The authors would like to thank Prof. Sze-Bi Hsu and Ms. Zhenzhen for useful discussions and help in the theoretical analysis and numerical simulation. Y. T. Y. would like to acknowledge funding support from the Ministry of Science and Technology under grant numbers MOST 103-2220-E-007-026 and MOST 104-2220-E-007-011, and from the National Tsing Hua University under grant numbers 103N2042E1, 104N2042E1, and 105N518CE1.

Morbidostatデバイスの1アセンブリおよび予備試験<ol><li> Morbidostatの組立<ol><li> 18 G注射針で培養バイアルのキャップ上の3つの穴をパンチ。 〜の長さは7センチポリエチレンチューブの3枚をカット。キャップにポリエチレン管のこれらの3枚を挿入します。 </li><li>ポリジメチルシロキサン（PDMS）の混合物のためのキャストとして機能するようにキャップの縁をラップするためにテープを使用してください。爪楊枝を使って手動で攪拌することにより、コンポーネントの5グラムと150ミリリットルのプラスチック容器中のPDMSのB成分の0.5グラムを混ぜます。 10ミリリットルの注射器に混合物をロードします。 <ol><li>シリンジでキャップにPDMSの混合物を注ぎます。 70℃のオーブン中で8時間、PDMSを硬化させるために全体のキャップを焼きます。 </li></ol></li><li>はんだごてを持つカーボン抵抗と24ゲージワイヤとLEDのカソードとLEDのアノードを半田付けします。 24 G線と680Ωカーボン抵抗を半田付けします。電気テープを使用して、ワイヤ接続を絶縁してください。 </li><li>そう24 G線と100kΩのカーボン抵抗と受光素子のエミッタと光検出器のコレクタlder。電気テープを使用して、ワイヤ接続を絶縁してください。 </li><li>培養バイアルホルダーに発光ダイオードを配置します。 補足 図2に回路図に従うことで、それはLEDはIR（ 例えば、携帯電話のカメラ）を検出することができるデジタルカメラを使用して動作することを確認し、5Vの電源で発光ダイオードと電源を接続します。 </li><li>文化バイアルホルダーに光検出器を配置します。 補足 図2の回路図に従うことにより、電子制御基板の両端の電圧を測定する光検出器抵抗器の両側にワイヤ5 V.接続の電力供給と光検出器と電源を接続します。 </li><li>磁気攪拌手段としての冷却ファンのシャフトの磁石を接着。 alignmことに注意してください培養バイアルにファンのシャフトの耳鼻咽喉科、培養バイアルと磁石の間の間隔は、磁気攪拌装置の適切な機能のために非常に重要です。 </li><li>マイクロポンプ、培地ボトル、および廃液ボトル用のシリコンチューブの対応する片に培養バイアルのポリエチレンチューブの各部分を接続します。 1時間ポンプの電源をオンにし、ポンプ速度を決定するために、培養バイアルに培地ボトルから圧送される総体積を測定。典型的なポンピング速度は、希釈率D = 0.33時間-1培養バイアルの12ミリリットル作業容量のために対応〜4ミリリットル/ hrで、でなければならないことに注意してください。 </li><li>中規模（34センチメートルのx 34センチメートルX 43.5センチメートル）シェーカーインキュベーターにアセンブリ全体を置きます。 </li></ol></li><li> Morbidostatを事前テスト<ol><li>磁気撹拌棒をテストするためのモーターを起動するために、5Vまで冷却ファンの電源電圧を設定します。攪拌動作が視覚的に検査することができることに注意してください。ドライブ電圧冷却ファンモータは正確にパルス幅変調（PWM）によって制御されていますよ。 </li><li>野生型E.一連の準備典型的には、キャリブレーションのための0.07から0.3までの範囲の600nmでの光学密度を有する大腸菌サンプル 。テストEを置きます培養バイアル中の大腸菌サンプルとは、光検出器の電圧を記録します。 <ol><li>場所〜100プレートリーダーで同じサンプルμlの光学密度を記録。検量線をプロットするために、光学濃度データ対の光検出器電圧を使用してください。一般的に、光学濃度の1単位の変化は、ここで使用されるバイアス方式と光検出器で〜7.6 Vの変化に対応していることに注意してください。 </li></ol></li></ol></li></ol> 2. Morbidostatを実行します<ol><li>毎日の実験を開始する前の準備<ol><li>セクションのようなデバイスを形成するための注入口用の内径1/32インチ、外径3/32インチと3超耐薬品性のタイゴンチューブで培養バイアルを準備1.1と補足図1インチ </li><li> M9最少培地（0.2％グルコース）およびトリメトプリム（TMP）は、薬物含有培地を準備します。培地、培地ボトル、薬の媒体ボトル、廃液ボトル、および121℃のオートクレーブ中で培養バイアルを滅菌します。 注：TMPは、後の表1に濃度を達成するために使用される媒体を含むTMP薬物がオートクレーブ処理されていないストック溶液です。 </li></ol></li><li> Morbidostatを実行します<ol><li>実験の初日に、凍結された野生型の1ミリリットルE.を解凍室温で5分間コリ MG1655細胞及びM9の成長培地の〜12ミリリットルを含む培養バイアルに細胞120μlのを転送します。最初の日の後、凍結したE.の1ミリリットルを解凍大腸菌 5分と転送含む新しい培養バイアルに細胞を120μlのために前日からの細胞〜M9の増殖培地12ミリリットル。 </li><li>シェーカーインキュベーターをオンにして、テンペを設定<html>無振とうしながら30℃までrature。 </li><li>マイクロポンプ、磁気攪拌装置、および光学密度測定をオンにしてmorbidostat操作を開始します。 注：動作中、フィードバック閾値アルゴリズムは、薬剤注入を制御するために使用されます。測定された光学濃度が予め設定された閾値を超えた場合に、薬剤注入ポンプがオンになると培地ポンプはオフになります。それ以外の場合は、薬剤注入ポンプがオフのままと培地ポンプがオンになっています。 </li><li>微生物の増殖があることを確認する時間までの時間からの電圧を監視します。 注：ため凍結融解サイクルの、E。大腸菌細胞は、毎日の成長時〜4時間の遅延を示します。 </li><li> 〜23時間後、その供給電圧をオフにすることで、マイクロポンプを停止し、培養バイアルを取り出します。培養バイアルに15％グリセロールを添加し、貯蔵のために-80℃で試料を凍結します。 </li><li>繰り返しは、2.2.5に2.2.1を繰り返します。 </li></ol></li></ol>96ウェル形式でル &quot;&gt; 3。抗生物質感受性測定注：抗生物質耐性のレベルを決定するために、96ウェル形式でプレートリーダーで成長速度測定を行います。 <ol><li> 5分間、室温で毎日凍結サンプルを解凍し、〜15ミリリットルM9培地を含む培養バイアルにセルの15μLを移します。彼らは〜0.1に到達した600nmでの光学密度まで増殖することを可能にします。 1 mlのボトルに15 mlで分割し、 表1に示すように、所望の薬物濃度を得るために、この段階でTMPの薬剤を加えます。 </li><li>ステップ3.1からのサンプルの200μLを取り、プレートリーダーで各ウェルに配置し、それらを12時間5のために成長することができます。波長600nmにおける光学密度を測定中に自動的に記録されます。 注：各データ点は、三連の測定値を記録し、データの整合性を確保するために平均化されます。時間に対する光学濃度のデータはbはできEは、成長速度を得るために使用されます。 </li><li> IC 50を得るために、薬物濃度に対する成長速度データをプロットします。 IC 50は、成長率が最大増殖速度5の五十パーセントされる薬剤濃度として定義されることに留意されたいです。 </li></ol> 4.単一細胞形態とマイクロ流体デバイスにおける抗生物質感受性測定<ol><li>他の場所で15説明したように、PDMSから多層ソフトリソグラフィーを介してマイクロ流体デバイスの製造を行います。 <ol><li>制御フロー層用のフォトレジスト金型を作るために、フォトリソグラフィを使用してください。制御層のために、ベアシリコンウェーハ上に金型を製造するためにネガ型フォトレジスト（〜15μmの高さ）と、ポジ型フォトレジスト（〜8μmの高さ）を使用することに注意してください。 </li><li>それぞれ、制御層と流動層のために1：1と20：5の架橋比を有するPDMSの混合物を準備します。任意の気泡を除去するために真空下でデシケーター中に混合物を入れて、1時間。 </li><li> （TMCS）蒸気をトリメチルクロロシランするために、シリコンウエハ上に制御層レジストの金型を公開します。 PDMS混合物を鋳造することにより~6ミリメートルの厚さの制御層を作る（架橋比5：1）制御層フォトレジスト型のシリコンウェハ上に。オーブン中で1時間、80℃で40分間、制御層を焼きます。 </li><li> （：1、スピン速度60秒間3,000rpmで架橋比20で）流れ層のフォトレジストモールド上のPDMS混合物をスピンコートすることにより流動層を作ります。オーブン中で1時間、80℃で30分間流動層を焼きます。 </li><li>希望のチップサイズに制御層（一般的に3センチメートル×2センチ）を切断し、手動制御層を剥離するカミソリの刃を使用してください。流動層の上に制御層を配置し、実体顕微鏡下でそれらを揃えます。オーブンで一晩中80℃で整列アセンブリを治します。その後、残余のTMCSを溶解するために5時間プラスチック容器に脱イオン水250ml中にアセンブリを浸漬。 </li><li>その後、20gの針を用いて入口穴パンチブランクPDMS層でコーティングされたガラススライドに全体のエラストマーを結合：で40秒間空気プラズマ処理によって（PDMS比5架橋30秒間、1の回転速度を2,000rpmで） 1.2トルの空気圧。 </li><li> PDMSからの細胞毒性効果を減少させるために80℃で36時間、大規模なベーキングを行います。この焼成工程は、細菌細胞への毒性を最小限にするために重要であることに注意してください。 </li></ol></li><li>チップ生育実験について<ol><li>マイクロ流体デバイスにロードする前に、1時間1時間とM9培地を脱イオン水で洗い流します。 </li><li> 5分間、室温で毎日凍結サンプルを解凍し、新鮮なM9培地で新しい試験管に接種します。 37℃で一晩振とう培養器内のサンプルを配置します。 </li><li> M9培地で微生物サンプル10倍に希釈し、5 psiで微生物サンプル容器を加圧することにより、マイクロ流体デバイスにロードします。そして、TMP薬をロード5 psiで薬剤培地容器を加圧することにより、チップへの媒体。 15分間のマイクロ流体チップ上の2つのチャンバ間のマイクロメカニカルバルブを作動させることにより、微生物と薬物との間の混合を実行します。 </li><li>倒立顕微鏡に搭載された顕微鏡インキュベーター内でマイクロ流体チップを置きます。成長チャンバー内の倒立顕微鏡に搭載されたCCDカメラで8時間毎に時間の細胞画像を取得します。 </li><li>細胞画像データ18上の閾値アルゴリズムを介して細胞数を計算するために、カスタムプログラムのスクリプトを使用します。セル番号データが3マイクロ流体チャンバーの上に一般的に平均化されることに注意してください。 </li></ol></li></ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+a+model+of+two+competitors+in+a+chemostat+with+an+external+inhibitor.">Analysis of a model of two competitors in a chemostat with an external inhibitor.</a> SIAM J. Applied Math. , 528-540 (1992).</li><li>Fu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maximizing+biomass+productivity+and+cell+density+of+Chlorella+vulgaris+by+using+light-emitting+diode-based+photobioreactor.">Maximizing biomass productivity and cell density of Chlorella vulgaris by using light-emitting diode-based photobioreactor.</a> J. Biotech. 161, 242-249 (2012).</li><li>Peabody, V. G. L., Winkler, J., Kao, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+developing+tolerance+to+toxic+chemicals+in+microbial+systems+and+perspectives+on+moving+the+field+forward+and+into+the+industrial+setting.">Tools for developing tolerance to toxic chemicals in microbial systems and perspectives on moving the field forward and into the industrial setting.</a> Curr Opin in Chem Eng. 6, 9-17 (2014).</li></ol>

