データベース検索で彼女の指導の MGH トレッドウェル仮想ライブラリのリサ Philpotts に感謝したいと思います。この仕事に支えられた嚢胞性線維症財団 (YONKER16G0 および HURLEY16G0) および NIH の NIAID (BPH と ADE: R01 A1095338)。

金融利害の衝突を報告しません。Eliana Drenkard とフレデリック Ausubel は、PA14 非冗長トランスポゾン変異ライブラリーの作成に参加しました。ブライアン ハーリーと Lael Yonker 現在家、マサチューセッツ総合病院で小児科学の一部として変異ライブラリーを配布します。

P。緑膿菌PA14NR 科学のコミュニティのための貴重なリソースです。Clarivate 分析指標データベース、Liberatiらから 2017 年 3 月のデータセットによると(2006 年)37、PA14NR セットの構造を記述するは、微生物学の出版物の上位 1% にランクされています。Google Scholar は Liberatiらの以上 600 の引用を報告します。2017 年 8 月現在 (2006 年) の元の原稿。ライブラ?...

緑膿菌は、多様な表現型と際と適応性グラム陰性菌で土壌、水、最も人間環境と皮膚の細菌叢に存在です。膿は、5.5-7 Mbp 高 G + c コンテンツ (65 〜 67%) の比較的大規模なゲノム多くの菌種に比べると。さらに、その遺伝子の大部分は代謝適応性に関与しているし、環境ストレス1への応答に大きな柔軟性を可能にする規制のネットワークの一部であります。膿病原因子の茄多を表現、フォーム バイオ フィルムに性癖を展示、複数クオラムセンシングで経路を介して応答を調整する能力を持って、抗生物質耐性を開発する顕著な容量が表示されます。許容値2,3,4,5,6,7,8。これらの属性は、膿によって引き起こされる感染症を治療するための重要な課題を提示します。
慢性的な膿菌感染は多くの病気の状態で発生することが。嚢胞性線維症 (CF)、嚢胞性線維症膜コンダクタンス レギュレータ (CFTR)遺伝子の変異によって引き起こされる遺伝病の結果、気道内で濃縮、感染した分泌物進歩的な気管支拡張症と、最終的には、死呼吸不全9。成人では、CF の患者の大半慢性膿、罹患率と死亡率のこの疾患10に関連付けられている重要な役割を果たしていると感染しています。さらに、重症熱傷の傷害11、気管切開12、関節置換術13、または留置カテーテル14患者フォームする細菌の能力に関連するp. 緑膿菌感染のリスクがバイオ フィルムとエスケープ ホスト炎症反応15。多抗生物質耐性や寛容の人口は広域スペクトル、連続的抗菌薬治療12,16,17によって選択後に、植民地化の競争がなければを発生するさらに、,18.膿の病態の理解を深めるお越しの数多くの病気の状態に大きな影響を与えます。
PAO1、PA103、PA14、朴セリは、系統を含む、 P. 緑膿菌臨床分離株が盛ん膿病因のさまざまな機能を調査します。ひずみ PA14 は臨床分離株はない広範囲継研究室で最も一般的なクローンのグループ世界19,20の 1 つに属しています。PA14is 顕著なエンドトキシンによる感染症の脊椎動物モデルにおける高度病原性プロファイル21、線毛構造22、病原性島23、タイプ III の分泌システム (TTSS)、哺乳類への毒性細胞24と抗生物質抵抗性と永続性25のプロファイル。さらに、PA14 はまた多数の宿主-病原体モデル システムにおける高度病原性植物葉を含む浸潤モデル26,27,線虫感染モデル28, 29昆虫モデル30,31, と同様にマウス肺炎モデル32,33皮の焼跡モデル34。
ゲノム変異ライブラリ不要な遺伝子ゲノム規模での遺伝子機能の分析により有機体の生物学を理解するための非常に強力なツールを構成する同質遺伝子変異体のコレクションです。膿に建設された 2 つの飽和近くトランスポゾン挿入変異ライブラリは現在配布です。トランスポゾンの挿入サイトは、両方のライブラリの決定されています。これらのいわゆる非冗長ライブラリ時間がかなり短縮されて細菌菌株のゲノム研究を容易にし、コストに関わるスクリーニング, ランダムなトランスポゾン変異体。膿の PAO1 トランスポゾン変異ライブラリー、MPAO1 に建設された分離はトランスポゾンを用いた歪み PAO1phoA/ほら、lacZ/ほら35がワシントン大学の Manoil 研究室によってキュレーションです。ライブラリのコレクションで構成、シーケンス確認 9,437 トランスポゾン変異体のゲノム広いカバレッジを提供し、ほとんどの遺伝子36の 2 つの突然変異体が含まれています。膿PAO1 トランスポゾン変異ライブラリーに関する情報が http://www.gs.washington.edu/labs/manoil/libraryindex.htm で公共のインターネットにアクセスできる Manoil ラボのウェブサイトでご利用いただけます。