ゲノミクスと進化科学を推進するファージハンター（SEA-ファージ）プログラム - 著者は、Active教育のためのゲノムコンソーシアム（GCAT）とHHMI-科学教育連盟とマルコム・キャンベルとトッドEckdahlを承認したいと思います。 このプロジェクトは、研究資源のための国立センター（P20RR016460）と国立衛生研究所から一般医科学研究所（P20GM103429）からの助成金によってサポートされていました。この研究は、助成金＃のIIA-1457888の下で国立科学財団によって部分的にサポートされていました。さらに制度（ウォシタ・バプティスト大学）資金はJDパターソン夏の特別研究員を介して提供されました。

1.適切な粘着末端を有するオリゴヌクレオチドを​​設計し、注文<ol><li>オンラインで自由に利用可能なプログラムを用いてゲノム解析を通じて潜在的なローに依存しないターミネータを識別します。7 </li><li>二本鎖DNAを操作する場合は、テストされるターミネーターの方向を決定する。7 PGR-ブループラスミドは上部のみ（フォワード鎖）の5 &#39;から3&#39;方向に連結したターミネータを検証します。 <ol><li>底に変換し、無料のオンラインソフトウェア、 例えば 、APEを使用して、PGR-ブルーでテストするためのシーケンスを再配向するために、その逆相補体に鎖ターミネーター（リバース）。 </li></ol></li><li>向きが正しいと判断された後、上部の鎖の5 &#39;末端に粘着末端「5&#39;-CGAC-3 &quot;&#39;を追加します。下の鎖の5 &#39;末端に粘着末端「5&#39;-CCGC-3 &quot;&#39;を追加。8これが連結したインサートが適切に挿入されることを保証しますGGAを使用して向き。 </li><li>配列が決定されると、商業的に一本鎖オリゴヌクレオチドとして注文します。 </li></ol> 2.アニーリングオリゴヌクレオチドは、（凍結乾燥するDNAをのみ適用されます） <ol><li>再懸濁、100μMの濃度にヌクレアーゼフリー水で個々のオリゴヌクレオチド。 </li><li> 1 M NaClおよび100 mMトリス - 塩酸pHが7.4：10×アニーリング緩衝液を作ります。アニーリング緩衝液は、数週間4℃で保存することができます。アニーリングバッファーが行われ、オリゴヌクレオチドが再懸濁された後、アニール8を開始します。 </li><li>各終端抵抗をテストするためには、1.5ミリリットルのマイクロチューブに超純水16μlを添加します。 </li><li>各マイクロチューブに10倍アニーリング緩衝液2μLを加えます。 </li><li>希望ターミネータから上の鎖オリゴヌクレオチドの1μlの（100μM）を追加します。 </li><li>希望ターミネータからボトム鎖オリゴヌクレオチド（100μM）の1μLを加えます。 </li><li>沸騰したお湯のBAを作成します。ホットプレート上で水道水と場所の約400ミリリットルを含む千ミリリットルのビーカーを使用して目。 </li><li>フロートのステップ2.6からマイクロ遠心チューブを置き、4分間沸騰。 4分後、熱板をオフにするが、チューブを残し、水浴にゆっくりとクールなO / N（18時間）8に浮きます。 -20℃でアニールターミネータを保管してください。 </li></ol> 3.ゴールデンゲートアセンブリ（標準ミックス - コスト効率のよいです） <ol><li>アニールターミネーターの40 nMの希釈を行います。アニールターミネーターミックス（100μM）の1μlにヌクレアーゼフリー水124μlを添加します。 <ol><li>氷上で30万×gで秒、店舗用の遠心分離機サンプル。 </li></ol></li><li>使用されているサーモサイクラーに適した新たなチューブに、ヌクレアーゼを含まない水の6μlを添加します。 </li><li>市販のDNAリガーゼバッファー[300mMのトリス塩酸（pHが7.7から7.8）、100 mMのMgCl 2を、100mMのDTT、および10mM ATP]の1μLを加えます。氷の上で後続のすべての手順を実行します。 </li><li>加えますPGR-ブルー（35-50 ngの/μl）をマイクロ遠心チューブに先プラスミドの1μlの。 </li><li>マイクロチューブに40 nMの焼鈍ターミネーターの1μLを加えます。 </li><li>マイクロチューブに0.5μlの市販の高忠実度のBsaI制限エンドヌクレアーゼを追加します。 </li><li>マイクロ遠心チューブに市販のDNAリガーゼの0.5μlを添加します。 </li><li> 120秒間万×gで反応混合物を遠心分離します。そして、サーマルサイクラーに直接チューブを配置します。 </li><li>サーモサイクラープログラムを実行する：37℃で1分間の20サイクル、37℃で15分間の1サイクル、続いて16℃で1分間続きます。使用サンプル直ちにまたは-20℃で凍結保存する。8 </li></ol> 4.ゴールデンゲートアセンブリ（商業マスターミックス - 時間効率的な） <ol><li>これは、40 nmのターミネーターDNA濃度を希釈するステップ2で作成したターミネータをアニールし、100μMの1μlにヌクレアーゼフリー水124μlを添加します。新たに、このステップを実行し、1.5ミリリットルのマイクロチューブは、標識されました。 </li><li>サーモサイクラー用の新しいチューブの適切に、ヌクレアーゼフリー水14μlを添加します。新しいチューブはそれに応じてラベル付けされていることを確認します。 </li><li>マイクロチューブに商業マスターミックス緩衝液の2μlを添加します。 </li><li>マイクロチューブにPGR-ブループラスミド（35-50 ngの/μL）の1μLを加えます。 </li><li>マイクロチューブに40 nMの焼鈍ターミネーターの2μLを加えます。 </li><li>マイクロチューブに商業マスターミックスの1μLを加えます。 </li><li> 120秒間万×gで40 nMのチューブを遠心。そして、サーマルサイクラーに直接チューブを配置します。 </li><li> 55℃で15分間の1サイクル、続いて37℃で60分間の1サイクル：サーモサイクラープログラムを実行します。使用してすぐにサンプルまたは-20℃で凍結して保存。 </li></ol> 5.形質転換およびコロニーの選択<ol><li>準備の1mM concentratを含むペトリ皿の寒天ルリアベース（LB）アンピシリンのイオン（アンプ）とアラビノースの10 mMの濃度（ARB）。このストック溶液は、後の工程で使用されるため、1 MのL -arabinose原液の少なくとも10 mlで調製します。 </li><li>任意の高効率使用（8月10日から9月10日までの形質転換体/μgのを）化学的コンピテントE.変換のための大腸菌 （JM109）細胞。最良の結果を得るために9、コンピテント細胞を50μlに連結反応の3-5μlを添加します。化学的コンピテント細胞を使用する場合、細胞が使用されている固有の形質転換プロトコルに従います。 </li><li>無菌のL字型の「ホッケースティック」を使用して寒天プレート予熱したLB（アンプ/ ARB）に形質転換された細胞の半分（25μl）を広げ、37℃でO / Nインキュベートします。分解のアンピシリンの急速なレートに、24時間にわたって37℃でインキュベートしません。 </li><li>目視による色に基づいて、コロニーの選択を行います。成功したライゲーションは、可視光で黄色/白と緑のアンクルを蛍光を発する必要がありますr個の青（450 nmの）またはUV光。 20時間 - 失敗したライゲーションは18の後に、可視光下での色は青であるコロニーを生成します。 </li></ol> 6.検証とターミネーターの定量<ol><li> 1 mMのアンピシリンおよびアラビノースの10mMで5ミリリットル滅菌LBブロスチューブを準備します。必要な管の数は、試験されるターミネータープラスコントロール（細胞アンピシリン耐性を持つ蛍光を発しないとノーカットPGR-ブループラスミドを含む細胞）の数に依存しています。 </li><li>可視光下で滅菌ループを使用して2-3の黄色/白色コロニー（ステップ5.4）を選択します。試料は37℃、16-18時間、160rpmでシェーカー上ブロスでインキュベートすることを可能にします。以上24時間これらのサンプルをインキュベートしないでください。 <ol><li>必要に応じて、適切な細胞の成長を確実にするために、600nmの（OD 600）での光学セル密度を決定するために分光光度計を使用します。一般的に、〜0.8〜1.0のOD 600は、成長の16-18時間後に観察されます。 </li></ol></li><li>つかいます残りのステップのための滅菌96ウェルプレート。無菌環境と三重にこれらのステップを実施します。各ウェルに滅菌LB +アンプ/アラビノースブロス（1 mMのアンピシリン/ 10 mMのアラビノース）の199μLを加えます。 3つの別々のコントロールのプレート上でルームなどがあります。 <ol><li>次の3つのコントロールを使用します。ウェル（1）を含むLB +アンプ/アラビノースブロスのみとウェル（2）（空白）非形質転換コンピテントセルは空白として機能します。定量化のために（3）ターミネータ強度の、非終結配列と連結したノーカットオリジナルPGRを含むまたはPGR-ブルーを含む細胞は、（定量化のための基準制御）に含まれるべきです。プレートリーダーのレイアウトについては、 図1を参照してください。 </li></ol></li><li> 96ウェルプレートの個々のウェルにO / N培養液から細胞溶液のピペット1μlの。 </li><li>通気性のカバーとシールプレートとは、37℃で18-20時間、および160 rpmで振とうします。 </li><li>プレートリーダーを用いて読み取ることにより、OD 600を決定します600 nmの吸光度。次のようにGFP蛍光を測定する：励起395及び発光509メジャーRFP蛍光を次のように：励起575および発光605を18から20時間後、OD 600が約0.5であるべきです。 </li><li>成長の変化を標準化するために、式を使用します。 <img alt="式（1）" src="/files/ftp_upload/54064/54064eq1.jpg" /> GFPとRFPの両方のために。 </li><li>正規化の後、式を用いて相対的なターミネーターの強度を決定します<img alt="式（2）" src="/files/ftp_upload/54064/54064eq2.jpg" /></li></ol>