The authors have nothing to disclose.

低コストの構成要素からの低フットプリントmorbidostat装置が示されています。デバイスが登録された薬剤耐性レベルの増加は、以前の報告5のものと一致しています。薬剤耐性の進化の研究のために設計され、デバイスは、他の多くの実験に潜在的に適用可能です。まず、薬剤誘発性の変異の包括的なデータベースは、臨床的に関連する抗生物質の大規模なセットのために確立するこ?...

最初の抗生物質ペニシリンの導入以来、微生物の抗生物質耐性は、世界的な健康問題1へと発展しています。抗生物質耐性の獲得が遡及的in vivoで研究することができるが、これらの実験の条件は、多くの場合、全体の進化2を通じて制御されていません。あるいは、適応実験室進化は抗生物質3から、環境ストレスまたは選択圧下微生物種の分子進化を明らかにすることができます。最近、抗生物質耐性の多くのよく制御された進化実験は、抗生物質耐性の出現を明らかにしています。たとえば、オースティンのグループが適切に設計されたマイクロ流体区画環境4の急速な出現を示しました。最近開発されたmorbidostatは、薬剤選択圧5,6の下で体系的突然変異を誘発します。 morbidostat、微生物セレク連続してほぼ一定の人口を維持するために抗生物質濃度を調整ションデバイスは、微生物学7,8で使用される変動試験から大きな進歩です。変動試験では、抗生物質を高濃度で注入し、生存変異体をスクリーニングし、計数します。その代わりに、morbidostat中の微生物は、常に挑戦し、複数の変異を獲得しています。 morbidostatはケモスタット、微生物集団9を希釈しながら継続的に栄養を供給することにより一定の人口を維持し、1950年にNovickとSzliardによって発明された培養装置と同様に動作します。導入以来、ケモスタットが進められ、改善されました。現在のマイクロ流体ケモスタットは、ナノリットルおよび単一セルの容量に達しています。しかしながら、これらのデバイスは、多くの変異事象10,11を有する大きな細胞集団を必要とする適応進化の実験には適していません。最近、ミニ〜10ミリリットルの作業容量でケモスタットもリットル規模のバイオリアクターおよびマイクロ流体ケモスタット12,13の間のギャップを埋めるために開発されてきました。 ここでは、抗生物質耐性の研究のための低コスト、自動化されたmorbidostatの設計および使用を提示します。提案されたモジュールは、最小限のハードウェア要件を有する微生物実験室でシェーカーインキュベーター内で使用することができます。オープンソースのファームウェアも簡単に、このような代謝工学3として適応進化の特定の用途に合わせて調整されます。最後に、morbidostatは、抗生物質感受性試験14用の多重マイクロ流体プラットフォームに統合されています。

