この作品は、生物医学研究の卓越性のためのウェストバージニア州のアイデアネットワークにアメリカ航空宇宙局ウェストバージニアスペースグラントコンソーシアム（NASA WVSGC）、嚢胞性線維症財団（CFF-YU11G0）およびNIH P20RR016477とP20GM103434によってサポートされていました。我々は、この作品について技術的な支援のためにVonya M·アイジンガーに感謝します。

1。細菌株の準備と二親共役<ol><li> E.接種 37℃で一晩振盪インキュベーター中ゲンタマイシンと場所の15μg/ mlのを補ったルリアブロス（LB）5ml中大腸菌 SM10/λpir/pFAC </li><li> Pを接種緑膿菌の菌株のLB 5ml中PAO1、そして42℃で一晩振とう培養器内の場所</li><li> OD一晩培養の600を測定し、PAO1とEの等量を混ぜる最終容量は1〜1.4ミリリットルの間であることを大腸菌 PFACは、。 </li><li> 5分間6000×gで遠心分離混合物。 </li><li>一方、結合のためにLBプレートを乾燥さ：10〜15分間、37℃のインキュベーター内で開いたプレートを残す。 </li><li>細胞混合物からの上清50μlが、すべてを削除します。 </li><li> 1コンパクト滴の乾燥をLBプレートに上清とピペットの残り50μlの細胞ペレットを再懸濁します。 </li><li>慎重に（IMPOを乾燥させるためにドラフト内でプレートを置きRTANT注：プレート上に広げ、細胞が、これは大幅に）結合の効率が低下しないようにしてください。 </li><li>液滴が乾燥した後、LBプレートを反転し、4〜6時間37℃でインキュベートする。 </li><li>細胞混合物をインキュベートしている間、ゲンタマイシン300μgの/ mlの大（150外径×15ミリメートル）PIAプレートを準備します。使用前に、15〜20分間、37℃のインキュベーター内に置くことにより、任意の残留水分を除去。 </li><li>細胞混合物のインキュベーションが完了した後、無菌の接種ループを用いて細胞を採取し、1mlのLB、ボルテックス、又はピペットを含有する微小遠心管中で、十分に混合する。 </li><li> 300μg/ mlのゲンタマイシンを含む大きなPIAプレートに細胞を均一に広げる。 （重要：この手順では、それは別々のプレートに細胞混合物の増加ボリュームを追加することをお勧めし（ 例えばプレート1：10μL、プレート2：50μL、プレート3：100μL、プレート4：500μL、 など ） 。 </li><li> 37℃で一晩インキュベートする。℃、 </li></ol> 2。ムコイドコロニーの検出と分離<ol><li>インキュベーターからプレートを取り外し、粘液状のコロニーについて検討する。 </li><li>小皿PIAとゲンタマイシン300μgの/ mlの（100外径×10ミリメートル）での分離のために、単一の粘液状コロニーと連勝を隔離する。 </li><li> 37℃で一晩インキュベートする。 </li><li>前の晩培養物にステップ2.2を繰り返します。 </li><li>繰り返します粘液性分離株の安定性を決定するために、2.2から2.4までさらに2回繰り返します。 </li><li>最終的な分離株の工程の後に、単一の粘液状コロニーをLBの4.5ミリリットルを接種し、37℃で一晩シェーカーでインキュベート</li><li>次の日、それぞれの粘液性変異体のラベル3マイクロチューブ。 </li><li>ゲノムDNA抽出およびトランスポゾン挿入部位を同定するための2管（プロトコル3を参照）に一晩培養の2 1.25ミリリットルずつを準備します。 </li><li>さらに、appropriatに1ミリリットル一晩培養をピペッティングすることにより、各粘液性変異体をアーカイブ10パーセントスキムミルクを等量含むイーリーで標識されたクライオバイアル。 -86℃での​​ストア</li></ol> 3。 gDNAを制限消化および連結<ol><li>任意の好適な方法（ 例えば 、フェノール-クロロホルム、スピンカラムなど ） を使用して、粘液性変異株からのgDNAを抽出します。 </li><li> gDNAの濃度を決定するため、制限エンドヌクレアーゼSalIで2μgの1μlの総ダイジェスト、ウシ血清アルブミンをSalI酵素緩衝液（100mMのNaCl、50mMのトリス-HCl、10mMのMgCl 25μlの0.5μlの、1mMのジチオスレイトール（DTT）、室温でのpH 7.9）および50μlの全容量を達成するためのdH 2 Oの適切な体積。 </li><li> 37℃で一晩DNAを消化（重要：制限酵素の効率に応じて、一晩ダイジェストに追加1μLを加え、1〜2時間、37℃でインキュベートする必要があるかもしれないこれは一晩消化するために追加されます。） </li><li> DIGEを浄化任意の好適な方法（ 例えば 、アガロースゲル精製、スピンカラム）とバッファの25μlの溶出を使用してSTEDのDNA。 </li><li> 、〜10月20日NG /μLの好ましい濃度で精製したDNAの11μLを混合して消化されたDNAを連結ライゲーションバッファー（330 mMトリス - 酢酸pH7.5の、660 mMの酢酸カリウム、100 mM酢酸マグネシウム、5 mMと1.5μL DTT）、100 mMのATPと1μlのDNAリガーゼ1.5μL。 </li><li>室温で15分間混合物をインキュベートし、次いで70℃で15分間インキュベートすることによって酵素を不活性化（注：長いを室温で混合物をインキュベートすると、ライゲーションの成功を増加させることができる）。 </li></ol> 4。逆PCR（のiPCR）と配列解析<ol><li>以下のフォワードおよびリバースプライマーおよび熱サイクル条件を使用してライゲーション産物でのiPCRを実行します。 フォワードプライマー（Gm3OUT）：GGGCATACGGGAAGAAGTGA - リバースプライマー（Gm5OUT）：GACTGCCCTGCTGCGTAACA サーモサイコンディtions： 1。 1分間94℃に。 2。 1分間94℃に。 3。 2分間58℃に。 4。 2分間72℃に。 5。 8分間72℃に。 6。 10℃のホールド。 繰り返します34サイクルの2-4を繰り返します。 （注：増幅iPCRの製品は4℃で保存することができます） </li><li>成功のiPCR増幅を確認するために、アガロースゲル電気泳動を行う。 </li><li> Gm3OUTとGm5OUTプライマーを用いてのiPCR製品にシーケンシングを実行します。挿入部位とhimar1の向きが外にマッピングされます。 </li></ol>