膿ひずみ PA14 非冗長トランスポゾン挿入変異ライブラリー (PA14NR セット) トランスポゾンMAR2xT7テネシー州phoAを用いた歪み PA1437に建設された現在の配布は小児科学マサチューセッツ総合病院。PA14NR 設定には、不要な遺伝子37単一トランスポゾン挿入 5,800 以上の突然変異体のコレクションが装備されています。PA14NR 設定の構成の詳細については、PA14NR の使用を促進するオンライン検索ツールのさまざまなを含む公共のインターネットからアクセスできるサイト http://pa14.mgh.harvard.edu/cgi-bin/pa14/home.cgi?section=NR_LIB のとおり設定します。
オリジナル PA14NR セット約 34,000 ランダム トランスポゾン挿入変異体の 4,596 予測 PA14 遺伝子すべて予測 PA14 遺伝子37の 77% を表すに対応する包括的なライブラリから選択された 5,459 変異株で構成されます。新しい突然変異体が追加された 2006 年に図書館の建設以来、現在約 4,600 PA14 遺伝子を表す 5,800 以上の変異体38含まれている PA14NR の設定。PA14 トランスポゾン変異体の大半は、野生型背景37に生成されました。遺伝的背景など、変異ライブラリーの各メンバーに関する詳細については、オンライン データベースを検索または非冗長ライブラリ スプレッドシート PA14 ウェブサイト (http:// で利用できる両方の機能をダウンロードすることによりpa14.mgh.harvard.edu/cgi-bin/pa14/home.cgi)。突然変異体の大部分は、MAR2xT7 を使用して作成された (MrT7) トランスポゾンが TnPhoA (phoA) トランスポゾン37を使用して作成された小さなセット。各トランスポゾンが抗生物質耐性カセット、ゲンタマイシン (MrT7) またはカナマイシン (phoA) を使用して突然変異体の選択を可能にします。突然変異体の PA14NR セット 63-96-ウェル プレートに格納され、2 つの追加の 96 ウェル制御プレート、野生型 PA14 から成っている接種が含まれています、プリセット パターンに挟在非接種の井戸。オンライン検索ツールとペアに大きく 96 ウェル プレート形式は、スクリーニング アッセイ突然変異体の表現型に関わる遺伝子を簡単に識別できるのカスタム開発を促進します。オンライン検索ツールには、検索してさらに研究に必要な追加の関連する変異体の選択も容易します。
PA14 と PAO1 トランスポゾン変異ライブラリは科学的なコミュニティのための非常に重要なグローバル リソースであり、彼らは未知の遺伝子の機能とこの細菌の病原体の経路を検証する際にお互いを補完します。偶然にも、PAO1 と PA14 トランスポゾン変異ライブラリの構築、以来多くの緑膿菌の全ゲノム DNA シーケンス解析を示している PAO1 と PA14 P. aeruginosa の別の主要なサブクレードに属しています。系統7,39,40,41。臨床緑膿菌株が発見されてサブグループ系統、PAO1 と PA14 異なる膿に属している事実全体に分散比較 2 トランスポゾン変異ライブラリーの価値の向上研究。
文書構造を記述して膿ライブラリ35,を含む細菌の突然変異体ライブラリのスクリーニング3742,,、文献で容易に利用できます。しかし、我々 の知識の限りで公開プロトコルの詳細な手順と、レプリケーションに使用される技術を記述する保守、および細菌変異ライブラリの検証がありません。
この文書に記載されている方法では、使用を容易にする 3 つのプロトコルのセットと PA14NR セットのメンテナンスについて説明します。最初のプロトコルでは、PA14NR 設定の受信者にお勧めのライブラリのレプリケーションについて説明します。2 番目のプロトコルには、ストリー キング、成長、および PA14NR のセットを使用して識別される個々 の変異体を格納するためのガイドラインが含まれています。3 番目のプロトコルでは、トランスポゾン変異体からの断片の PCR の拡大や突然変異体の身元を確認するそれに続くシーケンスなど、品質管理手法について説明します。この一連のプロトコルは、レプリケーションおよび他の細菌の突然変異体のライブラリやコレクションの維持の適応もあります。細菌変異ライブラリやコレクションのレプリケーションは「マスター コピー」の整合性を維持するために非常にお勧め (原本を受け取った)。ルーチン検査用 PA14NR セットの複数のコピーのレプリケーションでは、マスター コピーの間の汚染の可能性を最小限に抑えます。

