著者の研究室での作業は、ドイツ・フォルシュングスゲミンシャフト(http://www.dfg.de によってサポートされました。CO 1139/1-1)、フォンズ・デア・ケミッシェン・インダストリー(http://www.vci.de/fonds)、ゲッティンゲン分子生物学センター(GZMB)。著者たちは、ヨルグ・シュテルケに対する有益なコメントと原稿の批判的な読み取りを認めたいと考えています。

寒天プレート、培養メディア、クライオストック、プレカルチャーの製造
<ol><li>成長培地と必要な試薬を準備します(材料および試薬の表を参照)。</li>
	<li>B.サチリス株をストリーク(例えば.BP40(rocG+ gudBCR amyE::PgudB-yfp)および BP52(rocG+ gudB+ amyE::PgudB-cfp) は、それぞれ1つと2つのアクティブなGDHを発現します)2 SPミディアガルプレートの競技実験で使用される単一コロニーを得る。プレートを一晩37°Cでインキュベートする。</li>
	<li>単一コロニーを取り、4 ml LB液体培地を含む滅菌培養管を接種する。28°Cおよび220のrpmで一晩細菌を成長させる。</li>
	<li>細胞がライスまたは胞子を避けるために28°Cで一晩培養を成長させます。</li>
	<li>標準分光光度計を用いて、波長600nm(OD600)で培養液の光学濃度を測定します。</li>
	<li>培養物が2.0のOD600 に達した場合、1.5ml反応チューブ中の無菌50%グリセロール溶液の0.75mlと培養物の0.75mlを混合する。最終OD600 は、約108 細胞/mlを含む凍結ストックを得るために1.0でなければなりません。</li>
	<li>チューブは-80°Cで保管してください。</li>
	<li>フルオロフォア遺伝子をコードする cfp および yfp で標識された各株の3つの前培養を行う。-80°Cのクライオストックから1μl細胞で前培養(4ml LB液体培地を含む無菌培養チューブ)を接種する。培養物を28°Cおよび220rpmで一晩インキュベートする。</li>
</ol>2. 細菌の共同栽培、サンプル採取、試料保存
<ol><li>100 ml の C-Glc および CE-Glc の最小限の培地 (試薬と材料の表を参照) を準備し、各培地の 20 ml を滅菌 100 ml の振るフラスコに移します。</li>
	<li>一晩栽培したプレカルチャーの0.1mlを取り、1.5mlキュベットで0.9ml LB培地で希釈し、OD600を決定する。</li>
	<li>競技実験では、1.0~1.5の間に類似のOD600 を有する異なる株の前培養を行う。</li>
	<li>混合細胞集団を得るために、適切なOD600 を有した培養前培養の細胞を、20mlC-GlcおよびCE-Glc最小培地中の0.05のOD600 に希釈し、100mlの揺れフラスコで補充した。競技実験では、2つの株を1:1の比率で混合する必要があります。</li>
	<li>フラスコから10mlのサンプルを取り、15mlのプラスチックチューブに移し、標準的なテーブル上部遠心分離機で4,000 x gで10分間遠心分離して細胞を収穫し、上清を捨てます。</li>
	<li>0.5 ml新鮮なLB培地で細胞を再懸濁し、細胞を無菌1.5 ml反応カップに移します。0.5 mlの50%無菌グリセロールを加え、厳密なボルテックスで懸濁液を混合し、さらに処理するまで-80°Cの冷凍庫に保存します。</li>
	<li>37°Cおよび220rpmで最大24時間、振るフラスコに残された細胞をインキュベートする。蛍光色素の写真の漂白を防ぐために、暗闇の中で揺れるフラスコを保ちます。</li>
	<li>7時間および24時間の成長の後に0.1 mlのさらなるサンプルを取る。サンプルの1:10希釈液(C-GlcまたはCE-Glc最小媒体)のOD600 を測定して注意してください。</li>
	<li>各培養物から適切な量の細胞を取り出し、OD600 が1.0の凍結ストックを作ります。さらに処理するまで-80°Cでサンプルを保管してください。</li>
</ol>3. 定量分析のためのサンプル処理、めっき、インキュベーション
<ol><li>すべてのサンプルを採取した後、凍結ストックを解凍し、細胞を0.9%の生理食塩水(試薬および材料の表を参照)で10-3まで希釈した。</li>
	<li>SP培地寒天プレート上の10-3 希釈液の0.1mlをプレートし、滅菌ガラスピペットを使用して細胞を分配する。</li>
	<li>単一のコロニーが出現するまで、暗闇の中で37°Cで一晩プレートをインキュベートします。</li>
</ol>4. 定量分析のための立体蛍光顕微鏡による被爆者数