<table><tbody><tr><td>Environmental Shaker Incubator</td><td>BioSan</td><td>ES-20</td><td></td></tr><tr><td>Arduino Leonardo board</td><td>Arduino</td><td>Leonardo</td><td></td></tr><tr><td>680 Ohm Carbon Resistor</td><td>Digikey</td><td></td><td>Bias resistor for LED</td></tr><tr><td>100k Ohm Carbon resistor</td><td>Digikey</td><td></td><td>Bias resistor for phototransistor</td></tr><tr><td>940 nm light emitting diode</td><td>Bright LED Electronic</td><td>BIR-BM13E4G-2</td><td>Optical density measurement</td></tr><tr><td>940 nm phototransistor</td><td>Kodenshi&amp;nbsp;</td><td>ST-2L2B</td><td>Optical density measurement</td></tr><tr><td>Darlington pair IC Toshiba</td><td>Mouser</td><td>ULN2803APG</td><td>this IC drives micropumps and magnetic stirring unit</td></tr><tr><td>5 V DC brushless fan</td><td>ADDA</td><td>AD0405LX-G70</td><td>spec: 5 V supply voltage and 80&amp;nbsp;mA; available www.jameco.com</td></tr><tr><td>Piezoelectric micropump</td><td>CurieJet</td><td>PS15I-FT-5L</td><td>Pressure &gt; 3&amp;nbsp;kPa; Flow rate &gt;5 ml/min</td></tr><tr><td>Tygon 3350 Tuning</td><td>Saint Gobain</td><td>ABW00001</td><td>ID: 1/32&quot; OD: 3/32&quot; L:50&#039;&amp;nbsp;</td></tr><tr><td>Magnetic Stir bar</td><td>COWIE</td><td></td><td>tapered shape dim: 10&amp;nbsp;mm x 4 mm</td></tr><tr><td>Glass scintillation 20 ml vial</td><td>DGS</td><td></td><td>Pyrex glass 28 mm (diameter) x 61 mm (height)</td></tr><tr><td>Culture vial holder</td><td></td><td></td><td>Custom made from Polyformaldehyde&amp;nbsp;</td></tr><tr><td>Silicone</td><td>Dow Corning</td><td>Sylgald 184</td><td>used to seal the glass vial</td></tr><tr><td>Medium bottle</td><td>VWR</td><td>66022-065</td><td></td></tr><tr><td>Difco M9 minimal salt 5x</td><td>BD</td><td></td><td>Medium</td></tr><tr><td>Cadamino Acid</td><td>BD</td><td></td><td>Medium</td></tr><tr><td>glucose</td><td>Sigma</td><td></td><td></td></tr><tr><td>Agar Bateriological</td><td>Oxoid</td><td></td><td>for agar plate</td></tr><tr><td>Luria Bertani medium</td><td></td><td></td><td></td></tr><tr><td>Inverted microscope</td><td>Leica Microsystems</td><td>Leica DMI-LED</td><td>used for microfluidic measurement Use 40X objective NA = 0.55</td></tr><tr><td>Microscope Incubator</td><td>Live Cell Instrument</td><td>CU-109</td><td>used for microfluidic measurement</td></tr><tr><td>Solenoidal valves</td><td>Pneumadyne</td><td>S10MM-31-12-3</td><td>Normally open 1.3 Watt 12 Vdc</td></tr><tr><td>USB interface card</td><td>Hobby Engineering</td><td>USBIO24-R Digital I/O Module&amp;nbsp;</td><td>for microfluidics measurement</td></tr><tr><td>Air compressor</td><td>Rocker Scientific</td><td>ROCKER 440</td><td>Pressure source for microfluidcs; Max. Pressure 80 psi</td></tr><tr><td>Male luer-lock fittings to 1/8&quot; barb</td><td>ValuePlastics.com</td><td>MTLL230-1</td><td>used for microfluidic control</td></tr><tr><td>1/8&quot; barb to 10-32 threaded port</td><td>ValuePlastics.com</td><td>B-1</td><td>used for microfluidic control</td></tr><tr><td>Female luer-lock fittings to 10-32 threaded port</td><td>ValuePlastics.com</td><td>KFTL-1</td><td>used for microfluidic control</td></tr><tr><td>NPN darlington transistor 500 mA, 40&amp;nbsp;V (2N6427)</td><td>DigiKey.com</td><td>2N6427GOS-ND</td><td>used for microfluidic control</td></tr><tr><td>10 kOhm, carbon film resistor, 0.25 W</td><td>DigiKey.com</td><td>P10KBACT-ND</td><td>used for microfluidic control</td></tr><tr><td>Tantalum capacitor, 10&amp;nbsp;&amp;mu;F, 25 V, 10%</td><td>DigiKey.com</td><td>478-1841-ND</td><td>used for microfluidic control</td></tr><tr><td>Andor CCD camera</td><td>Andor</td><td>Zyla 4.2 Plus SCMOS</td><td>used for microfluidic on chip imaging</td></tr><tr><td>ELISA plate reader</td><td></td><td></td><td></td></tr><tr><td>two component Silicone&amp;nbsp;</td><td>Momentive</td><td>RTV 615</td><td>used for microfluidic chip fabrication</td></tr><tr><td>SU-8 photoresist</td><td>Micrchem</td><td>SU8 2015</td><td>used for microfluidic chip fabrication</td></tr><tr><td>AZ4620 photoresist</td><td>Clariant</td><td>AZ 4620</td><td>used for microfluidic chip fabrication</td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC 32G</td><td>used for microfluidic chip fabrication</td></tr><tr><td>20 Gauge Syringe Needle</td><td>BD</td><td></td><td>used for microfluidic chip fabrication</td></tr><tr><td>Labcycler</td><td>Sensoquest</td><td>Labcycler</td><td>PCR</td></tr><tr><td>DNA polymerase</td><td>Toyobo</td><td>KDO Plus</td><td>PCR amplification</td></tr><tr><td>Trimethoprim</td><td>Sigma</td><td></td><td></td></tr><tr><td>Plate reader</td><td>Biotek</td><td>Synergy H1 hybrid&amp;nbsp;</td><td>antibiotic resistane measurement</td></tr></tbody></table>