<ol>
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著者Hongwei D. YuはProgenesisテクノロジーズ、LLCの最高科学責任者および共同設立者です。

この方法は、これらの変化と他のシュードモナス種に使用することができることに留意することが重要である：P.インキュベートフルオレッセンスおよびP. 30℃でプチダ 、およびP. 42℃でのストチェリ ; P. stuzeriはゲンタマイシンの150μg/ mLのを追加したLBプレート上で培養しなければならない。ステップ1.11、Pにスタッツェリ細胞をLB5...

アルギン酸塩を過剰にする日和見、グラム陰性病原体緑膿菌の能力は、バイオフィルムを確立する能力において主要な因子である。アルギン酸塩の過剰産生は、多くの場合、mucoidyと呼ばれる表現型である。嚢胞性線維症（CF）に罹患した個体の痰からムコイドのコロニーの単離は、慢性感染の指標であり、直接的に患者の健康1における全体的な減少と関連している。現在、それは理解されるP.におけるアルギン酸塩の調節および生産緑膿菌は、主に2つのオペロンで行われます。全体のアルギン酸塩ポリマーの合成と輸出を担当している（ 藻類 - ALG8 - alg44 - algK - 代数 - algG - algX - algL - algI - algJ - - algF algD）最初は、12の遺伝子が含まれているアルギン酸生合成オペロン、である外environmeにペリプラズムNT 2-5。二オペロンは遺伝子のクラスター代替シグマ因子algU / Tで始まり、 ムカ、mucB、およびMUCDと続いている。 mucAB-Dはアルギン酸塩の生産6-8の負の調節因子として分類されている間AlgU / Tは、正の調節因子である。さらに、異化代謝産物抑制制御、キナーゼ活性（KinB）と内タンパク質分解によってそのようなAlgB、AlgQ、AlgR、およびRpoNだけでなく、転写後および翻訳後修飾のような転写調節因子も、アルギン酸塩に関与することが示されているレギュレーション9月14日 。 PFACとして知られているミニhimar1トランスポゾンベクターは、もともとハーバード大学医学部15博士Mekalanos &#39;研究室で作成されました。 PFACプラスミドは2 27 BPSの逆方向反復と中央のゲンタマイシン耐性カセット（aacC1：534 BP）に挟まれた転移因子で構成され、hyperactをコードする遺伝子IVEはマリナートランスポザーゼ 16、および遺伝子をコードするβ-ラクタマーゼ（BLA）（ 図1）。 DQ366300 13：PFACの転移因子についての配列情報は、GenBankアクセッション番号で入手できます。 PFACにおいて、インバースPCRを用いて染色体挿入部位の同定のために使用されるaacC1遺伝子の背後にある複数のクローニング部位（MCS）が存在する。ミニhimar1トランスポゾンを使用する場合の1つの主要な利点は、特定の宿主因子を移調（誘発）のために必要ありませんです。また、Pのゲノムで発見TAジヌクレオチド挿入部位の高い存在があります緑膿菌 。それぞれのゲノムを、例えば、TAジヌクレオチド挿入部位はPAO1に94404と100229回発生（6264404 BPS、GenBankアクセッション番号NC_002516.2）とPA14（ジェンバンクアクセス番号NC_008463 6537648 BPS）。なぜなら、ゲノム中のTAジヌクレオチドの豊富さ、ミニhimar1 </em&gt;トランスポゾンは、毒性遺伝子の分析および高度に調節される遺伝子に特に適している高密度のランダム突然変異誘発を引き起こすことができる。理論的には、ミニhimar1トランスポゾンは、Pのゲノム内の任意の非必須遺伝子に挿入することができます緑膿菌 。これはPAO1ゲノムにおけるオープンリーディングフレームあたり約18のTA挿入部位を提供しています。 ここでは、P.中mucoidyの新規制御因子を特定するために、ミニhimar1トランスポゾンによる突然変異誘発を使用して、プロトコルを記述緑膿菌 。具体的には、我々はbiparentally Eからミニhimar1トランスポゾンを含むPFACベクトル共役非ムコイド栄養PAO1株に大腸菌 SM10/λpir。トランスポゾンは、ゲノムに組み込まれた後、レシピエント株は、 大腸菌の増殖を阻害するトリクロサンを含有する、シュードモナス分離寒天（PIA）上で培養する大腸菌 。このように、の変異体のライブラリー<em&gt; P.緑膿菌は、ゲンタマイシンおよびムコイド表現型の存在を補充したPIAプレート上の増殖により選択することができる。真のトランスポゾン媒介統合変異対ゲンタマイシン耐性およびカルベニシリン感受性表現型を持つことになります、注意してください。本研究では、PAO1の約8万挿入変異体は、4つの別々の動詞によって単離した。次に、ムコイド分離株についてスクリーニングし、制限酵素消化、ライゲーション、逆PCRにより挿入部位を決定した。私たちは、ゲノム当たりの挿入の数を確認するためのプローブとしてゲンタマイシン耐性カセットを用いてサザンブロット解析を行った。我々は、このプロトコルを使用してコンジュゲート当たりの得た変異体の90％以上がゲノム中にhimar1の単一コピーを持っていたし、ゲンタマイシン耐性およびカルベニシリンに敏感な表現型を示していると判断。 32粘液性分離株の合計が同定された、そのうちの22は、Pの異なる遺伝子座にマップされた緑膿菌 PAO1染色体。挿入このレートは、アルギン酸塩過剰生産のいくつかの新規制御因子を特定するために十分なカバレッジを提供します。

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		<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

