数字を再印刷するスプリンガーとエルゼビアの許可が大幅に認められている。

E.の単離大腸菌共同形質転換体<ol><li>適切なE.のエレクトロコンピテント細胞を準備大腸菌株は形質転換される。選択した菌株の単一コロニーを1mlのLB培地（トリプトン、酵母エキス、10時のNaCl、5、および10gのそれぞれ/ L）に移し、180rpmでの条件を振盪しながら37℃でインキュベートする。新鮮なLB培地25ml中で500し、37℃で培養物をインキュベートする：前培養1希釈する。 </li><li>対数期（OD 0.6）で20分間、5000×gで）で細胞懸濁液を遠心分離し、元の培養体積の半分に10％（v / v）の氷冷滅菌グリセロール、水にペレットを再懸濁する。たびに再懸濁体積を半分に、この手順を4回繰り返します。最後に、グリセロール/水にペレットを再懸濁し、50μlのアリコート中の細胞懸濁物を分割する。 6ヶ月までのCまで-80℃でアリコートに保管してください。 </li><li> 0.5 mMのEDTAを添加し、滅菌水で所望のプラスミドに溶解する。 IC上に解凍電子エレクトロコンピテント細胞のアリコートとベクトルの適切な量（2.5〜NG）と混合。エレクトロポレーションに適した0.1cmキュベットに混合物を分配し、1.8 kVのを適用します。 </li><li>直ちに（w / vグルコース0.2％、10のMgCl 2、2.5mMのKClを補充したLB培地）を、振とうしながら1時間インキュベートSOC培地1ml中にエレクトロポレーションした細胞を転送し、最終的に含むペトリ皿に100μlのアリコートを転送する適切な抗生物質を含むLB寒天。 37℃でO / Nインキュベートします。 </li><li>ペトリ皿上で単一のコロニーをストリーキングによって形質転換体を精製する。 </li><li>一次形質転換体のエレクトロコンピテント細胞を準備し、繰り返しをさらにプラスミドで形質転換するために1.1から1.5のステップ。 </li><li>共同形質転換体のグリセロールストックを準備します。適切な抗生物質を添加したLBで単一コロニーを移し、振とうを37℃でインキュベートし、対数期（0.6 OD）遠心分離機20分間5000×gで細胞懸濁物で。 R培地を15％（v / v）のグリセロールを含むLB-抗生物質ペレットをesuspend。一定分量で分注し、で保存して - 80°C。 </li></ol> 2. Eのαおよびεサブユニットの共発現大腸菌のDNAポリメラーゼIII <ol><li>無菌ループで適切なストレイングリセロールストックを少量転送（ 例えば、TOP10 / PGOOD-ε243及びTOP10 / PGOOD-ε243-θ/pBADα1160、 図1を参照）LB培地と抗生物質を含むペトリ皿に。ペトリ皿に細胞懸濁液の乾燥をしましょう​​、とストリークは、細胞は、液滴。 37℃CO / Nでインキュベートする。 </li><li>無菌の爪楊枝、LB-抗生物質培地1mlに単一コロニーを使用して転送、。 37℃で8時間インキュベートする。新鮮な培地に500と15時間30℃でインキュベート：前培養1希釈する。 </li><li> IPTG、アラビノース、またはアラビノースおよびIPTGを追加し、1 mMの各。 2.5時間30℃でインキュベートする。細胞を回収し、-20℃でのペレットを保存する。 </li><li>溶解緩衝液（50mMトリス-HCl、50mMのNaCl、1mMのEDTA、2.5mMのジチオスレイトール、1mMのフェニルメチルスルホニルフルオリド（PMSF）、pH8）中で再懸濁し、氷上に解凍ペレット。静かに冷たいガラスポッターと細胞懸濁物を均質化する。 </li><li> 15秒の冷却間隔（4サイクル）、続いて15秒間15 W、で超音波処理。 </li><li>可溶性画分を回収し、4℃で、20分間、10,000×gでの総タンパク質抽出物を集める。 </li><li> SDS-PAGE（12.5％アクリルアミド）各可溶性タンパク質抽出物のアリコートにより分析する。この目的のために、H 2 O60μlのローディングバッファー（250mMのトリス-HClを20μlを含むエッペンドルフチューブにそれぞれの可溶性タンパク質抽出物20μLを移す6.8、10 mMのβメルカプトエタノール、10％（w / v）のSDS、50％（v / v）グリセロール、0.25％ブロモフェノールブルー（w / vの））と、5分間沸騰させる。ロード各サンプルの18μlの約1.5時間、140 Vで電気泳動を行う。 </li></ol> 3. Deinococのαサブユニットの同時発現デュランスDNAポリメラーゼIIIおよびεEのサブユニットCUS 大腸菌のDNAポリメラーゼIII <ol><li> Eの前培養を準備LB-アンピシリンテトラサイクリン培地の1ミリリットル中に大腸菌株TOP10 /のpBAD-αDR / PGOOD-ε243し、37℃で8時間インキュベートする。