この研究は、自然科学からディスカバリーグラント＆カナダのエンジニアリング評議会（NSERC）によってサポートされていました。

<ol>
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J., Whitaker, N., Gonz&#225;lez-Rivera, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+and+structure+of+the+bacterial+type+IV+secretion+systems.">Mechanism and structure of the bacterial type IV secretion systems.</a> Biochim Biophys Acta. 1843 (8), 1578-1591 (2014).</li><li>Cascales, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+type+VI+secretion+toolkit.">The type VI secretion toolkit.</a> EMBO Rep. 9, 735 (2008).</li><li>Silverman, J. M., Brunet, Y. R., Cascales, E., Mougous, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas:+New+Tools+for+Genetic+Manipulations+from+Bacterial+Immunity+Systems.">CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems.</a> Annu Rev Microbiol. 69 (1), 209-228 (2015).</li><li>Dahlberg, C., Bergstrom, M., Andreasen, M., Christensen, B. B., Molin, S., Hermansson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interspecies+bacterial+conjugation+by+plasmids+from+marine+environments+visualized+by+gfp+expression.">Interspecies bacterial conjugation by plasmids from marine environments visualized by gfp expression.</a> Mol Biol Evol. 15 (4), 385-390 (1998).</li></ol>

著者は、彼らが競合する金融利害関係を持たないことを宣言します。

細菌接合プロセスは、細菌は、抗生物質耐性マーカーの普及など厳しい環境での成長のための進化上の利点を提供する遺伝子を広げることができる手段を提供します。接合性プラスミドの多くは非常に大きいので、接合プラスミド自体に標的遺伝子の突然変異によって転写装置の組み立てに関与するタンパク質上の12機能的研究は扱いにくいです。本明細書で詳細なプロトコルは、一つにはより容易に、より小さく扱い発現プラスミド（ 図1）を使用することによって、目的の標的遺伝子を評価することができる手段を提供します。我々は、Fプラスミド媒介性の結合を研究するためにFプラスミドの誘導体pOX38-TC（ 表1）を使用します 。他の接合性プラスミドはここで詳述プロトコルと適切な派生プラスミドを使用して研究することができます。概説交配アッセイは、いくつかのmodificatiとフロストや同僚、42から適応されていますアドオン。以前の研究では、pOX38-TCΔgene:: Cmを構築物の8,33,42作成は、適切な制限酵素を用いて目的の遺伝子を切断し、pOX38-TCに増幅CATカセットを挿入することによって達成されました。現在の方法では42、我々は、標的遺伝子をノックアウトし、CATカセットと交換するリコンビニアリング大腸菌株 DY330R 33,34における相同組換えを使用します。 F-T4SSはpK184遺伝子回復プラスミドを用いた転写の回収を介して接合伝達を媒介外これはpOX38-TCΔgene:: Cmは、遺伝子特異的ノックアウトのための対照として作用するように構成保有得DY330R株を可能にするという利点を有します。それは野菜室-Cas9方法論を用いてノックアウトを生成することが可能かもしれないが、43は、現時点ではこの可能性を調査していません。 