design and use of a low cost, automated morbidostat for adaptive evolution of bacteria under antibiotic drug selection

私たちは、抗生物質耐性の進化の経路を特徴付けるための低コスト、設定morbidostatについて説明します。 morbidostatは、継続的に細菌の増殖を監視し、動的にそれらが薬剤耐性を獲得するように進化として常に細菌に挑戦する薬物濃度を調整した細菌培養装置です。デバイスは、〜10ミリリットルの作業容量を備え、完全に自動化し、光学密度測定、中および薬物送達のためのマイクロポンプが装備されています。プラットフォームを検証するために、我々はMG 1655 大腸菌でトリメトプリム耐性の段階的買収を測定し、細胞の形態や抗生物質感受性を調査するために多重化されたマイクロ流体プラットフォームとデバイスを統合しました。アプローチは、抗生物質耐性の実験室での研究にスケールアップ、および代謝工学および他の細菌培養実験における歪みの改善のために進化を適応するために拡張することができます。

上記のmorbidostatは、図1に図式化されている。実験的進化、抗生物質感受性試験および細胞形態のチェックなどの一般的なmorbidostat操作を、Eに検証されましたトリメトプリム（TMP）にさらさコリ MG1655文化、一般的に使用される抗生物質5,6。 TMPは、薬物耐性に非常に特徴的な段階的な増加を誘導し、そして突然変異はジヒドロ葉酸還元?...