新鮮な培地中で千をし、37℃でO / Nインキュベート：1希釈する。 </li><li>アラビノース及びIPTG、1mMのそれぞれ3時間誘導した後、3時間37℃でインキュベート、新鮮な培地200mlに100：1に希釈する。細胞を採取し、-20℃でペレットを保存する。 </li><li> 50mMトリス - 塩酸pHが8、150mMのNaCl、1mMのEDTA、1mMのPMSFでペレットを再懸濁する。ホモジナイズし、2.4と2.5で説明したように、細胞を破壊。 </li><li>すぐに細胞が20分間万×gで溶解物遠心する。ペレットを捨てる。ペーパーフィルターの3層を装備したブフナー漏斗で上清をフィルタリングします。穏やかな真空を適用します。過剰な泡立ちを避けるために、濾過の間、氷上で真空三角フラスコに保つ。 </li><li>ブラッドフォード14に記載のタンパク質濃度を決定します。 </li></ol> 4.ゲルろ過クロマトグラフィー<ol><li> 50mMトリス-HCl、150mMのNaCl、1mMのEDTA、pHをカラムに負荷8の可溶性タンパク質抽出物を有する水ジャケット付き16x70（200）ゲルろ過カラムを平衡化する。最適な解像度の場合は、1ミリリットルサンプルループを使用しています。 /分0.6ミリリットルでクロマトグラフィーを行います。全体で4℃でカラム温度を保つ。 </li><li> 0.9ミリリットル画分を回収し、SDS-PAGEによってそれらを分析する。ローディングバッファーのH 2 Oおよび20μlの（250mMのトリス-HCl pH6.8の、10mMのβメルカプトエタノール、（v）のSDS、50％（重量/ 10％の60μLを含むエッペンドルフチューブに移し、各関連する画分を20μl v / v）グリセロール、0.25％（w / v）のブロモフェノールブルー）で5分間沸騰。ロード各サンプルの18μlの約1.5時間、140 Vで電気泳動を行う。 </li><li> 3&#39;-5 &#39;エキソヌクレアーゼと96我々の各画分のDNAポリメラーゼ活性を決定するマイクロプレートLL。基板15としてチミジン5&#39;-一リン酸p-ニトロフェニルエステル（p個の NP-TMP）を使用して、エキソヌクレアーゼ活性をアッセイする。 PPX（ピロホスファターゼ、プリンヌクレオシドホスホリラーゼ、キサンチンオキシダーゼ）、酵素結合アッセイ16を使用して、DNAポリメラーゼ活性を推定し、2-（4-ヨードフェニル）-3-（4-ニトロフェニル）-5-フェニル-2H-テトラゾリウムクロリド（INT）電子受容体16として。 </li></ol>集団E.の共発現する野生型εサブユニットの5変異解析大腸菌 DNAポリメラーゼIIIおよび変異原εD12Aバリアント<ol><li> Eの単一コロニーを転送LB-抗生物質培地1mlにεをpBAD-とPGOOD1-εD12A11ベクターを含む大腸菌 TOP10。 37℃でO / Nインキュベートします。 </li><li> 、新鮮な培地を含む3フラスコ（10ミリリットル）で250適切な誘導物質（アラビノース、IPTG、またはアラビノース及びIPTG、1mMの各）を追加し、37℃でインキュベート：前培養1に希釈8時間。パラレル非誘導培養物中に準備します。 </li><li> 1ミリリットルのアリコートを収集し、その後1希薄：新鮮な培地（10ミリリットル）を含む新しいフラスコ中に500を適切な誘導物質を補給したりしない。 37℃でO / Nインキュベートします。 </li><li>繰り返します5.2と5.3ステップと1ミリリットルのアリコートを収集します。 </li><li>世代数は、各培養物で発生したかどうかを判別します。 LBプレート、接種材料と文化の適切な連続希釈液100μlに移す。 37℃でO / Nインキュベートします。 （Cを記録）の成長の終わりにLBプレート上のコロニーをカウントし、接種物中に存在する細胞の数の対数を計算する（ ログI）と文化。世代数 ​​を決定するには、式を使用します。（Cログ- 私のログを）/0.301。 </li><li> 20分間5000×gで遠心分離し、50mMのTris-HCl pH7.6で、50mMのNaCl 1 mlに細胞を再懸濁。細胞を透過するために、20秒間クロロホルムと渦の2〜3滴を追加します。 </li><li> β-を決定基板としてのpニトロフェニルβ-D-グルコピラノシド（PNPGluc）を用いて96ウェルマイクロプレートの各アリコートのグルコシダーゼ活性、。各ウェルに透過性細胞を100μlおよび100μlの基質（16ミリグラム/ H 2 O中のmlストック溶液）を加える。ウェル内の気泡を避けるように注意してください。マイクロプレートリーダーおよび適切なフィルターを使用して、420nmで吸光度を読み取る。 </li></ol>