<ol>
	<li>Li, J., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+promoter+sequence+and+its+strength+in+gene+expression.">Relationship between promoter sequence and its strength in gene expression.</a> Eur Phys J E Soft Matter. 37 (9), (2014).</li><li>Engler, C., Kandzia, R., Marillonnet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+one+pot,+one+step,+precision+cloning+method+with+high+throughput+capability.">A one pot, one step, precision cloning method with high throughput capability.</a> PLoS One. 3 (11), e3647 (2008).</li><li>Lampropoulos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GreenGate---a+novel,+versatile,+and+efficient+cloning+system+for+plant+transgenesis.">GreenGate---a novel, versatile, and efficient cloning system for plant transgenesis.</a> PLoS One. 8 (12), e83043 (2013).</li><li>Chen, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+582+natural+and+synthetic+terminators+and+quantification+of+their+design+constraints.">Characterization of 582 natural and synthetic terminators and quantification of their design constraints.</a> Nat Methods. 10 (7), 659-664 (2013).</li><li>Woodin, T., Carter, V. C., Fletcher, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vision+and+change+in+biology+undergraduate+education,+a+call+for+action--initial+responses.">Vision and change in biology undergraduate education, a call for action--initial responses.</a> CBE Life Sci Educ. 9 (2), 71-73 (2010).</li><li>Vasaly, H. L., Feser, J., Lettrich, M. D., Correa, K., Denniston, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vision+and+change+in+the+biology+community:+snapshots+of+change.">Vision and change in the biology community: snapshots of change.</a> CBE Life Sci Educ. 13 (1), 16-20 (2014).</li><li>Naville, M., Ghuillot-Gaudeffroy, A., Marchais, A., Gautheret, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARNold:+a+web+tool+for+the+prediction+of+Rho-independent+transcription+terminators.">ARNold: a web tool for the prediction of Rho-independent transcription terminators.</a> RNA Biol. 8 (1), 11-13 (2011).</li><li>Campbell, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pClone:+Synthetic+Biology+Tool+Makes+Promoter+Research+Accessible+to+Beginning+Biology+Students.">pClone: Synthetic Biology Tool Makes Promoter Research Accessible to Beginning Biology Students.</a> CBE Life Sci Educ. 13 (2), 285-296 (2014).</li><li>Rhee, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+the+medium+composition+and+plasmid+combination+on+the+growth+of+recombinant+Escherichia+coli+JM109+and+on+the+production+of+the+fusion+protein+EcoRI::SPA.">Influence of the medium composition and plasmid combination on the growth of recombinant Escherichia coli JM109 and on the production of the fusion protein EcoRI::SPA.</a> J Biotechnol. 55 (2), 69-83 (1997).</li><li>Dirla, S., Chien, J. Y., Schleif, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+mutations+in+the+Escherichia+coli+AraC+protein.">Constitutive mutations in the Escherichia coli AraC protein.</a> J Bacteriol. 191 (8), 2668-2674 (2009).</li><li>Schleif, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+L-arabinose+operon+of+Escherichia+coli.">Regulation of the L-arabinose operon of Escherichia coli.</a> Trends Genet. 16 (12), 559-565 (2000).</li><li>Kosuri, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composability+of+regulatory+sequences+controlling+transcription+and+translation+in+Escherichia+coli.">Composability of regulatory sequences controlling transcription and translation in Escherichia coli.</a> Proc Natl Acad Sci U S A. 110 (34), 14024-14029 (2013).</li><li>Sharon, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inferring+gene+regulatory+logic+from+high-throughput+measurements+of+thousands+of+systematically+designed+promoters.">Inferring gene regulatory logic from high-throughput measurements of thousands of systematically designed promoters.</a> Nat Biotechnol. 30 (6), 521-530 (2012).</li></ol>

The authors have nothing to disclose.