<table>
	<tbody>
		<tr>
			<td>Materials for Library Replication</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 96-well Tissue-culture treated, case of 50</td>
			<td>Corning Life Sciences</td>
			<td>353072</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Sterile 96 Well Clear V-Bottom 2000&#956;L Deep Well Plates, case of 25</td>
			<td>Corning Life Sciences</td>
			<td>3960</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Nunc OmniTray (rectangular plates), case of 60</td>
			<td>Thermo Scientific Rochester</td>
			<td>242811</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Rectangular Ice Pan, Midi (4L)</td>
			<td>Corning Life Sciences</td>
			<td>432104</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Secure-Gard Cone Mask, case of 300</td>
			<td>Cardinal Health</td>
			<td>AT7509</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>AluminaSeal, pack of 100</td>
			<td>Diversified Biotech</td>
			<td>ALUM-100</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Breathe-Easy membrane, pack of 100</td>
			<td>Diversified Biotech</td>
			<td>BEM-1</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Sterile, individually wrapped, 50mL Solution Trough/Reagent Reservoir, case of 100</td>
			<td>Sorenson</td>
			<td>S50100</td>
			<td>via Westnet Incorporated</td>
		</tr>
		<tr>
			<td>Plate roller</td>
			<td>VWR</td>
			<td>60941-118</td>
			<td>via VWR</td>
		</tr>
		<tr>
			<td>Cryo Laser Labels - CRYOLAZRTAG 2.64&#34; x 0.277&#34;, pack of 16 sheets</td>
			<td>GA International</td>
			<td>RCL-11T1-WH</td>
			<td>via Labtag.com (template for printing also available from Labtag.com)</td>
		</tr>
		<tr>
			<td>96-well replicator</td>
			<td>V &#38; P Scientific, Inc.</td>
			<td>Custom 407C, 3.18mm pin diameter, 57mm long</td>
			<td>via V &#38; P Scientific, Inc.</td>
		</tr>
		<tr>
			<td>Multitron Pro, 3mm Shaking incubator</td>
			<td>Infors HT</td>
			<td>l10003P</td>
			<td>via Infors HT</td>
		</tr>
		<tr>
			<td>Picus 12 Channel 50-1200&#956;L Electronic Pipette</td>
			<td>Sartorius</td>
			<td>735491PR</td>
			<td>via Sartorius</td>
		</tr>
		<tr>
			<td>Filter Tips 50-1200&#956;L, pack of 960</td>
			<td>Biohit</td>
			<td>14-559-512</td>
			<td>via Fisher Scientific; use electronic multichannel-compatible tips</td>
		</tr>
		<tr>
			<td>Dry Ice</td>
			<td>User-specific vendor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials for Individual Mutant Storage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Premium Microcentrifuge Tubes: 1.5mL</td>
			<td>Fisher Scientific</td>
			<td>05-408-130</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Pipettes (P1000, P200, P20, P2)</td>
			<td>Gilson</td>
			<td>F167370</td>
			<td>via Gilson</td>
		</tr>
		<tr>
			<td>Materials for Quality Control PCR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Premium Microcentrifuge Tubes: 1.5mL</td>
			<td>Fisher Scientific</td>
			<td>05-408-130</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Scientific</td>
			<td>ND-2000</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>PCR Thermocycler</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Omnistrips PCR Tubes with domed lids</td>