<ol>
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Bacteriol. 181 (9), 2693-2695 (1999).</li><li>Perrino, F. W., Harvey, S., McNeill, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+functional+domains+of+the+&#949;+subunit+of+DNA+polymerase+III.">Two functional domains of the &#949; subunit of DNA polymerase III.</a> Biochemistry. 38 (48), 16001-16009 (1999).</li><li>Kim, D. R., McHenry, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+assembly+of+overproduced+DNA+polymerase+III.+Overproduction,+purification,+and+characterization+of+the+&#945;,+&#945;-&#949;,+and+&#945;-&#949;-&#952;+subunits.">In vivo assembly of overproduced DNA polymerase III. Overproduction, purification, and characterization of the &#945;, &#945;-&#949;, and &#945;-&#949;-&#952; subunits.</a> J. Biol. Chem. 271 (34), 20681-20689 (1996).</li><li>Maul, R. W., Ponticelli, S. K. S., Duzen, J. M., Sutton, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+binding+of+Escherichia+coli+DNA+polymerase+to+the+&#946;-sliding+clamp.">Differential binding of Escherichia coli DNA polymerase to the &#946;-sliding clamp.</a> Mol. Microbiol. 65 (3), 811-827 (2007).</li><li>Greener, A., Callahan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XL1-red:+highly+efficient+random+mutagenesis+strain.">XL1-red: highly efficient random mutagenesis strain.</a> Strategies. 7, 32-34 (1994).</li><li>Chanda, P. K., Edris, W. A., Kennedy, J. 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The authors declare that they have no competing financial interests.

タンパク質は、その三次構造の立体配座25の限られた数に制限されていない領域が特徴、本質的に無秩序であることができる。これらの無秩序のタンパク質は、通常、25を凝集する傾向があり、それらの単離および特徴付けは困難なタスクを表すこと。 Eのεサブユニット大腸菌 DNAポリメラーゼIIIの機能つの異なるドメイン、すなわち26,27、は、i）N-スタ?...