プロセスは、F派生プラスミドpOX38-TC（Figurに遺伝子ノックアウトの世代で始まりますE 1）。これはDY330R細胞（ 表1）中での相同組換えを介して達成され、例えば、DY329、DY331およびDY378と同様の機能を有する他の株も使用することができます。プライマーは、最初pBAD33プラスミド32からCATカセットを増幅し、標的遺伝子（ 表2）に特異的なオーバーハング塩基を含むPCRように設計されています。 PCR産物を、次いでpOX38-TCを保有DY330R細胞にエレクトロポレーションされます。相同組換えは、CATのカセットがCm耐性を提供しながら、効果的にT4SSアセンブリおよび結合を破壊し、標的遺伝子内にインフレームで挿入されているノックアウトプラスミドpOX38-TCΔgene:: Cmとを生成します。これと同時に、標的遺伝子はpOX38-TCからPCR増幅し、小発現プラスミドに挿入し、このプロトコルでは、我々は、必要に応じてしかし1は、標的遺伝子の誘導発現とプラスミドを選んだ可能性があり、構成的発現のためにpK184を使用します。 pK184-遺伝子プラスミドは、その後、形質転換されますDY330R pOX38-TCΔgene:: Cmを細胞に、これらの細胞は、次いでpOX38-TCΔgene:: Cmは交配アッセイのためXK1200細胞への結合を介して構築転送するためのドナーとして使用されます。交配アッセイでは、ドナーとレシピエント細胞は、それぞれXK1200 pOX38-TCΔgene:: CMとMC4100、です。 pK184遺伝子回収プラスミド、ならびに標的遺伝子（欠失または点変異）の一連の変異体は、MC4100のレシピエント細胞（ 図2を用いて、交配を復元し、その効率を決定する能力を評価するために、ドナー細胞に提供されます。 ;表3）。 手順にとって重要適切なドナーおよびレシピエント株（ 表1）、および使用されるプライマーの設計を使用することです。設計された各プライマーについて、従うべき一般的なガイドラインがあります。我々は厳密に40から60パーセントのGC含量に遵守しようとするが、これは常に可能ではないかもしれません。このような場合には、プライマーWをテストするために実験者の判断でありますわずかにこの範囲を下回るまたは上記i番目のGC含量。順方向および逆方向プライマーの融解温度（T m）は類似していなければならない、アニール温度が（T a）の値は常にPCR 2-5゜CによるT mよりも低くなければなりません。ホモおよびヘテロ二量体化のT m &#39;はsは（より-10キロジュールと30°C以下）非常に低くなければならない一方でヘアピンが開発できるようにするために利用可能な自由エネルギーは、Tよりもはるかに低くする必要があります。プライマーは、所望のように欠失、挿入または点変異を調べるために設計することができます。なお、得られた遺伝子構築物が増幅され、それが適切に発現されるようにインフレームで回収プラスミドに連結されることが当然重要です。コンピテント細胞の形質転換は、それぞれ、エレクトロまたは化学的コンピテント細胞を用いてエレクトロポレーションまたは熱ショックを介して行うことができます。私たちは、エレクトロポレーションは、このようなpOX38-TCと相同組換えoligonucleとして大きな構成のためのより効率的であることがわかりotide、例えばpK184-TRAFとしてより小さい発現プラスミドは、容易に熱ショック方法を用いて、細胞に形質転換することができるが。最後に、両方のドナーとレシピエント株は接合およびリカバリのプラスミド上で使用するものとは異なる別の抵抗を必要とするように、プロトコル全体で使用されている複数の抗生物質が存在するであろうことを覚えておくことが重要です。 別にここで説明交配アッセイ技術から、彼らのアプローチで若干変動する細菌接合を研究するために使用されている他の方法が、あります。水平遺伝子伝達は、細菌が種間の受信者を含む、レシピエント細胞へのプラスミドを転送するプロセスです。 44例えばダールバーグと同僚44による研究は、種間の水平遺伝子伝達の程度を決定するために、細菌接合を使用していました。それらは、KT2442細胞にクローン化したプラスミドに、緑色蛍光タンパク質（GFP）の取り込みを使用しました。染色体リットルKT2442細胞における交流I q遺伝子は、GFPの発現を抑制する。 GFPを担持するプラスミドをlacリI q遺伝子なし種に転送されると、蛍光が観察されます。 44は、種々の種におけるタンパク質の特定の機能を提供するために、その制限にもかかわらず、種間の結合実験は、おそらく異なる種間のタンパク質-タンパク質相互作用のための進化の予測を行うためにここに提示されたプロトコルを用いて結合することができます。