Watch this Scientific Journal Video about Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection at JoVE.com

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

設計と低コストの使用、自動Morbidostat細菌の適応進化のための抗生物質選択下

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

design-use-low-cost-automated-morbidostat-for-adaptive-evolution

Universidad San Sebastian

Research

JoVE Journal

Genetics

9.6K ビュー.  National Tsing Hua University. We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

ビデオ: Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) refers to the experimental situation in which evolution is observed using living organisms under controlled conditions and stressors; organisms are thereby artificially forced to make evolutionary changes. Microorganisms are subject to a variety of stressors in the environment and are capable of regulating certain stress-inducible proteins to increase their chances of survival. Naturally occurring spontaneous mutations bring about changes in a microorganism's genome that affect its chances of survival. Long-term exposure to chemostat culture provokes an accumulation of spontaneous mutations and renders the most adaptable strain dominant. Compared to the colony transfer and serial transfer methods, chemostat culture entails the highest number of cell divisions and, therefore, the highest number of diverse populations. Although chemostat culture for ALE requires more complicated culture devices, it is less labor intensive once the operation begins. Comparative genomic and transcriptome analyses of the adapted strain provide evolutionary clues as to how the stressors contribute to mutations that overcome the stress. The goal of the current paper is to bring about accelerated evolution of microorganisms under controlled laboratory conditions.

Here, we present a protocol to obtain adaptive laboratory evolution of microorganisms under conditions using chemostat culture. Also, genomic analysis of the evolved strain is discussed.

ケモスタットを用いた微生物の適応研究所進化のための手順

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

遺伝学

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

抗生物質耐性の進化のマルチコピー プラスミドのロールのテスト

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring fitness phenotypes and improving our understanding of how genotypes are shaped by selection. Extensive recent efforts to develop and apply niche continuous culture devices have revealed the benefits of conducting new forms of cell culture control. This includes defining custom selection pressures and increasing throughput for studies ranging from long-term experimental evolution to genome-wide library selections and synthetic gene circuit characterization. The eVOLVER platform was recently developed to meet this growing demand: a continuous culture platform with a high degree of scalability, flexibility, and automation. eVOLVER provides a single standardizing platform that can be (re)-configured and scaled with minimal effort to perform many different types of high-throughput or multi-dimensional growth selection experiments. Here, a protocol is presented to provide users of the eVOLVER framework a description for configuring the system to conduct a custom, large-scale continuous growth experiment. Specifically, the protocol guides users on how to program the system to multiplex two selection pressures &#8212; temperature and osmolarity &#8212; across many eVOLVER vials in order to quantify fitness landscapes of Saccharomyces cerevisiae mutants at fine resolution. We show how the device can be configured both programmatically, through its open-source web-based software, and physically, by arranging fluidic and hardware layouts. The process of physically setting up the device, programming the culture routine, monitoring and interacting with the experiment in real-time over the internet, sampling vials for subsequent offline analysis, and post experiment data analysis are detailed. This should serve as a starting point for researchers across diverse disciplines to apply eVOLVER in the design of their own complex and high-throughput cell growth experiments to study and manipulate biological systems.

The eVOLVER framework enables high-throughput continuous microbial culture with high resolution and dynamic control over experimental parameters. This protocol demonstrates how to apply the system to conduct a complex fitness experiment, guiding users on programming automated control over many individual cultures, measuring, collecting, and interacting with experimental data in real-time.

EVOLVER を用いた自動、高スループット、連続細胞増殖実験の設計

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring ...

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

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

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

実験的進化論におけるプラスミド媒介抗生物質耐性の定量化

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

Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of Escherichia coli as they have adapted to a simple laboratory environment for more than 35 years and 77,000 bacterial generations. The setup and procedures used in the LTEE epitomize reliable and reproducible methods for studying microbial evolution. In this protocol, we first describe how the LTEE populations are transferred to fresh medium and cultured each day. Then, we describe how the LTEE populations are regularly checked for possible signs of contamination and archived to provide a permanent frozen "fossil record" for later study. Multiple safeguards included in these procedures are designed to prevent contamination, detect various problems when they occur, and recover from disruptions without appreciably setting back the progress of the experiment. One way that the overall tempo and character of evolutionary changes are monitored in the LTEE is by measuring the competitive fitness of populations and strains from the experiment. We describe how co-culture competition assays are conducted and provide both a spreadsheet and an R package (fitnessR) for calculating relative fitness from the results. Over the course of the LTEE, the behaviors of some populations have changed in interesting ways, and new technologies like whole-genome sequencing have provided additional avenues for investigating how the populations have evolved. We end by discussing how the original LTEE procedures have been updated to accommodate or take advantage of these changes. This protocol will be useful for researchers who use the LTEE as a model system for studying connections between evolution and genetics, molecular biology, systems biology, and ecology. More broadly, the LTEE provides a tried-and-true template for those who are beginning their own evolution experiments with new microbes, environments, and questions.