過剰発現技術の導入は、低コピー数の酵素の生化学的研究および薬理学的に活性なタンパク質（ 例えば、インスリン）の工業的製造のいずれかをブースト。これらの技術の出現以来、重要な進歩は、組み換えタンパク質の収量と品質を高めるために達成された。さらに、原核生物および真核1,2 3過剰発現システムは、タンパク質バイオテクノロジー、 すなわち、大腸菌の「作業馬」に有用な選択肢を提供し、長年にわたって開発された。 E.の代替プラットフォーム特に、可用性大腸菌は、翻訳後修飾を有する組換えペプチドまたはタンパク質の産生をもたらした。しかし、E.ことが言及されるべきである大腸菌は依然として組換えタンパク質生産のための選択の生物を表す。これは最も関連があることができ、その中のいくつかの要因によるものであると考えた：i）過剰発現システム（発現ベクターおよび菌株のかなりの数の可用性が）E.のために大腸菌 1,2。 ii）の短い世代時間を、高いバイオマス収量、E.の豊かで合成様々なメディアで大腸菌 。 ⅲ）生化学的で、その微生物の遺伝子レベルでのいずれかの容易な操作。 iv）の毒性タンパク質4の産生が可能な株の単離を、 v）が集団レベル5,6で均質誘導を搭載株の構築。また、最近E.生産に適した発現系が示された翻訳後修飾タンパク質の大腸菌が考案2を構成することができる。 現在では、タンパク質の過剰発現は、主にそのhypersynthesis適切なプラスミドにクローニングし、単一の遺伝子を用いて行うことができる単量体またはホモオリゴマータンパク質を得るために使用される。ただし、注意が最近だったEの建設に支払わヘテロオリゴマー複合体2の大腸菌タンパク質の共発現系、 インビボで 、生産に挑戦。興味深いことに、タンパク質の同時発現の初期の実験では、シアノバクテリアのリブロース-1,5-ビスリ ​​ン酸カルボキシラーゼ/オキシゲナーゼ7,8、及びHIV-1の切断型および全長型の関連性の大小サブユニットの種間組立体を取り上げ転写酵素9の逆。これらの先駆的な研究は、タンパク質の共発現は、in vitro再構成における伝統的に強力な代替手段を表していることを実証した。加えて、タンパク質の共発現E.で大腸菌は、非天然アミノ酸2を含有するタンパク質を得るために、翻訳後修飾2を有する異なるタンパク質を生成するために、及び過剰発現した膜タンパク質2の収量を増大させるために使用した。また、ツールとしてのタンパク質共発現の電位はEに付与するコリが競うタンパク質分泌におけるNCEは、アクティブな捜査2の下にある。 E.におけるタンパク質共発現の二つの主な戦略大腸菌を追求することができます：ⅰ）単一のプラスミドの使用が過剰発現されることが異なる遺伝子をホストする。 ii）は、単一セル内の複数のプラスミドの使用は、標的タンパク質を同時発現する。タンデムプロモーター/オペレーター要素を含む特定のプラスミドを共発現10のために構築されたが、最初のケースでは、プラスミドの選択の基準は、従来の単一のタンパク質の過剰発現実験のものと異ならない。この最初のアプローチは、したがって、非常に簡単です。しかし、異なるタンパク質を共発現する単一プラスミドの使用は、二つの主要な困難に直面していることが言及されるべきである：i）の共発現されたタンパク質の数を制限するホストされる遺伝子の数を有するベクトルが増加すると、分子量;複数の遺伝子は、単一プロモーターの制御下にクローニングされた場合ii）は、極性がdecreasできプロモーターから遠位の遺伝子の発現を電子。シングルE.でデュアルまたは複数のプラスミドの使用大腸菌細胞は、従って、プラスミドの適格な組み合わせに制約を課し、選択したベクターとの適合性を達成しなければならない。しかし、この第2の共発現戦略は、ベクターの分子量を含むという利点を備えており、極性が制限される。我々は最近、同時発現プラスミド11との間の遺伝子の往復を容易にするように設計されたタンパク質の共発現系を構築した。特に、我々はPGOODベクトルシリーズを構築し、関連する機能には、そのうちのは、i）p15A複製起点は、市販のベクター（ 例えば、のpBADシリーズ12）のColE1起点を含むとPGOODプラスミドの互換性を提供する。 ⅱ）テトラサイクリン耐性カセット。 ⅲ）LACの存在は調節エレメント、 すなわち、プロモーター-オペレーター（O 1）夫婦とのlacIの由来</eM&gt; q遺伝子 。適切なのpBAD-PGOODのカップルを使用して、我々はEの触媒コアを過剰発現することができました三つの異なるサブユニット、 すなわち α（5&#39;-3 &#39;ポリメラーゼ）、ε（3&#39;-5&#39;エキソヌクレアーゼ）とθ（εを安定化する）13から構成される大腸菌 DNAポリメラーゼIII、。特に、我々はαεθ複合体の共発現はE.のほかに厳密に依存することを実証したIPTGおよびアラビノースの両方の大腸菌培養培地、PGOODとのpBAD、それぞれ（ 図1A）からトリガ過剰発現。 今回の報告では、タンパク質の共発現を効率的にタンパク質複合体のサブユニットの乏しい溶解性にリンクされて困難を解決することができる方法を示しています。さらに、我々は、in vivoでのタンパク質相補性試験を行うことができ、我々は最終的にE.で調整変異頻度のタンパク質の共発現の使用について報告する方法を示しコル私は。この目的のために、私たちはそれぞれのケーススタディの関連する例を説明するために、適切なPGOOD-のpBADのカップルを使用していました。