ミクロ生物レベルでの遺伝子やタンパク質の転送は、細菌の生存と発展だけでなく、感染の過程において中心的な役割を果たしています。細菌の間、または細菌と細胞間のDNAの交換は、形質転換、接合またはベクターの形質導入によって達成することができます。 1,2結合は、複雑な高分子システムがドナーとレシピエント細胞を接続することによりDNAの転移がドナー・制御された様式で発生し、 大腸菌などのグラム陰性菌間の共役中にその中の形質転換および形質導入と比較してユニークです。結合はまた、細菌細胞は、ホストシステムの中での遺伝子、タンパク質または化学物質を注入するために、宿主細胞と相互作用する最も直接的な方法です。 図3は、かなり頻繁に、このような薬剤の転送は、進化と適応をホストするために発病し、発癌に至るまで、ホスト上の顕著な効果を持っています。その接合recombinatioが示されていますnは環境ストレスの条件下で高い突然変異率を持つ細菌に適応3倍の速度を増加させます。 4さらに、結合は、はるかに菌株における抗生物質耐性遺伝子が拡散され、それを通して、最も一般的な経路です。 5,6 微生物は細胞膜を横切る巨大分子の転送をサポートするために特殊な分泌系を進化させてきました。 T1SS、T2SS、T3SS、T4SS、T5SS、T6SS、T7SS、ならびに秒（分泌）およびTat（2-アルギニン転座）：記載されているグラム陰性菌における分泌系（のTSS）の9種類が現在あります経路。分泌系の7,8各タイプは、さらに、異なるサブタイプに起因するタンパク質の多様性と異なる細菌株で、関与する経路の特殊性への必要性を分割されています。例えば、IV型分泌系（T4SS）で、TiおよびCAGシステムはF-プラスミド、R27 Aに対し、エフェクター輸送を促進しますND pKM101 T4SSsは、接合性プラスミドの伝達を促進します。生物はその構成タンパク質からそれぞれの分泌系を組み立てると、受信者またはその周囲の環境と細胞内容を共有するメカニズムの7,9,10詳細な理解は、病原性微生物やプロセスに対抗するための目標と戦略の開発における重要な因子であります携帯感染。 レーダーバーグ＆テイタムによって大腸菌での最初の同定細菌接合に続いて、モバイルおよび接合性プラスミドの11多数同定され、特徴付けられています。 12このようなモバイルプラスミドは、かなりの範囲は（1から200以上のキロベース（キロバイト）まで）のサイズであることを示す、しかしすべてのモバイルプラスミドは、それによって、プラスミドの伝達を可能にする転送（oriTを）の原点を認識しrelaxaseを、含まれています。機能T4SSの組み立てだけでなく、タイプの接合プラスミドさらにエンコード遺伝子IV結合タンパク質。 12例えば、 大腸菌の100キロバイトFプラスミドは33.3 kBの転送（TRA）領域内のすべての接合の遺伝子をコードしています。 13ペア形成（MPF）、DNA転移と除外機能を交配線毛形成を促進する全てのタンパク質、接合性プラスミドの転送中にFプラスミドエンコードのTRA領域における遺伝子。 10,14,15は、知識の重要な身体は、接合T4SSsのために利用可能である、しかし、唯一のより最近利用可能になってきている共役タンパク質との複合体の構造研究を詳述しました。 16から28 接合プロセスの包括的なビューを組み立てるためには、接合伝達タンパク質の変異分析に詳細な構造研究のカップリングが必要です。これは、接合交配アッセイを介して達成することができます。 Fプラスミドは、TRA領域内にコードされる各タンパク質は、F媒介コにおいて役割を果たすnjugation;従って、転写遺伝子のノックアウト/欠失は、細胞の接合容量（ 図1）を廃止します。小さい携帯プラスミドは、標準的な削除の手続きをより助長しているが、このようなFのようなより大きな接合プラスミドのために、遺伝子ノックアウトは、より容易に標的遺伝子は明確な抗生物質耐性遺伝子を伝えるものに交換され、相同組換えを介して達成されます。現在のプロトコルでは、我々は55キロバイトFプラスミドの誘導体pOX38-TCでクロラムフェニコールアセチルトランスフェラーゼ（CAT）と利益の伝達遺伝子を置き換えるために、相同組換えを利用します。 29,30、得られたノックアウトプラスミド、pOX38-TCΔgene:: Cmが、増殖培地中のクロラムフェニコール（CM）の存在に対する抵抗性を促進します。 pOX38-TCΔgene:: Cmとを保有するドナー細胞は交配アッセイの使用を介して観察されるように接合DNA転送/交配に影響を与えることができません。 pOX38-TCΔgene:: Cmをドナー細胞と正常RECIPの接合効率ientは廃止され、より多くの場合、減少またはます。 pOX38-TCΔgene:: Cmをプラスミドの接合伝達を標的に転送遺伝子を保有する小型の回復プラスミドを経由して復元することができます。この回復プラスミドは、プラスミドpK184（pK184遺伝子）、31または限り、そのプラスミドが適切細胞（細胞質またはペリプラズム）内の正しい場所に遺伝子を標的として誘導性の発現を提供するものとして、構成的発現を提供するものであることができます。したがって、この新しいドナー間の交配アッセイ（pOX38-TCΔgene:: Cmを+ pK184遺伝子のプラスミドを有します） そして、レシピエント細胞は、接合効率はほぼその正常ドナー・レシピエントの交配アッセイのに復元することが期待されます。このシステムは、CM曲庇:: pK184-遺伝子構築物（欠失または点変異）の一連の生成を介してとpOX38-TCΔgeneの交配能力を回復するために各構築物の能力を試験するノックアウト遺伝子の機能を調べるために1を可能にしますドナー細胞秒。