<ol><li>ステレオ蛍光顕微鏡の下で生存者を数えながら、より良い方向のために4つの部分に黒いペンで寒天プレートの底を分割します。</li>
	<li>プレートを顕微鏡の下に逆さまに置き、冷たい光源を使用してコロニーを焦点にします。</li>
	<li>コロニーが焦点を合わせられたら、CFP用に適切なフィルタセットに切り替えて 、cfp標識株の生存細胞を視覚化します。ペンでコロニーにラベルを付けることによって、この株の生存者を数え、数をメモします。</li>
	<li>エタノールでラベルを取り除き、YFPフィルタセットに切り替えて 、yfpラベル付き歪みの生存細胞を可視化します。ペンでコロニーにラベルを付けることによって、再び生存者を数え、番号をメモします。</li>
</ol>5. 半定量分析のためのサンプル処理と顕微鏡
<ol><li>例示的な図では、SP寒天プレート上のステップ3.1から10-4 希釈液(約100細胞)の10μlをスポットする( 図3を参照)。</li>
	<li>単一のコロニーが出現するまで、暗闇の中で37°Cで一晩プレートをインキュベートします。</li>
	<li>顕微鏡の下に蓋をせずにペトリ皿を置き、冷たい光源を使用してスポットを焦点にします。</li>
	<li>例示の図のためのスポットの写真を撮ります。冷たい光源でコロニーの写真を撮るための適切な露出時間を選択してください。</li>
	<li>プレートを動かさずに、CFPフィルタセットに変更し、露光時間を調整して写真を撮ります。YFP フィルタセットでも同じ操作を行い、画像を保存してさらに解析を行います。</li>
</ol>6. データ分析
<ol><li>定量的データ分析には、Excel などのソフトウェアを使用します。カウントされたコロニーに基づいて、100%に設定されたコロニーの総数に対する黄色と青のコロニーの割合を計算します。</li>
	<li>計算された数値を使用して、積み上げバー図を作成します ( 図 4 および図 5を参照)。Adobe Photoshop などの画像処理プログラムを使用して、さまざまな場所から撮影した写真のマージされた画像を作成します。あるいは、自由に入手可能なソフトウェアImageJは <a href="http://rsbweb.nih.gov/ij/">、http://rsbweb.nih.gov/ij/</a> からダウンロード可能な画像処理に使用することができる。</li>
	<li>CFP フィルタ セットと YFP フィルタ セットで撮影した 1 つの場所から画像を開きます。メディアから任意のバックグラウンド蛍光を低減するためにコントラストと明るさを最適化します。</li>
	<li>いずれかの画像に移動し、すべてを選択します。図をコピーして、他の図に貼り付けます。</li>
	<li>「カラードッジ」機能を使用して画像をマージするか、チャンネルタブを使用して蛍光画像をオーバーレイします。異なる媒体および異なる時点からのコロニーの結合された写真は、液体培養中の異なる株の成長を表す。</li>
</ol>7. 具体的なヒント:色素スイッチ実験とcfpとyfpで標識された同種株の共同栽培
B.サブティリスにおける2つのフルオロフォアコード遺伝子のどちらかの発現は、適性および細菌の増殖速度に影響を与える可能性がある。したがって、栽培中の細胞集団からの1つの競合株の排除が単にフルオロフォアの悪影響によるものであることを排除するために、以下の実験を行うことを推奨する。
<ol><li>実験全体を繰り返し、逆に標識された株を共同栽培する(例えば、以前の cfp標識株は yfp 遺伝子で標識され、 その逆も同様である)。逆であるが、得られた結果は、一方の株が他の株よりも選択的な成長優位性を有するという以前の観察と同等であるべきである。</li>
	<li>実験全体を繰り返し、yfpおよびcfpでラベル付けされた同等体性株(例えばBP40(rocG+ gudBCR amyE::PgudB-yfp)およびBP41(rocG+ gudBCR amyE::PgudB-cfp))を共同栽培する。時間の経過に伴う細胞集団の組成に従うことによって、細菌の適合性に対する2つのフルオロフォアのどちらかの負の効果を推定する。</li>
</ol>

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著者らは、競合する財政的利益はないと宣言している。

細菌の競争力のある適合性を分析するためにいくつかの方法が開発されています16.多くの場合、細菌は異なる抗生物質耐性カセット17で標識された。我々のアプローチと同様に、抗生物質耐性カセットを用いた細胞の標識は、定義された成長条件下での共培養中の細菌の競争力の適合性の評価を可能にする。さらに、この方法は、染色体17上の特定の軌跡において互い...