<table border="0" cellpadding="0" cellspacing="0" width="627">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:125px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agar</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">A1296</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ampicillin</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">288306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">EDS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">G5516</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">INT</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">I8377</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">IPTG</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">I5502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">L-Arabinose</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">A3256</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MgCl2</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">M2670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">31434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PMSF</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PNP-gluc</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">N7006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pNP-TMP</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">T0251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tetracyclin</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">87128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trizma base&#160;</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tryptone</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">95039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yeast extract</td>
			<td style="width:125px;">Fluka</td>
			<td style="width:119px;">70161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acrylamide solution 30%</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0158</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ammonium persolphate</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0700</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycine</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0718</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0302</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TEMED</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0800</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0719</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cuvettes 0.1 cm</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">1652089</td>
			<td>For electroporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EQUIPMENT</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge 5415R</td>
			<td style="width:125px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge Allegra 21R</td>
			<td style="width:125px;">Beckman</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Chromatography apparatus GradiFrac</td>
			<td style="width:125px;">Pharmacia Biotech</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gene Pulser II electroporation</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microplate Reader 550</td>
			<td style="width:125px;">Biorad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MiniProtean 3 cell</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Power Supply</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sonicator 3000</td>
			<td style="width:125px;">Misonix</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

the multifaceted benefits of protein co-expression in escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

Eのεサブユニット大腸菌のDNAポリメラーゼIIIは、243個のアミノ酸からなり、残基187から243が17を削除しない限り、乏しい溶解17,18を備えています。しかし、我々は以前に、完全長のα、εおよびθサブユニットの共発現は、可溶性DNAポリメラーゼIII触媒コア（ 図1）が得られることを示している11。 （ 図1A）は、それぞれ、i）のα?...

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The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

タンパク質の共発現の多面的メリットで<em&gt;エシェリヒア·コリ</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

the-multifaceted-benefits-of-protein-co-expression-in-escherichia-coli

Research

JoVE Journal

Biology

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

11.8K ビュー.  University of Bologna. Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells. 

ビデオ: The Multifaceted Benefits of Protein Co-expression in Escherichia coli

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

の活性を測定するアッセイ大腸菌誘導性リジンDecarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

生物学

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

における真菌エンドグルカナーゼ活性のハイスループットスクリーニング大腸菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

細菌機能ネットワークとPathwaysでのマッピング<em&gt;大腸菌</em&gt;合成遺伝子配列を用いて

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

のタンパク質複合体の同定<em&gt;大腸菌</em&gt;タンデム質量分析との組み合わせでシーケンシャルペプチドアフィニティ精製を用いた

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

エレクトロ作成のための迅速なプロトコル<em&gt;エシェリヒア·コリ</em&gt;と<em&gt;コレラ菌</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

ライブの検出<em&gt;エシェリヒア·コリ</em&gt; O157：PMA-qPCRによりH7細胞

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

からのペプチドおよびタンパク質との組換えエラスチン様ポリペプチドおよびその融合の非クロマトグラフィー精製<em&gt;エシェリヒア·コリ</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

ギャップ結合細胞間コミュニケーション：毒物および毒素の有害効果を評価するための機能バイオマーカー、および天然物の健康上の利点

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.