<table border="0" cellpadding="0" cellspacing="0" width="762">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">GeneJet Plasmid Mini-Prep Kit</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">K0503</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">GeneJet Gel Extraction Kit</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">K0692</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">GeneJet PCR Purification Kit</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">K0702</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Q5 Site-Directed Mutagenesis Kit</td>
			<td>New England Biolabs</td>
			<td>E0554S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Broad Range DNA Ladder</td>
			<td>New England Biolabs</td>
			<td>N0303A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Petri Dishes</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">FB0875713</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Electroporator</td>
			<td style="width:165px;">Eppendorf</td>
			<td style="width:131px;">4309000027</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Electroporation cuvettes</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">FB101</td>
			<td style="width:212px;">Cuvettes have a 1 mm gap.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Enzymes</td>
			<td style="width:165px;"></td>
			<td style="width:131px;"></td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">AvaI</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0152S</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">EcoRI</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0101S</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">HindIII</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0104L</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">NdeI</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0111S</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Phusion DNA Polymerase</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">M0530L</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">T4 DNA Ligase</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">M0202S</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">DpnI</td>
			<td style="width:165px;">New England Biolabs</td>
			<td style="width:131px;">R0176S</td>
			<td style="width:212px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Antibiotics</td>
			<td style="width:165px;"></td>
			<td style="width:131px;"></td>
			<td style="width:212px;">Final Concentrations</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Chloramphenicol (Cm)</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">BP904-100</td>
			<td style="width:212px;">20 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Kanamycin (Km)</td>
			<td style="width:165px;">BioBasic Inc.</td>
			<td style="width:131px;">DB0286</td>
			<td style="width:212px;">50 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Nalidixic acid (Nal)</td>
			<td style="width:165px;">Sigma-Aldrich</td>
			<td style="width:131px;">N8878-25G</td>
			<td style="width:212px;">10 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Rifampicin (Rif)</td>
			<td style="width:165px;">Calbiochem</td>
			<td style="width:131px;">557303</td>
			<td style="width:212px;">20 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tetracycline (Tc)</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">BP912-100</td>
			<td style="width:212px;">10 &#181;g/mL</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Streptomycin (Sm)</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:131px;">BP910-50</td>
			<td style="width:212px;">50 &#181;g/mL</td>
		</tr>
	</tbody>
</table>