			<td>Thermo Scientific</td>
			<td>AB0404</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>ART Barrier low-retention pipette tips (10 uL, 100 uL, 1000 uL)</td>
			<td>Molecular BioProducts, Inc.</td>
			<td>Z676543 (10 uL), Z676713 (100 uL), Z676802 (1000 uL)</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Pipettes (P1000, P200, P20, P2)</td>
			<td>Gilson</td>
			<td>F167370</td>
			<td>via Gilson</td>
		</tr>
		<tr>
			<td>Fisherbrand Premium Microcentrifuge Tubes: 1.5mL</td>
			<td>Fisher Scientific</td>
			<td>05-408-130</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>MasterPure DNA Purification Kit</td>
			<td>Epicentre</td>
			<td>MCD85201</td>
			<td>via Epicentre Technologies Corp</td>
		</tr>
		<tr>
			<td>GeneRuler 1 kb Plus DNA Ladder, ready-to-use</td>
			<td>Thermo Scientific</td>
			<td>SM1333</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>RediLoad Loading Buffer</td>
			<td>Invitrogen</td>
			<td>750026</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals for Library and Individual Mutant Storage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol MB Grade, 1L</td>
			<td>Sigma Aldrich</td>
			<td>G5516</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td></td>
			<td></td>
			<td>Per 1L dH2O: 10g tryptone, 5g yeast extract, 5g NaCl, 1ml 1N NaOH (Current Protocols in Molecular Biology.&#160; Wiley, 1994.)</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Sigma Aldrich</td>
			<td>T7293</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Sigma Aldrich</td>
			<td>Y1625</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma Aldrich</td>
			<td>S8045</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>LB&#160; agar</td>
			<td></td>
			<td></td>
			<td>See preparation above, add 15g Bacto Agar</td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>Sigma Aldrich</td>
			<td>A5306</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Gentamicin sulfate, 10g</td>
			<td>BioReagent</td>
			<td>1405-41-0</td>
			<td>via Sigma-Aldrich</td>
		</tr>
		<tr>
			<td>Kanamycin sulfate</td>
			<td>Gibco</td>
			<td>11815024</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>Ethanol, 190 proof</td>
			<td>Decon</td>
			<td>04-355-221</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Chemicals for Quality Control PCR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primers</td>
			<td>User-preferred vendor</td>
			<td></td>
			<td>See primers listed in Table 3</td>
		</tr>
		<tr>
			<td>Corning cellgro Molecular Biology Grade Water</td>
			<td>Corning</td>
			<td>46000CV</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Taq Polymerase Buffer</td>
			<td>Invitrogen</td>
			<td>10342020</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>Taq DNA Polymerase, recombinant</td>
			<td>Invitrogen</td>
			<td>10342020</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>Invitrogen</td>
			<td>10297018</td>
			<td>via ThermoFisher</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9539</td>
			<td>via Sigma-Aldrich</td>
		</tr>
	</tbody>
</table>