土壌細菌は、通常、柔軟な規制ネットワークと広範な代謝能力に恵まれています。両方の特徴は、細胞が与えられた生態学的ニッチ1で利用可能である栄養素のために彼らの仲間や他の微生物と競合するために、それらの同化および同化経路を調整することを可能にする。しかし、細菌が環境に適応できない場合、他のメカニズムは種の生存を説明する可能性があります。確かに、多くの細菌が急速に増殖し、集団が高い細胞密度に達することができるように、亜集団は自発的に細胞に選択的な成長の利点を提供し、したがって、その適性を高める有益な突然変異を蓄積している可能性があります。さらに、突然変異ホットスポットおよびストレス誘発適応性変異発生は、不適合細菌2,3の進化を促進することができる。したがって、連続選択下での突然変異および増殖の蓄積は、同一属4,5内であっても、巨大な微生物多様性の起源である。自然界と同様に、細菌ゲノムの形成は、選択下の連続栽培のために実験室でも起こる。これは、基礎研究および産業界で世界的に使用されているグラム陽性細菌B.サブティリスの家畜化によって例示される。1940年代にB.サチリスをDNA損傷X線で処理し、その後、特定の成長条件6で培養した。それらの家畜化中に細菌に蓄積された突然変異は、多くの成長特性の喪失を占める、すなわちB.サブチリス実験室株168は、複雑なコロニー7,8を形成する能力を失った。
今日では、最もよく研究されたモデル細菌 大腸菌 と B.サブティリスのために、特定の科学的な質問に対処するためにゲノムを遺伝的に操作するための様々な強力なツールが利用可能です。時には、目的の遺伝子の不活性化は、重篤な成長欠陥を引き起こし、その後、標準的な成長培地9ではっきりと見える。対照的に、弱い成長欠陥を引き起こし、ひずみの適性にわずかに影響を与える突然変異はしばしば無視される。しかしながら、いずれの場合も、数世代にわたって変異株の培養および経架を長時間化すると、通常は親株2,9の表現型を回復したサプレッサー変異体の蓄積をもたらす。サプレッサー変異体の特徴付けと、親突然変異株の成長欠陥を回復した突然変異の同定は、重要かつしばしば新しい細胞プロセス10,11の解明を可能にする非常に有用なアプローチである。
我々はB.サチリス12におけるグルタミン酸恒常性の制御に興味を持っている。大腸菌と同様に、B.サチリスは、サプレッサー突然変異体の蓄積によるグルタミン酸恒常性(すなわちグルタミン酸分解2のブロック)の摂動に応答する。自発的突然変異によって獲得されたこれらのサプレッサー変異体におけるゲノム変化は、グルタミン酸恒常性9,13を迅速に回復させることが示された。したがって、細菌の家畜化中の特定の成長条件に対するB.サチリスの適応が酵素合成および進化した酵素活性においてミラーリングされ、グルタミン酸代謝12に関与していることは驚くべきことではない。家畜化過程中の成長培地における外因性グルタミン酸の欠如が、実験室株1682,14における暗号性グルタミン酸脱水素酵素(GDH)gudB CR遺伝子の出現および固定化の原動力であったことが示唆されている。この仮説は、実験室株におけるGDH活性の減少量が外因性グルタミン酸が不足しているときに選択的増殖の利点を細菌に提供するという我々の観察によって支持される2。また、B.サチリス株の培養は、GDH GudBを合成し、外因性グルタミン酸がない場合には、gudB遺伝子2を不活性化したサプレッサー変異体の蓄積をもたらす。明らかに、同化活性GDHの存在は、他の方法で使用できる内因的に産生されたグルタミン酸がアンモニウムおよび2-オキソグルタル酸に分解されるため、細胞にとって不利である(図1)。対照的に、グルタミン酸が培地によって提供される場合、高レベルGDH活性を有するB.サチリス株は、1つの機能GDHのみを合成する株に対して選択的な増殖性を有する。高レベルのGDH活性により、細菌は培地2によって提供される他の炭素源に加えて、第2の炭素源としてグルタミン酸を利用することを可能にする(図1を参照)。したがって、GDH活性は、外因性グルタミン酸の入手可能性に応じて、細菌の適性に強く影響を及ぼす。
ここでは、染色体上の単一の遺伝子座で異なる2つの B.サチリス 株間の種内競争を監視し、可視化するための非常に例示的な方法を提示する(図2)。この2株を、フルオロフォアYFPおよびCFPをコードする yfp および cfp 遺伝子で標識し、異なる栄養条件下で共栽培した。時間をかけてサンプリングし、寒天プレートに適切な希釈液をめっきすることにより、各培養物の生存者は、一般的なステレオ蛍光顕微鏡を使用して容易に監視することができます。この論文に記載されている手順は、細胞集団における有益および有害な突然変異の急速なクローン拡張および除去を、時間の経過とともに容易に行い、視覚化するのに適している。