conjugative mating assays for sequence-specific analysis of transfer proteins involved in bacterial conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Fプラスミド駆動型細菌接合プロセスは、線毛の合成と接合DNAの転送を容易にするT4SSを組み立てるF-プラスミドのTRA領域内に伝達タンパク質を伴う協調プロセスです。タンパク質TRAF（GenBankアクセッション番号BAA97961、UniProtのID P14497）は、接合F線毛形成に必要とされます。 10,14,35 -それは、触媒CXXCモチーフを持っていませんが、37タンパク質は、C末端チオレドキシン様ドメインが含まれています。それは、そのN末端ドメインを介してTraHタンパク質と相互作用すると予測されているが35,38、39地域は他にあまりない37は 、タンパク質の構造的特徴について知られている、そのC末端ドメインよりも、よりダイナミックであることが示されました。構造研究と併せてTRAFタンパク質の機能的側面を評価するために、我々はまず、相同経由pOX38-TCにTRAF遺伝子をノックアウト組換え大腸菌 DY330R細胞におけるpOX38-TCΔtraF:: Cmのプラスミド（ 表1）を生成します 。また、生成された33,34は、結合の回復を提供し、タンパク質の配列をプローブ有効にするTRAF特異的プライマー（ 表2）からpK184-TRAFプラスミドでした。 pK184-TRAFがトランスで提供されていたXK1200細胞DY330R細胞から40へpOX38-TCΔtraF:: Cmをプラスミドの転送を示している（のトランスは、10 / mlのTcのおよび20μg/ mLのCmが10μgの/ mLのNalで上に成長させました） pOx38-TCΔtraF:: CMで、その（a）は、TRAFノックアウトは、インフレームCATカセットを提供し、（b）はそのpK184-TRAFは、接合機能を回復させることができます。 TRAF変異体のシリーズはXK1200 pOX38-TCΔtraF:: Cmを+ pK184-TRAFΔXを使用して接合交配アッセイ37を使用して、分析のために生成されましたそれぞれのドナーとレシピエントのような細胞およびMC4100細胞41。一つの代表的な変異体TRAF 55から247（ 表1）、タンパク質の領域はTraHと相互作用することが予測さ削除N末端欠失変異体です。完全長TRAFタンパク質がドナー細胞に提供されている場合、空のプラスミドpK184んではない（ 表3）を提供しながら、接合伝達は、（ 図2）復元されます。プラスミドpK184-TRAF 55から247（ 表3）が設けられたとき同様に、XK1200ドナー細胞における接合機能が復元されません。これは、タンパク質の切断部位は、おそらくTraHとの相互作用を介して、接合装置内のTRAFの機能にとって重要であることを示し、さらに変異解析のために標的化するためにタンパク質の領域を提供します。 <img alt="図1" src="/files/ftp_upload/54854/54854fig1.jpg" /> <strong&gt;図1：接合交配アッセイの模式図。ステップ1において、標的遺伝子は、PCRは、標的遺伝子に相同なオーバーハングを有するCATカセットを生成し使用して、DY330R細胞、効率的な組換え株において相同組換えによりノックアウトされています。結果として得られるDY330Rクローンは、プラスミドノックアウト遺伝子が回復プラスミド（pK184-遺伝子）を介してトランスで提供されていない限り、結合を促進することができませんpOX38-TCΔgene:: Cmとし、保有します。得pOX38-TCΔgene:: CmをプラスミドはMC4100の受信者（工程3）と交配アッセイを用いて遺伝子のさらなる評価のためXK1200株（ステップ2）に転送されます。 <a href="http://ecsource.jove.com/files/ftp_upload/54854/54854fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/54854/54854fig2.jpg" /> 図2： マット結合中の標的遺伝子の機能を評価するためのアッセイをる。ドナーとレシピエント細胞は、大腸菌 XK1200 pOX38-TCΔtraF:: Cmは、それぞれ、pK184-TRAFとMC4100で形質転換しました。得られたトランス（MC4100 pOX38-TCΔtraF:: Cmが）接合機能の正常な回復を示し、Sm及びCmとを含むプレート上で成長します。実験は、単一の寒天プレート上に二重に行われ、連続希釈を修復遺伝子変異体（ 表2）の接合効率を計算するために使用されています。 <a href="http://ecsource.jove.com/files/ftp_upload/54854/54854fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 細菌株/プラスミド </td><td> 関連する特性 </td><td> 選択マーカー（複数可）† </strong&gt; </td><td> 参照 </td></tr><tr><td> 菌株 </td><td></td><td></td><td></td></tr><tr><td> DY330R </td><td> W3110ΔlacU169gal490λc1857Δ（CRO-のbioA）リフR </td><td>解雇</td><td> 33,34 </td></tr><tr><td> XK1200 </td><td> F - lacΔU124Δ（バイオジャイラattλ灘aroGギャル） </td><td> NAL </td><td> 40 </td></tr><tr><td> MC4100 </td><td> araD139Δ（ARGF-LAC）U169 rpsL150 relA1 flbB3501 deoC1 ptsF25 rbsR </td><td> Smの</td><td> 41 </td></tr><tr><td> ベクターおよび構築 </td><td></td><td></td><td></td></tr><tr><td> pBAD33 </td><td> P のaraBADから下を発現するプラスミド</td><td> Cm </td><td> 32 </td></tr><tr><td> pK184 </td><td>ベクトル、P15Aレプリコンをクローニング2.4キロバイト</td><td>キロ</td><td> 31 </td></tr><tr><td> pK184-TRAF‡&lt;/ TD&gt; <td> pK184でpOX38からF TRAF </td><td>キロ</td><td> 35 </td></tr><tr><td> pK184-TRAF 56から247 </td><td> pK184-TRAFからF TRAFアミノ酸56から247 </td><td>キロ</td><td></td></tr><tr><td> 接合プラスミド </td><td></td><td></td><td></td></tr><tr><td> pOX38-TC </td><td> IncFI、トラ+、RepFIA +、F、ミニのTnのF1 HindIII断片</td><td> Tcの</td><td> 29,30 </td></tr><tr><td> pOX38-TCΔtraF:: Cmを</td><td> CATとpOX38-TcはTRAFに挿入します</td><td> TcのCmを</td><td> 35 </td></tr><tr><td colspan="4"> †Cmを、クロラムフェニコール;キロ、カナマイシン。 Nalで、ナリジクス酸;リフ、リファンピシン; Smを、ストレプトマイシン; Tcを、テトラサイクリン</td></tr><tr><td colspan="4"> ‡すべてのTRAF構築物は、細胞膜周辺腔への局在化を確実にするために、19残基のリーダー配列を含んでいます。 </td></tr></tbody></table> 表1： <st栄&gt; E。 coli 株およびプラスミド本 研究で使用しました。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 構築します </td><td> プライマー* </td></tr><tr><td> TRAF-CM-について</td><td> 5&#39;- GATCGAGGCTGGCAGTGGTATAACGAGAAAATAAATCCGAAGGA - CTGTGACG GAAGATCACTTC -3 &#39; </td></tr><tr><td> TRAF-CM-REV </td><td> 5&#39;- TCTTCAGAAACGTTCAGGAACTGTTTTGCCAGGTCGTCCT - CTTATTCAGGCGT AGCACCAG -3 &#39; </td></tr><tr><td> pK184-TRAF-について</td><td> 5&#39;- TTTTTT GAATTC TATGAATAAAGCATTACTGCCAC -3 &#39; </td></tr><tr><td> pK184-TRAF-REV </td><td> 5&#39;- TTTTTT AAGCTT TAAAAATTGGGTTTAAAATCTTCAGAAA -3 &#39; </td></tr><tr><td> pK184-TRAF 55から247 -For </td><td> 5&#39;- TACG CATATG ATGGCCGCACTGCAGACGG -3 &#39; </td></tr><tr><td> pK184-TRAF 55から247 </sub&gt; -rev </td><td> 5&#39;- TACG CATATG TCCTGACGCCGGAAAAATAAAGCAGCAGAGTAA -3 &#39; </td></tr><tr><td colspan="2"> †レントらから適応表。許可を得て、2016年35 </td></tr><tr><td colspan="2"> * TRAF張り出し領域はイタリック体です。 （：AAGCTTをEcoRI：GAATTCをNdeI：CATATG HindIIIで）制限酵素部位には下線が引かれています</td></tr></tbody></table> 表2：本研究で使用したプライマーのリスト。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> ドナープラスミド† </td><td> 回復プラスミド </td><td> トランス接合‡§（細胞ミリリットル-1） </td><td> 交尾Efficiency§|| </td></tr><tr><td> pOX38-TCΔtraF:: Cmを</td><td>なし</td><td> 0 </td><td> 0 </td></tr><tr><td> pOX38-TΔtraF:: CmをC </td><td> pK184 </td><td> 0 </td><td> 0 </td></tr><tr><td> pOX38-TCΔtraF:: Cmを</td><td> pK184-TRAF </td><td> 5×10 3 </td><td> 0.0167 </td></tr><tr><td> pOX38-TCΔtraF:: Cmを</td><td> pK184-TRAF 55から247 </td><td> 0 </td><td> 0 </td></tr><tr><td colspan="4"> *レントらから適応表。許可を得て、2016年35 </td></tr><tr><td colspan="4"> †ドナー細胞は、3×10 7細胞mlの平均濃度で、 大腸菌 XK1200た-1 </td></tr><tr><td colspan="4"> ‡レシピエント細胞は、 大腸菌 MC4100ました。ポジティブコントロール用のトランスの数は10-5希釈からです。 0は10-2希釈からのトランス接合体がないことを示します。 </td></tr><tr><td colspan="4"> 2〜4個の交配実験の§An平均は、各構築物について実施しました。 </td></tr><tr><td colspan="4"> ||交尾効率がdefinです100ドナー細胞当たりのトランスとして編。 0交配効率は10-2希釈からのトランス接合体がないことを示します</td></tr></tbody></table> 表3：TRAF 欠失構築物により廃止接合効率。

