferred method/vendor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastLink II DNA Ligation Kit</td>
			<td>Epicentre Technologies</td>
			<td>LK6201H</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Accu block Digital Dry Bath&#160;</td>
			<td>Labnet</td>
			<td>NC0205808</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sal1, restriction endonuclease</td>
			<td>New England BioLabs</td>
			<td>R0138L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EasyStart Micro 50</td>
			<td>Molecular BioProducts</td>
			<td>6020</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taq DNA Polymerase</td>
			<td>New England BioLabs</td>
			<td>M0267L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iCycler, Thermocycler</td>
			<td>Bio-Rad</td>
			<td>170-8740</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LE agarose</td>
			<td>Genemate</td>
			<td>3120-500</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamycin Sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP918-1</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">2.0 mL Cryogenic Vials</td>
			<td>Corning</td>
			<td>430659</td>
			<td>via Fisher Scientific</td>
		</tr>
	</tbody>
</table>

identification of novel genes associated with alginate production in pseudomonas aeruginosa using mini-himar1 mariner transposon-mediated mutagenesis

铜绿假单胞菌是一种革兰氏阴性，环境细菌具有多功能的代谢能力。P.绿脓杆菌是一种条件致病菌细菌确立患者的囊性纤维化（CF）的慢性肺部感染。荚膜多糖称为藻酸盐，也被称为mucoidy的生产过剩，促进粘液的生物膜是比浮游细胞对抗生素的化疗和宿主防御更能抵抗的形成。此外，从nonmucoid到粘液表型的转化是一个临床标记的发作的慢性感染的CF。海藻酸钠生产过剩由P.绿脓杆菌是一种吸能的过程，大量征税细胞能量。因此，海藻酸钠生产是高度管制的P。铜绿假单胞菌 。为了更好地理解海藻酸钠调控，我们描述了一个协议，使用mini-himar1转座子诱变的新型海藻酸调节器的识别S IN一个原型株PAO1。该过程包括两个基本步骤。首先，我们从主机E.转让迷你himar1转座子（PFAC） 大肠杆菌 SM10/λpir到受体P。通过双亲结合来创建一个高密度的插入突变体库，并于辅以庆大霉素假单胞菌分离琼脂平板上选择的铜绿假单胞菌 PAO1。其次，我们筛选和分离出的粘液状菌落，通过反向PCR用DNA引物从盒式庆大霉素和DNA测序指着向外绘制的插入位点。使用此协议，我们已经确定了两个新海藻酸钠监管，MUCE（PA4033）和kinB（PA5484），在PAO1菌株与野生型mucA编码抗σ因子MucA对主海藻酸钠调节AlgU（ALGT，σ22） 。这种高通量诱变协议可以修改为其他毒力相关基因的鉴定引起的变化在结肠NY形态。

如图1所示 ，微型himar1水手转座子载体，PFAC，包含两个27碱基的反向重复序列与TA的插入位点侧翼的aacC1庆大霉素抗性盒，其σ70依赖型启动子和多克隆位点（MCS）。此外，PFAC载体含有编码基因的高活性himar1转座，β-内酰胺酶（BLA），和TRA传递操纵子。电讯局长插入位点允许一个有效的融入P的基因组铜绿假单胞菌菌株。在识别和?...

Watch this Scientific Journal Video about Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis at JoVE.

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

这里我们描述了使用微型himar1水手转座子介导的诱变以产生高密度的插入突变体文库，以屏幕的协议，分离和鉴定中的原型的铜绿假单胞菌菌株PAO1新颖藻稳压器。

在伴有海藻酸钠生产新基因的鉴定<em&gt;铜绿假单胞菌</em&gt;使用小型<em&gt; himar1</em&gt;水手转座子介导的诱变

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

identification-novel-genes-associated-with-alginate-production

Research

JoVE Journal

Immunology and Infection

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

12.2K 次观看.  Marshall University. 这里我们描述了使用微型himar1水手转座子介导的诱变以产生高密度的插入突变体文库，以屏幕的协议，分离和鉴定中的原型的铜绿假单胞菌菌株PAO1新颖藻稳压器。 

视频: Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

科研

免疫与感染

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new infections each year. Despite decades of research, there is still no vaccine against malaria, highlighting the need for novel control strategies. One innovative approach is the use of genetically modified mosquitoes to effectively control malaria parasite transmission. Deliberate alterations of cell signaling pathways in the mosquito, via targeted mutagenesis, have been found to regulate parasite development 1. From these studies, we can begin to identify potential gene targets for transformation. Targeted mutagenesis has traditionally relied upon the homologous recombination between a target gene and a large DNA molecule. However, the construction and use of such complex DNA molecules for generation of stably transformed cell lines is costly, time consuming and often inefficient. Therefore, a strategy using locked nucleic acid-modified oligonucleotides (LNA-ONs) provides a useful alternative for introducing artificial single nucleotide substitutions into episomal and chromosomal DNA gene targets (reviewed in 2). LNA-ON-mediated targeted mutagenesis has been used to introduce point mutations into genes of interest in cultured cells of both yeast and mice 3,4. We show here that LNA-ONs can be used to introduce a single nucleotide change in a transfected episomal target that results in a switch from blue fluorescent protein (BFP) expression to green fluorescent protein (GFP) expression in both Anopheles gambiae and Anopheles stephensi cells. This conversion demonstrates for the first time that effective mutagenesis of target genes in mosquito cells can be mediated by LNA-ONs and suggests that this technique may be applicable to mutagenesis of chromosomal targets in vitro and in vivo.

Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.

酸修饰的寡核苷酸锁定核酸转染和蚊虫细胞的靶基因突变

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

铜绿假单胞菌和酿酒酵母流细胞生物膜

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

RNA的分离绿脓杆菌殖民小鼠胃肠道

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

绿脓杆菌引起的肺损伤模型

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

复制的有序, 非冗余图书馆的铜绿假单胞菌应变 PA14 座子插入突变体

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

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 ...