replication of the ordered, nonredundant library of pseudomonas aeruginosa strain pa14 transposon insertion mutants

緑膿菌は、人間環境のユビキタス多様な表現型と際と適応性グラム陰性菌です。膿はバイオ フィルムを形成、抗生物質耐性、病原性因子を生成、慢性感染の過程で急速に進化することができます。したがって膿が両方急性と慢性、感染症を治療することは困難特定の患者集団で重大な合併症の結果として行われます。膿株 PA14 はさまざまな PA14 この病原体を研究するための魅力的なひずみを作る哺乳類と nonvertebrate のホストに感染する保存されたゲノム構造をもつ人間の臨床分離株です。2006 年、4,596 予測 PA14 遺伝子に対応する 5,459 変異体を含む非冗長トランスポゾン挿入変異ライブラリが生成されました。それ以来、PA14 ライブラリの配布は、個々 の遺伝子の機能と緑膿菌の複雑な経路を理解する研究コミュニティを許可しています。レプリケーション プロセスによるライブラリの完全性の維持には、適切な処理と正確な技術が必要です。そのために、この原稿はライブラリのレプリケーション、ライブラリの品質管理および個々 の突然変異体の適切なストレージに関連する手順の詳細を記述するプロトコルを示します。

PA14NR セットの 12 の新しいコピーは、生成される新しいコピーの品質管理評価・議定書 III を使用して実施されたプロトコルを使用してレプリケートされていました。
PA14NR セット野生型 PA14 から成っている制御板と共にミュータント プレート接種し接種コントロール プリセット パターン (図 4 a) に?...

Watch this Scientific Journal Video about Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants at JoVE.com

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

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

緑膿菌感染症は、脆弱なホストで重大な合併症を引き起こします。膿ひずみ PA14、PA14NR を設定すると、指定の非冗長トランスポゾン挿入変異ライブラリー多数プロセスの遺伝子機能の解析が容易になります。ここに提示 PA14NR セット変異ライブラリーの高品質のコピーを生成するためのプロトコルです。

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

replication-ordered-nonredundant-library-pseudomonas-aeruginosa

Research

JoVE Journal

Immunology and Infection

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

8.0K ビュー.  Massachusetts General Hospital. 緑膿菌感染症は、脆弱なホストで重大な合併症を引き起こします。膿ひずみ PA14、PA14NR を設定すると、指定の非冗長トランスポゾン挿入変異ライブラリー多数プロセスの遺伝子機能の解析が容易になります。ここに提示 PA14NR セット変異ライブラリーの高品質のコピーを生成するためのプロトコルです。

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

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

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 ...

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 ...

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

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/&#955;pir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, &#963;22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

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

Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.

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 ...

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. ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

発生とのマルチ表現型ハイコンテンツスクリーニング<em&gt; Q熱リケッチア</em&gt;トランスポゾン変異体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

密なトランスポゾン挿入ライブラリを使用して細菌活用腸内細菌系統など、大腸菌や赤痢菌の作成

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness during infection of Golden Syrian hamsters. Tn-Seq combines random transposon mutagenesis with the power of high-throughput sequencing technology. Animals are challenged with a pool of transposon mutants (input pool), followed by harvesting of blood and tissues a few days later to identify and quantify the number of mutants in each organ (output pools). The output pools are compared to the input pool to evaluate the in vivo fitness of each mutant. This approach enables screening of a large pool of mutants in a limited number of animals. With minor modifications, this protocol can be performed with any animal model of leptospirosis, reservoir host models such as rats and acute infection models such as hamsters, as well as in vitro studies. Tn-Seq provides a powerful tool to screen for mutants with in vivo and in vitro fitness defects.

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

We describe here a technique that combines transposon mutagenesis with high-throughput sequencing to identify and quantify transposon leptospiral mutants in tissues after a challenge of hamsters. This protocol can be used to screen mutants for survival and dissemination in animals and can also be applied to in vitro studies.

レプトスピラトランスポゾン挿入変異体中に黄金シリアのハムスター感染の測定フィットネスにハイスループット並列配列

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

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 ...