<table><tbody><tr><td>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Roth, Germany</td><td>3746</td><td></td></tr><tr><td>Agar</td><td>Difco, USA</td><td>214010</td><td></td></tr><tr><td>Ammonium ferric citrate (CAF)</td><td>Sigma-Aldrich, Germany</td><td>9714</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&amp;nbsp;&lt;/sub&gt;</td><td>Roth, Germany</td><td>5239</td><td></td></tr><tr><td>Glucose</td><td>Applichem, Germany</td><td>A3617</td><td></td></tr><tr><td>Glycerol</td><td>Roth, Germany</td><td>4043</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;3H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Roth, Germany</td><td>6878</td><td></td></tr><tr><td>KCl</td><td>Applichem, Germany</td><td>A3582</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth, Germany</td><td>3904</td><td></td></tr><tr><td>KOH</td><td>Roth, Germany</td><td>6751</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Roth, Germany</td><td>P027</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Roth, Germany</td><td>T881</td><td></td></tr><tr><td>MnSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck Millipore, Germany</td><td>102786</td><td></td></tr><tr><td>NaCl</td><td>Roth, Germany</td><td>9265</td><td></td></tr><tr><td>Nutrient broth</td><td>Roth, Germany</td><td>X929</td><td></td></tr><tr><td>Potassium glutamate</td><td>Applichem, Germany</td><td>A3712</td><td></td></tr><tr><td>Tryptone</td><td>Roth, Germany</td><td>8952</td><td></td></tr><tr><td>Tryptophan</td><td>Applichem, Germany</td><td>A3445</td><td></td></tr><tr><td>Yeast extract</td><td>Roth, Germany</td><td>2363</td><td></td></tr><tr><td>1.5 ml Reaction tubes</td><td>Sarstedt, Germany</td><td>72,690,001</td><td></td></tr><tr><td>2.0 ml Reaction tubes</td><td>Sarstedt, Germany</td><td>72,691</td><td></td></tr><tr><td>15 ml Plastic tubes with screw cap</td><td>Sarstedt, Germany</td><td>62,554,001</td><td></td></tr><tr><td>Petri dishes</td><td>Sarstedt, Germany</td><td>82.1473</td><td></td></tr><tr><td>1.5 ml Polystyrene cuvettes</td><td>Sarstedt, Germany</td><td>67,742</td><td></td></tr><tr><td>15 ml Glass culture tubes&amp;nbsp;</td><td>Brand, Germany</td><td>7790 22</td><td></td></tr><tr><td>100 ml Shake flasks with aluminium caps</td><td>Brand, Germany</td><td>928 24</td><td></td></tr><tr><td>Sterile 10 ml glass pipettes</td><td>Brand, Germany</td><td>278 23</td><td></td></tr><tr><td>Incubator (28 and 37 &amp;deg;C)</td><td>New Brunswick</td><td>M1282-0012</td><td></td></tr><tr><td>Standard pipette set (2-20 &amp;mu;l, 10-100 &amp;mu;l, 100-1,000 &amp;mu;l)</td><td>Eppendorf, Germany</td><td>4910 000.034, 4910 000.042,</td><td></td></tr><tr><td>Table top centrifuge for 1.5 and 2 ml reaction tubes</td><td>Thermo Scientific, Heraeus Fresco 21, Germany</td><td>75002425</td><td></td></tr><tr><td>Table top centrifuge for 15 ml plastic tubes</td><td>Heraeus Biofuge Primo R, Germany</td><td>75005440</td><td></td></tr><tr><td>Standard spectrophotometer</td><td>Amersham Biosciences Ultrospec 2100 pro, Germany</td><td>80-2112-21</td><td></td></tr><tr><td>Stereofluorescence microscope&amp;nbsp;</td><td>Zeiss SteREO Lumar V12, Germany</td><td>495008-0009-000</td><td></td></tr><tr><td>Freezer (-20 and -80 &amp;deg;C)</td><td>-</td><td>-</td><td></td></tr><tr><td>Fridge (4 &amp;deg;C)</td><td>-</td><td>-</td><td></td></tr><tr><td>Autoclave</td><td>Zirbus, LTA 2x3x4, Germany</td><td>-</td><td></td></tr><tr><td>pH meter</td><td>pH-meter 766, Calimatic, Knick, Germany</td><td>766</td><td></td></tr><tr><td>Vortex</td><td>Vortex 3, IKA, Germany</td><td>3340000</td><td></td></tr><tr><td>Balance</td><td>CP2202S, Sartorius, Germany</td><td>replaced by</td><td></td></tr><tr><td>Black pen (permanent marker)</td><td>Staedler, Germany</td><td>317-9</td><td></td></tr><tr><td>Powerpoint program</td><td>Microsoft, USA</td><td>-</td><td></td></tr><tr><td>Office Excel program</td><td>Microsoft, USA</td><td>-</td><td>Program for data processing</td></tr><tr><td>Adobe Photoshop CS5</td><td>Adobe, USA</td><td>replaced by CS6, download</td><td>Computer program for image processing</td></tr><tr><td>Computer</td><td>PC or