このプロトコルの中で最も重要なステップは、発注​​前に、適切なオリゴヌクレオチドの設計です。オリゴヌクレオチドは、適切な粘着末端は、GGAの取り込みが可能であることを保証するために、両方の上部と下部の鎖の5 &#39;末端に付加されている必要があります。 GFPおよびRFP式が（トップ鎖に向かって右にあるのでさらに、右向きのものに左向きターミネーター（下の鎖上で転写を停?...

Large synthetic biology projects necessitate the use of highly effective transcription terminators to help regulate gene expression. Identification of novel terminators requires bioinformatic analysis of novel genomes. However, as increasing amounts of bioinformatic software are developed, each with a unique algorithm utilized for prediction, more discrepancy between putative results occurs. Because this process is somewhat subjective and is done in silico, these predictions need biological confirmation.1 Additionally, the volume of putative terminators identified through in-silico analysis requires the use of cloning strategies that can be completed in a relatively short time frame. 
The PGR-Blue plasmid is a modification of the PGR plasmid that has been redesigned to use Golden Gate Assembly (GGA) to simplify the cloning procedure by allowing for all reaction steps to be simultaneously performed in one micro-centrifuge tube.2,3 Color selection was incorporated into the plasmid to increase the ease of identifying positive colonies. A successful ligation should be white/yellow in visible light and fluoresce green under blue (450 nm) or ultraviolet (UV) light when grown on plates containing arabinose. Because uncut pGR-blue contains a blue chromo protein (amilCP), colonies containing an unmodified plasmid are blue under visible light. This simplification along with the streamlined protocol allows researchers to proceed from bioinformatic identification to biological confirmation in three to four days. The design nature of this system can be beneficial both in the research lab and in educational settings.
The pGR-Blue plasmid allows for quantification of terminator strength.4 A single arabinose inducible promoter is used to produce green fluorescent protein (GFP) and red fluorescent protein (RFP). The terminator is cloned into the plasmid after the GFP sequence but before the RFP sequence, thus stopping the transcription of the RFP protein. The strength of the terminator is determined by the ratio of GFP produced to RFP produced. 
The Vision and Change5 report suggested that Science, Technology, Engineering and Math (STEM) education incorporate research based experiences into the classroom.6 However, this requires the development of protocols that can be done by students with limited skill sets in a defined time frame. While the protocol can be accomplished in as little as three days, it was also designed so that each major step could be accomplished in a separate weekly (2-3 hr) lab period to create a Course Research Experience (CRE). When used in this manner, the procedure will take between three and six weeks and is appropriate for both introductory and advanced courses in Genetics, Cell Biology or Bioinformatics.

<table border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">pGR-Blue Plasmid</td>
			<td style="width:243px;">Addgene</td>
			<td style="width:115px;">68374</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pGR-Plasmid</td>
			<td>Addgene</td>
			<td style="width:115px;">46002</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AeraSeal-(Sterile Sheets)</td>
			<td>Excel Scientific</td>
			<td>BS-25</td>
			<td style="width:235px;">Sterile Sheets only</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X T4 DNA ligase Buffer</td>
			<td>NEB</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">BsaI-HF</td>
			<td>NEB</td>
			<td>R3535S</td>
			<td style="width:235px;">The non-HF enzyme will work but is less heat stable.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">NEB Golden Gate Assembly Mix</td>
			<td>NEB</td>
			<td>E1600S</td>
			<td style="width:235px;">Commerial Master Mix refered to in the protocol.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Round Microcentrifuge Floating Rack</td>
			<td>Nova Tech International</td>
			<td>F18875-6401</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Ampicillin sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>A9518</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">L-(+)-Arabinose</td>
			<td>Sigma Aldrich</td>
			<td>A-3256</td>
			<td style="width:235px;">D-Arabinose will not induce the pBAD promoter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria Base (LB) - Broth, Miller</td>
			<td>Sigma Aldrich</td>
			<td>L1900</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria Base (LB) - Agar , Miller&#160;</td>
			<td>Sigma Aldrich</td>
			<td>L2025</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tecan-Infinite M200 Plate Reader</td>
			<td>Tecan</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Mix &#38; Go Competent Cells - Strain JM109</td>
			<td>Zymo Research</td>
			<td>T3005</td>
			<td style="width:235px;">Use company recommended transformation protocol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ApE: A plasmid editor-software</td>
			<td style="width:243px;">http://biologylabs.utah.edu/jorgensen/wayned/ape/</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl, Molecular Grade</td>
			<td>Promega</td>
			<td>H5121</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Sodium Chloride (Crystalline/Biological, Certified)</td>
			<td>Fisher Chemical</td>
			<td>S671</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Comercial Oligonucleotide synthesis</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td style="width:235px;">http://www.idtdna.com/site</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Microtest Tissue Culture Plates- 96 well (Sterile)</td>
			<td>Falcon</td>
			<td>35-3072</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mycobacteriophage &#34;Bernal13&#34;</td>
			<td>Genebank</td>
			<td>KJ510413</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease Free Water</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td>IDT004</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile, L-shaped Hockey-Stick Cell</td>
			<td>Life Science Products</td>
			<td>6444-S1</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nano-Drop 2000c UV-Vis Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>2000c</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">ARNold: a web tool for the prediction of Rho-independent transcription terminators.</td>
			<td style="width:243px;">http://rna.igmors.u-psud.fr/</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
	</tbody>
</table>