Watch this Scientific Journal Video about Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation at JoVE.com

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

細菌接合に関与する転写タンパク質の配列特異的分析のための接合交配アッセイ

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

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Genetics

13.7K ビュー.  York University. Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations. 

ビデオ: Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

in vitro転写アッセイおよび創薬への応用

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

遺伝学

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little is known about the molecular control that governs their development. This is in part due to physical, resource and time limitations often associated with the study of secondary growth processes. A number of in vitro techniques have been used involving either plant part or whole plant system in both woody and non-woody plant species. However, questions about their applicability for the study of secondary stem growth processes, the recalcitrance of certain species and labor intensity are often prohibitive for medium to high throughput applications. Also, when looking at secondary stem development and wood formation the specific traits under investigation might only become measurable late in a tree&#39;s lifecycle after several years of growth. In addressing these challenges alternative in vivo protocols have been developed, named Induced Somatic Sector Analysis, which involve the creation of transgenic somatic tissue sectors directly in the plant&#39;s secondary stem. The aim of this protocol is to provide an efficient, easy and relatively fast means to create transgenic secondary plant tissue for gene and promoter functional characterization that can be utilized in a range of tree species. Results presented here show that transgenic secondary stem sectors can be created in all live tissues and cell types in secondary stems of a variety of tree species and that wood morphological traits as well as promoter expression patterns in secondary stems can be readily assessed facilitating medium to high throughput functional characterization.