Mac</td><td>-</td><td></td></tr><tr><td>ZEN pro 2011 software for the stereofluorescence microscope</td><td>Zeiss, Germany</td><td>410135 1002 110</td><td>AxioCam MRc Rev. Obtained through Zeiss</td></tr><tr><td colspan="4">&lt;strong&gt;Specific solution recipes&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;SP Medium&lt;/strong&gt;</td></tr><tr><td>8 g Nutrient broth</td><td></td><td></td><td></td></tr><tr><td>0.25 mg MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>1 g KCl</td><td></td><td></td><td></td></tr><tr><td>15 g agar for solid SP medium</td><td></td><td></td><td>if required</td></tr><tr><td>add 1 L with H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td>1 ml CaCl&lt;sub&gt;2&lt;/sub&gt; (0.5 M), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>1 ml MnCl&lt;sub&gt;2&lt;/sub&gt; (10 mM), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>2 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;LB Medium&lt;/strong&gt;</td></tr><tr><td>10 g Tryptone</td><td></td><td></td><td></td></tr><tr><td>5 g Yeast extract</td><td></td><td></td><td></td></tr><tr><td>10 g NaCl</td><td></td><td></td><td></td></tr><tr><td>15 g agar for solid LB medium</td><td></td><td></td><td>if required</td></tr><tr><td>add 1 L with H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td colspan="4">&lt;strong&gt;C-Glc Minimal Medium&lt;/strong&gt;</td></tr><tr><td>200 ml 5 x C salts</td><td></td><td></td><td></td></tr><tr><td>10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>10 ml III&amp;rsquo; salts</td><td></td><td></td><td></td></tr><tr><td>25 ml Glucose (20%)</td><td></td><td></td><td>autoclaved for 20 min at 121 &amp;deg;C</td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;CE-Glc Minimal Medium&lt;/strong&gt;</td></tr><tr><td>200 ml 5 x C salts</td><td></td><td></td><td></td></tr><tr><td>10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>10 ml III&amp;rsquo; salts</td><td></td><td></td><td></td></tr><tr><td>20 ml Glutamate (40%)</td><td></td><td></td><td></td></tr><tr><td>25 ml Glucose (20%), autoclaved for 20 min at 121 &amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>5 x C salts&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>20 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>80 g K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;3H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>16.5 g (NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td>III&amp;rsquo; salts</td><td></td><td></td><td></td></tr><tr><td>0.232 g MnSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>12.3 g MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O, autoclave for 20 min at 121 &amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>40% Glutamate solution</td><td></td><td></td><td></td></tr><tr><td>200 g L-Glutamic acid</td><td></td><td></td><td></td></tr><tr><td>adjust the pH to 7.0 by adding approximately 80 g KOH</td><td></td><td></td><td></td></tr><tr><td>add 0.5 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td colspan="4">&lt;strong&gt;0.9% Saline (NaCl) Solution&lt;/strong&gt;</td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td>50% Glycerol solution</td><td></td><td></td><td></td></tr><tr><td>295 ml Glycerol (87%)</td><td></td><td></td><td></td></tr><tr><td>add 0.5 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td colspan="4">Bacteria (All strains are based on the &lt;em&gt;Bacillus subtilis&lt;/em&gt; strain 168)</td></tr><tr><td>&lt;em&gt;Bacillus subtilis&lt;/em&gt; BP40 (rocG&lt;sup&gt;+&lt;/sup&gt; gudB&lt;sup&gt;CR&lt;/sup&gt; amyE::PgudB-yfp)&amp;nbsp;</td><td>Laboratory strain collection</td><td></td><td></td></tr><tr><td>&lt;em&gt;Bacillus subtilis&lt;/em&gt; BP41 (rocG&lt;sup&gt;+&lt;/sup&gt; gudB&lt;sup&gt;CR&lt;/sup&gt; amyE::PgudB-cfp)&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Bacillus subtilis&lt;/em&gt; BP52 (rocG&lt;sup&gt;+&lt;/sup&gt; gudB&lt;sup&gt;+&lt;/sup&gt; amyE::PgudB-cfp)</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Bacillus subtilis&lt;/em&gt; BP156 (rocG&lt;sup&gt;+&lt;/sup&gt; gudB&lt;sup&gt;+&lt;/sup&gt; amyE::PgudB-yfp)</td><td></td><td></td><td></td></tr></tbody></table>