rapid verification of terminators using the pgr-blue plasmid and golden gate assembly

The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed specifically for this protocol and allows for the quantification of terminator efficiency. As a proof of concept, six terminators were bioinformatically identified in the mycobacteriophage Bernal13. Once identified, terminators were then made as oligonucleotides with the appropriate sticky ends and annealed together. Using Golden Gate Assembly (GGA), terminators were then cloned into pGR-Blue. Under visible light, false positive colonies appear blue and positively transformed colonies are white/yellow. After induction of an arabinose inducible promoter (pBad) with arabinose, colony strength can be determined by measuring the ratio of green fluorescent protein (GFP) produced to red fluorescent protein (RFP) produced. With pGR-Blue, the protocol can be completed in as little as three days and is ideal in an educational setting. Additionally, results show that this protocol is useful as a means for understanding in silico predictions of terminator efficiency related to the regulation of transcription.

このプロトコルは、ゴールデンゲートアセンブリ（ 図2）を使用して、GFPとRFPの間に連結ターミネータでPGR-ブルーを含む細胞を生成します。ライゲーションされた挿入物を含む陽性コロニーの色に基づいて選択することができます。可視光では陽性コロニーは白/黄色になり、偽陽性は37°C（ 図3）でのインキュベーションの18から20時間後に青...

Watch this Scientific Journal Video about Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly at JoVE.com

Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly

PGR-ブループラスミドとゴールデンゲートアセンブリを使用してターミネータの迅速な検証

This protocol utilizes Golden Gate Assembly and the plasmid pGR-blue to rapidly quantify the strength of terminators found in silico.

The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed ...

rapid-verification-terminators-using-pgr-blue-plasmid-golden-gate

Research

JoVE Journal

Biology

7.6K ビュー.  Ouachita Baptist University. This protocol utilizes Golden Gate Assembly and the plasmid pGR-blue to rapidly quantify the strength of terminators found in silico.

ビデオ: Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

キアゲンミニプレップキットを用いて細菌のコロニーからプラスミドDNAを精製

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

生物学

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

ヒートショック法による大腸菌へのプラスミドDNAの形質転換

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

分別アフリカツメガエルの卵抽出を用いたin vitro核アセンブリで

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

アセンブリ、読み込み、および分析用超遠心器のサンプルセルの整列

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

初代培養およびコルチのマウス臓器のプラスミドエレクトロ。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Rapid PCR Thermocycling using Microscale Thermal Convection

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed1. Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format.
Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations2-9. Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument.
One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time10,11. Here we describe results of our efforts to probe the full 3-D velocity and temperature distributions in microscale convective thermocyclers12. Unexpectedly, we have discovered a subset of complex flow trajectories that are highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone the rate limiting step of PCR. Extremely rapid DNA amplification times (under 10 min) are achievable in reactors designed to generate these flows.

We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.

ミクロ熱対流を使用して迅速なPCRの熱サイクル

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

ハイスループット酵母プラスミドの過剰発現の画面

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters&#160;and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.

迅速な合成およびGFPベースの記者との化学的に活性化する転写因子のスクリーニング

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

ギブソンアセンブリを使用して、ヒト伸長因子1αプロモーターの制御下でFLAGタグ化タンパク質を発現するプラスミドベクターの作製

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸の濾過単離：簡易迅速DNA抽出方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...