Daily Transfers, Archiving Populations, and Measuring Fitness in the Long-Term Evolution Experiment with Escherichia coli

This protocol describes how to maintain the Escherichia coli Long-Term Evolution Experiment (LTEE) by performing its daily transfers and periodic freeze-downs and how to conduct competition assays to measure fitness improvements in evolved bacteria. These procedures can serve as a template for researchers starting their own microbial evolution experiments.

大腸菌を用いた長期進化実験における毎日の移動、集団のアーカイブ、および適応度の測定

The Long-Term Evolution Experiment (LTEE) has followed twelve populations of Escherichia coli as they have adapted to a simple laboratory environment for ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

内タンパク質の定方向進化のための変異導入と機能選択プロトコル E.大腸菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

生物学

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Cell growth rate is a regulated process and a primary determinant of cell physiology. Continuous culturing using chemostats enables extrinsic control of cell growth rate by nutrient limitation facilitating the study of molecular networks that control cell growth and how those networks evolve to optimize cell growth.

微生物システム生物学のケモスタットの使用

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

多重ケモスタットアレイの設計および使用

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

結核の宿主向け治療の前臨床試験に使用する自動培養システム

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...

免疫・感染学

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.

Bacteria may accumulate either detrimental or beneficial mutations during their lifetime. In a population of cells individuals that have accumulated beneficial mutations may rapidly outcompete their fellows. Here we present a simple procedure to visualize intraspecies competition in a bacterial cell population over time using fluorescently labeled individuals.&#160;

設計と低コストの使用、自動Morbidostat細菌の適応進化のための抗生物質選択下

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ケモスタットを用いた微生物の適応研究所進化のための手順

抗生物質耐性の進化のマルチコピープラスミドのロールのテスト

EVOLVER を用いた自動、高スループット、連続細胞増殖実験の設計

実験的進化論におけるプラスミド媒介抗生物質耐性の定量化

大腸菌を用いた長期進化実験における毎日の移動、集団のアーカイブ、および適応度の測定

内タンパク質の定方向進化のための変異導入と機能選択プロトコル E.大腸菌

微生物システム生物学のケモスタットの使用

多重ケモスタットアレイの設計および使用

結核の宿主向け治療の前臨床試験に使用する自動培養システム

蛍光標識株の共同栽培による細菌細胞集団における種内競争のモニタリング

設計と低コストの使用、自動Morbidostat細菌の適応進化のための抗生物質選択下

要約

さらに動画を探す

この動画の章

ケモスタットを用いた微生物の適応研究所進化のための手順

抗生物質耐性の進化のマルチコピー プラスミドのロールのテスト

EVOLVER を用いた自動、高スループット、連続細胞増殖実験の設計

実験的進化論におけるプラスミド媒介抗生物質耐性の定量化

大腸菌を用いた長期進化実験における毎日の移動、集団のアーカイブ、および適応度の測定

内タンパク質の定方向進化のための変異導入と機能選択プロトコル E.大腸菌

微生物システム生物学のケモスタットの使用

多重ケモスタットアレイの設計および使用

結核の宿主向け治療の前臨床試験に使用する自動培養システム

蛍光標識株の共同栽培による細菌細胞集団における種内競争のモニタリング

抗生物質耐性の進化のマルチコピープラスミドのロールのテスト