monitoring intraspecies competition in a bacterial cell population by cocultivation of fluorescently labelled strains

細菌などの多くの微生物は非常に急速に増殖し、集団は高い細胞密度に達する可能性があります。集団の細胞の小さい分数は、常に細胞にとって有害または有益な突然変異を蓄積している。突然変異の適性効果がサブ人口に強力な選択的成長優位性を提供するならば、この亜集団の個体は急速に競争し、さらには彼らの直近の仲間を完全に排除するかもしれない。したがって、有益な突然変異を獲得した細胞の小さな遺伝的変化および選択主導的蓄積は、細胞集団の遺伝子型の完全なシフトにつながる可能性がある。ここでは、グラム陽性モデル 細菌サブティリスの蛍光標識体の共培養によって、時間の経過とともに細菌細胞集団における有益および有害な突然変異の迅速なクローン拡張および消失を監視する手順を提示する。この方法は、細菌細胞集団における個体間の種内競争を表示する方法が容易かつ非常に例示的である。

ここで説明した方法は、フルオロフォアCFPおよびYFPをコードするcfp遺伝子およびyfp遺伝子で標識されたB.サチリス株からなる細胞集団における種内競争を可視化するために、それぞれうまく適用された。図3に示すように、この方法は、非常に例示的な方法で種内競争を視覚化するために使用することができる。小さな領域にサンプルを見つけることによって?...

Watch this Scientific Journal Video about Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains at JoVE.com

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

細菌は、その生涯の間に有害または有益な突然変異のいずれかを蓄積することができます。細胞の集団では、有益な突然変異を蓄積した個体は、急速に仲間を上回る可能性があります。ここでは、蛍光標識された個体を用いて、時間の経過とともに細菌細胞集団における種内競争を可視化する簡単な手順を提示する。

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

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

monitoring-intraspecies-competition-bacterial-cell-population

Research

JoVE Journal

Biology

8.5K ビュー.  Georg-August University. 細菌は、その生涯の間に有害または有益な突然変異のいずれかを蓄積することができます。細胞の集団では、有益な突然変異を蓄積した個体は、急速に仲間を上回る可能性があります。ここでは、蛍光標識された個体を用いて、時間の経過とともに細菌細胞集団における種内競争を可視化する簡単な手順を提示する。