緑膿菌は、汎用性の高い代謝機能を持つグラム陰性、環境細菌である。P.緑膿菌は、嚢胞性線維症（CF）患者における慢性肺感染症を確立日和見病原菌である。またmucoidyとして知られているアルギン酸と呼ばれる莢膜多糖の過剰産生は、抗生物質、化学療法および宿主防御に浮遊細胞よりも抵抗性がある粘液状のバイオフィルムの形成を促進する。また、粘液性の表現型への非ムコイドからの変換は、CFにおける慢性感染の発症のための臨床マーカーである。 P.によるアルギン酸塩過剰生産緑膿菌は重く細胞エネルギーを課税吸エルゴン過程である。したがって、アルギン酸塩生産はPに高度に規制されている緑膿菌 。より良いアルギン酸調節を理解するために、我々は、新規なアルギン酸塩レギュレータの同定のためのミニhimar1トランスポゾン突然変異誘発を使用して、プロトコルを記述原型PAO1株におけるS。手順は、2つの基本ステップで構成されています。まず、ホストEからミニhimar1トランスポゾン（PFAC）に転送受信者P.に大腸菌 SM10/λpirゲンタマイシンを補充したシュードモナス分離寒天プレート上で選択した高密度の挿入変異体ライブラリーを作成するための共役二親を介して緑膿菌 PAO1。第二に、我々は、スクリーニングおよびゲンタマイシンカセットおよびDNA配列決定から外向きDNAプライマーを用いた逆PCRを介して挿入部位をマッピングするムコイドのコロニーを単離した。 （AlgTは、22を σ）は、野生型ムカマスターアルギン酸レギュレータAlgU抗シグマ因子ムカをコードするPAO1株で、このプロトコルを使用して、我々は2つの新規アルギン酸レギュレータを特定し、mucE（PA4033）とkinB（PA5484） 。このハイスループット突然変異誘発プロトコルは、結腸の変化を引き起こす他の病原性関連遺伝子の同定のために修飾することができるNY形態。

図1に示すように、ミニhimar1マリナートランスポゾンベクターは、PFACは、TAの挿入部位は、そのσ70依存性プロモーターとマルチクローニングサイト（MCS）と、aacC1ゲンタマイシン耐性カセットに隣接した2 27 bpの逆方向反復が含まれています。さらに、PFACベクトルは、高活性himar1トランスポザーゼ 、β-ラクタマーゼ（BLA）、およびTRA転送?...

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

ここでは、画面に高密度の挿入変異体ライブラリーを生成するためのミニhimar1マリナートランスポゾンによる突然変異誘発を使用して、プロトコルを記述し隔離し、原型緑膿菌 PAO1株における新規アルギン酸レギュレータを識別します。

アルギン酸塩産生に関連する新規遺伝子の同定<em&gt;緑膿菌</em使用&gt;ミニ<em&gt; himar1</em&gt;マリナートランスポゾンによる突然変異誘発

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 ビュー.  Marshall University. ここでは、画面に高密度の挿入変異体ライブラリーを生成するためのミニhimar1マリナートランスポゾンによる突然変異誘発を使用して、プロトコルを記述し隔離し、原型緑膿菌 PAO1株における新規アルギン酸レギュレータを識別します。 

ビデオ: 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.

共培養モデルの緑膿菌</emライブヒト気道細胞上に成長した>バイオフィルム

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

免疫・感染学

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.

ロックされた核酸修飾オリゴヌクレオチドによるトランスフェクションおよび蚊細胞における標的遺伝子の変異

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).

フローセル内の緑膿菌と出芽酵母のバイオフィルム

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.

のRNAの単離緑膿菌マウス消化管に定着

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.

に対する抗生物質の有効性をテストするための人工痰培地の使用<em&gt;緑膿菌（Pseudomonas aeruginosa）</em&gt;肺嚢胞性線維症へのより適切な条件で

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.

長期的な慢性<em&gt;緑膿菌</emマウスにおける&gt;気道感染

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.

緑膿菌誘発肺傷害モデル

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.

サルモネラの分離-大食細胞のファゴゾームを含む

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.

発注済み、緑膿菌ひずみ PA14 トランスポゾン挿入変異体の非冗長ライブラリのレプリケーション

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.

緑膿菌におけるフレーム内遺伝子欠失変異体の生成と感染の単純マウスモデルにおける毒性減衰の試験

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