The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

木部形成に関与する遺伝子およびプロモーターを研究するための誘導性体細胞セクター分析（ISSA）の使用とセカンダリステム開発

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

生理的酸素条件に曝露されたヒト細胞におけるキャップ結合タンパク質の解析

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

新規トランスポゾンを介したアプローチを使用してエシェリヒア属大腸菌DNA 要素の最適な染色体の場所の決定

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

一本鎖 Rna の二重鎖 Rna の特定のシーケンスで選択的な認識化学修飾ペプチド核酸

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

細菌の活用頻度の高解像度の比較

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

酵母の過剰発現の交尾ベース ライブラリ スクリーニング

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

Cell Based Assays of SINEUP Non-coding RNAs That Can Specifically Enhance mRNA Translation

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. Here, we present a method to selectively up-regulate protein expression of desired genes in cultured cells by means of synthetic antisense non-coding RNAs known as SINEUPs. This positive control of gene expression is at the post-transcriptional level and exerted by an inverted short interspersed nuclear element (SINE) repeat at the 3&#697; end of SINEUPs that comprises its effector domain (ED). SINEUPs can specifically bind to any protein-coding mRNA of choice through its binding domain (BD), a region designed to complement the sequence within the 5&#697; untranslated region (5&#697; UTR) and around the start codon of the mRNA. Target-specific SINEUPs designed in this manner are transfected to cultured cells, and protein and RNA are extracted for downstream analyses, generally 24-48 h post-transfection. SINEUP-induced protein up-regulation is detected by Western-blot analysis and RNA expression is measured using real-time quantitative reverse transcription PCR. We have observed that BD design is critical for achieving optimum SINEUP activity and that testing different BD sizes and positions with respect to the start codon of the target mRNA is recommended. Therefore, we describe here a semi-automated high-throughput imaging method based on fluorescence detection that can be implemented to target mRNA fused with green fluorescent protein (GFP). SINEUPs specifically enhance translation within normal physiological range of the cell, without altering the target transcript level. This method has been successfully employed against a range of endogenous and exogenous targets, in a wide variety of human, mouse, and insect cell lines along with in vivo systems. Moreover, SINEUPs have been reported to increase antibody production and work as an RNA therapeutic against haploinsufficient genes. The versatile and modular nature of SINEUPs makes them a suitable tool for gene-specific translational control.

SINEUPs are synthetic antisense non-coding RNAs, which contain a binding domain (BD) and an effector domain (ED) and up-regulate translation of target mRNA. Here, we describe detection methods for SINEUPs in cultured cell lines, analysis of their translation-promoting activity by Western-blot and a semi-automated high throughput imaging system.

セルベースのアッセイ SINEUP 非コード Rna mRNA の翻訳を特に高めることができるの

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. ...

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast permitting in vivo phenotypic analysis. This method does not rely on maize transformation but exploits microbial genetics and secretory capabilities of pathogens. Herein, it allows inspection of in vivo delivered secreted proteins with high spatiotemporal resolution at different kinds of infection sites and tissues. The Trojan horse strategy can be utilized to transiently complement maize loss-of-function phenotypes, to functionally characterize protein domains, to analyze off-target protein effects, or to study onside protein overdosage, making it a powerful tool for protein studies in the maize crop system. This work contains a precise protocol on how to generate a Trojan horse strain followed by standardized infection protocols to apply this method to three different maize tissue types.

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

This work describes the cloning of an Ustilago maydis Trojan horse strain for the in situ delivery of secreted maize proteins into three different types of maize tissues.

トロイの木馬として経路を用いたトウモロコシ蛋白質場配信で

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast ...

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to agriculture. However, the Ralstonia model is considerably underexplored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae in Arabidopsis. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction in native host plants. In this scenario, we have developed a method to perform genetic analysis of Ralstonia infection of tomato, a natural host of Ralstonia. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi. As a proof of concept, we used this method to show that RNAi-mediated silencing of SlCESA6 in tomato roots conferred resistance to Ralstonia. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relatively short time and with small requirements of equipment and plant growth space.

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Here, we present a versatile method for tomato root transformation followed by inoculation with Ralstonia solanacearum to perform straightforward genetic analysis for the study of bacterial wilt disease.

細菌性ウィルト病の簡単な遺伝子解析のためのラストニア・ソラナセアラムによるトマト根形質転換

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to ...