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

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

微生物集団に定着表面に空間的分離の監視

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

遺伝学

Title Cell Encapsulation by Droplets

My name is Hang Moon. I'm working with the professor of condoms. He and me work with the XYJ stage to make a 3D structure of cell.Hi, I am PE and Lynn and I'm working with the cell printing project with cell encapsulation and media and gels. This is solenoid be for ejection of a cell and cell mixture of aros over cell media. The so valve is activated by two signal line.The signal line is came from a dry board here. Signal came from here. That line through the that line, that one is the function first generator.So first is generated by our programmed way. So if you want, you want to change the purse, you can change the program with the that switches line or you can control that process generator to computer. The cellular valve is open and closed by the electric signal, but the, the cell is supplied through that line.The cell and the media or agro gel is supplied through the syringe syringes. The syringes inside of the water, inside of syringe is the press light by the air. The pressurized air is supplied from a tank like here the line.So the little line and the ized air is fly through the air filter. The air filter is discriminate the dust and other kind of the dirt. That is pressure generator we call it, is generating the signal used for open the solar air bell or closed the solar air valve computer is the connected through P-I-B-U-S-V connector.So we can use the USB port by directly our computer, the laptop computer. So that one is connected to that side here. And then we can control through the control laptop here.So if we want to synchronize of the movement of the stage and opening the bell is can be controlled by the same controller as a laptop. So we can control the timing when it open, when it close, and when it moves and when it stops. And we can synchronize the motion and opening bell.That stage is the motorized stage. It is automatically automatically moved by through the death signal line and motor. So motorization is 0.1 micrometers.So it can ideally it can draw the line up to 100 millimeter line. So jet axis is moving, going up and down like that way. So it control the cap between the substrate and the solar belt.So it can control the height of ejection. That substrate is the connected to the XY stages here, X here and Y there. So it's moving around a plan XY plan.So it draws some feature demo right stages is three Xs, X, Y, and G.So the jet con jet axis or XY axis is controlled by signal here. So signal line is connected to the motion controller here. That motion controller doesn't have any manual switch at all because it cannot be controlled manually.It's controlled through the laptop computer. So laptop computer is connected. The backside of the controller, we have three xs for a printing cell XY jet.So it make three different kind of signal independently. And then motion controller is communicate through the rf 2, 3, 2 connectors is directly connected to the USV. And then USV is connected to laptop computer.So laptop controlled borrower. We use the AutoCAD program, we make a gen A path like that way bam. And then it change to the machine language like that.So machine language is transferred to the like that the simple character and numbers like that. There is each position related to the X, Y, Z stages here I choose the file, contain that kind of program and then open downloading program like that way. And then I choose from file like does exact the same, the text file.Just the text file is downloaded, choose text file and automatically download from laptop to com motion controller executive and then it going the BAM character as I mentioned before, BAM. In this flask are N IH 3G three mouse fiberblast cells. And I'm going to first aspirate the old media.This process is to passage the cells and the cells are adhered to the bottom of the flask. So after aspirating the media, I'm going to put in six RYS in which is an enzyme.Okay? So after the trypsin has been put into the flask, we need to wait three to five minutes for the cells to become detached from the bottom of the flask.Now we're ready to put in the media and passage the cells into a centrifuge tube. It's a one-to-one dilution. So I'm putting in six mil of this media right here and I'm gonna wash out the saws a little bit to make sure all of them are passaged.And now the cells are ready to be centrifuge in this tube. Now we're going to centrifuge these cells at a thousand RPM three to five minutes. Now that the cells have been spun down into a pellet, I'm going to aspirate the supernatant.Now I'm going to wash out the cells with PBS. And this is to, this is for staining the cells. The stains that we are using are esterase substrates.Okay, now we we're going to centrifuge again and spin down the cells into a palate. And I'm going to aspirate the PBS and I'm going to resin the cells. I'm in media to be counted.I'm mixing the cells so that when we take the a sample of it, the concentration will be even throughout the tube. Now I'm going to take a hundred microliters of the cells to measure the cell density. Now I'm putting in a hundred microliters of type and blue, which is a stain.We're going to wait three to five minutes in order for the stain to permeate dead cell membranes. When we count the cells, the blue ones we don't count. The cells are ready to be counted with the hemo cytometer, I'm going to transfer 50 microliters of the cell mixture to each side of the hemo cytometer.Okay, I'm going to cover it with a glass slide side. There's two sides to the hemo cytometer. I'm gonna count the first side and after counting both sides, I'm going to take the average and then multiply by two because we had a one-to-one dilution with the type in blue.So basically our cell density will be 135 times 10 to the four cells per mil. For our experiment, we will be using a cell solution with a concentration of half a million cells per mil. And so since we have 13.5 million cells, I will be resus suspending the cells in 27 mil of media.We are using two different types of stains. This is a live dead assay from molecular probes. Our first stain is called calcium am.Our second stain is called AUM homodimer. It stains dead cells. We use blue excitation to get green fluorescence and green excitation to get red fluorescence For these two stains, when we're imaging our dice solution will be prepared in PBS.So first I'm going to transfer five mil of PBS into a tube phosphate buffered saline. That's a saline solution. Now I'm going to add half a microliter of calcium am per mil.Now I'm adding 2.5 microliters of calcium am to the tube and two microliters of athe homodimer per mil of PBS. Here is the btex machine. It makes the cell and media well distributed through the whole volume.Now we load the cell 200 microliter onto the seven here. And then we put on the cap, this is a tight tightly sealed through like that way here I open the valve and then it's the pressure light air comes in and then I adjust the belt like that. And then it change the pressure.So I adjust. The pressure is five PSI. The inside of tubing pressure is five PSI.From here to here, it connected through the this inside the hole. So just we open the bell here and then the cell injected with the five PSI pressure and then starting open. And then Sona valve starting operate.And then we roll the substrate. It substrate is just a simple slide glass. And then we load the proper portion and I put some sketch tape to fix the substrate like that, right?And simply I just fix it. So we are setting whole thing the cell and ary valve and the substrate during the moving the ary working, start working. So we are set up whole thing and then open ary valve start.And then we move the stage like that, right? And then we make some pattern onto the substrate pattern onto the substrate and bam character. I'm going to immerse these droplets that we printed in this dye solution that we made earlier.And now the droplets will be incubated for 10 minutes and then imaged. First I'm going to adjust the image through the eyepiece. Our first image is just a bright field image.I'm gonna turn off the light and change the filter so that we have blue excitation, which will show green fluorescence. And the third image we'll be taking is the dead cells, which is the Thm Homodimer stain. And with green light, we will get red fluorescence.

滴タイトルセルのカプセル化

生物学

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

免疫手術でヒト胚性幹細胞の導出

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

T6SS媒介細菌競争を監視するには、Visualアッセイ

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

免疫・感染学

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

遺伝的にコードされたカルシウムのセンサを用いたアポトーシス時のミトコンドリア内カルシウム濃度の動的変更の監視

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. This method employs stable plasmids that allow each population to be differentially tagged with distinct antibiotic resistance capabilities and fluorescent proteins for selection and visual discrimination of each population, respectively. Here, we describe the preparation and coincubation of competing Vibrio fischeri strains, fluorescence microscopy imaging, and quantitative data analysis. This approach is simple, yields quick results, and can be used to determine whether one population kills or inhibits the growth of another population, and whether competition is mediated through a diffusible molecule or requires direct cell-cell contact. Because each bacterial population expresses a different fluorescent protein, the assay permits the spatial discrimination of competing populations within a mixed colony. Although the described methods are performed with the symbiotic bacterium V. fischeri using conditions optimized for this species, the protocol can be adapted for most culturable bacterial isolates.

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Bacteria encode diverse mechanisms for engaging in interbacterial competition. Here, we present a culture-based protocol for characterizing competitive interactions between bacterial isolates and how they impact the spatial structure of a mixed population.

ビブリオ・フィシェリ・アイシス間の競争的相互作用を定量化するためのコインキュベーション・アッセイ

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

単細胞間細菌相互作用の運動的可視化

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

化学的に媒介種間相互作用を検出するための共培養を使用して

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

単細胞蛍光イメージングによる細菌間競争の定量化

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...

Bacterial Co-Incubation Assay: A Fluorescence Microscopy-Based Technique to Visualize Intraspecific Bacterial Competition at the Single-Cell Level

Source: Smith, S., et al., <a href="https://app.jove.com/v/62851">Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging.</a> J. Vis. Exp. (2021).
This video demonstrates a fluorescence microscopy-based method to study bacterial competition at the single-cell level. This method provides insights into the structure and function of bacterial communities.