Autoren möchten Malcom Campbell und Todd Eckdahl mit dem Genome Consortium for Active Teaching (GCAT) und dem HHMI-Science Education Alliance bestätigen - Phage Hunters Advancing Genomics und Evolutions Science (SEA-Phagen) Programm. Dieses Projekt wurde durch Zuschüsse aus dem National Center for Research Resources (P20RR016460) und dem National Institute of General Medical Sciences (P20GM103429) von den National Institutes of Health. Diese Forschung wurde teilweise von der National Science Foundation unter Gewährung # IIA-1457888 unterstützt. Zusätzlich institutionelle (Ouachita Baptist University) Mittel wurden durch die JD Patterson Summer Research Fellowship zur Verfügung gestellt.

1. Planung und Bestellung von Oligonucleotiden mit den entsprechenden Sticky Ends <ol><li> Identifizieren Sie potenzielle rho-unabhängige Terminatoren durch genomische Analyse Programme , die online frei verfügbar sind. 7 </li><li> Wenn sie mit doppelsträngiger DNA arbeiten, bestimmen die Orientierung des Terminators zu testen. 7 PGR-Blue - Plasmid nur Abbrechern in der 5 &#39;zu 3&#39; - Richtung auf der Oberseite (Vorwärtsstrang) ligiert prüft. <ol><li> Konvertieren Sie einen Boden (rückwärts) Strang Terminator seine umgekehrt Komplement der Sequenz zum Testen in pGR-Blau mit dem kostenlosen Online - Software neu zu orientieren, zum Beispiel ApE. </li></ol></li><li> Nachdem die Ausrichtung bestimmt wird, korrekt zu sein, fügen Sie das sticky end &quot;5&#39;-CGAC-3 &#39;&quot; an das 5&#39;-Ende des oberen Stranges. Fügen Sie das sticky end &quot;5&#39;-CCGC-3 &#39;&quot; an das 5&#39; - Ende des unteren Strang. 8 Dadurch wird sichergestellt , dass ligierte Insert wird in den entsprechenden eingefügt werdenmit GGA Orientierung. </li><li> Sobald Sequenzen bestimmt werden, zu bestellen im Handel als Einzelstrang-Oligonukleotiden. </li></ol> 2. Ausglühen Oligonucleotides (Gilt nur für gefriergetrocknete DNA) <ol><li> Re-suspendieren einzelnen Oligonukleotide in Nuklease freiem Wasser zu einer Konzentration von 100 uM. </li><li> Machen 10x Annealing-Puffer: 1 M NaCl und 100 mM Tris-HCl pH 7,4. Annealing-Puffer kann bei 4 ° C für mehrere Wochen gelagert werden. Sobald Anelierungspuffer hergestellt und Oligonukleotide erneut suspendiert sind, beginnen die Glühen 8. </li><li> Für jeden Terminator getestet werden, 16 &amp; mgr; l ultrareinem Wasser in ein 1,5 ml Mikrozentrifugenröhrchen hinzufügen. </li><li> Fügen Sie 2 ul 10 fach Anelierungspuffer zu jedem Reaktionsgefäß. </li><li> 1 ul des Obertrums Oligonukleotid (100 uM) von dem gewünschten Terminator. </li><li> 1 ul des Untertrums Oligonukleotid (100 uM) von dem gewünschten Terminator. </li><li> Erstellen eines Siedewasser-bath unter Verwendung von auf einer heißen Platte einen 1000-ml-Becher etwa 400 ml Leitungswasser und Ort enthalten. </li><li> Legen Sie die Reaktionsgefäße aus Schritt 2.6 in einem Schwimmer und kochen für 4 min. Nach 4 min, drehen Sie die Heizplatte aber lassen Sie das Rohr und schweben im Wasserbad langsam abkühlen O / N (18 h) 8 ab. Speichern der geglühten Terminatoren bei -20 ° C. </li></ol> 3. Golden Gate Assembly (Standard Mix - Cost Efficient) <ol><li> Machen Sie eine 40 nM Verdünnung der angelagerten Terminatoren. In 124 ul Nuklease freiem Wasser auf 1 ul geglüht Terminatoren-Mix (100 &amp; mgr; M). <ol><li> Zentrifugieren Sie die Proben für 30 Sekunden bei 10.000 × g und auf Eis lagern. </li></ol></li><li> In ein neues Röhrchen geeignet für den Thermocycler verwendet werden, fügen Sie 6 ul Nuclease freies Wasser. </li><li> 1 &amp; mgr; l handelsüblichen DNA - Ligasepuffer [300 mM Tris-HCl (pH 7,7-7,8), 100 mM MgCl 2, 100 mM DTT und 10 mM ATP]. Führen Sie alle nachfolgenden Schritte auf dem Eis. </li><li> Hinzufügen1 ul PGR-Blue (35-50 ng / ul) Ziel Plasmid mit dem Reaktionsgefäß. </li><li> 1 &amp; mgr; l der 40 nM geglühten Terminatoren an den Mikrozentrifugenröhrchen. </li><li> In 0,5 ul kommerziellen High-Fidelity-BsaI Restriktionsendonuklease zum Reaktionsgefäß. </li><li> In 0,5 ul kommerzieller DNA-Ligase an das Reaktionsgefäß. </li><li> Zentrifugieren der Reaktionsmischung bei 10.000 × g für 120 Sekunden. Dann legen Sie den Schlauch direkt in den Thermocycler. </li><li> Führen Sie die Thermocycler-Programm: 20 Zyklen von 1 min bei 37 ° C, gefolgt von 1 min bei 16 ° C, gefolgt von 1 Zyklus von 15 min bei 37 ° C. Verwenden Proben sofort oder einfrieren und bei -20 ° C. 8 </li></ol> 4. Golden Gate Assembly (Commercial Master Mix - Zeit Efficient) <ol><li> In 124 ul nukleasefreiem Wasser zu 1 &amp; mgr; l der 100 &amp; mgr; M Terminatoren in Schritt erstellt geglüht 2. Dies schwächt die Terminator-DNA-Konzentration auf 40 nM.Führen Sie diesen Schritt in einem neuen, markierten 1,5 ml Mikrozentrifugenröhrchen. </li><li> In ein neues Röhrchen geeignet für den Thermocycler, fügen Sie 14 ul Nuklease freiem Wasser. Stellen Sie sicher, dass das neue Rohr entsprechend markiert ist. </li><li> In 2 ul Gewerbe Master Mix Puffer zum Reaktionsgefäß. </li><li> 1 ul PGR-Blau-Plasmid (35-50 ng / ul) zum Reaktionsgefäß. </li><li> In 2 ul der 40 nM geglühten Terminatoren an den Mikrozentrifugenröhrchen. </li><li> 1 &amp; mgr; l Gewerbe Master Mix zum Reaktionsgefäß. </li><li> Zentrifugieren Sie die 40 nM Röhrchen bei 10.000 × g für 120 Sekunden. Dann legen Sie den Schlauch direkt in den Thermocycler. </li><li> Führen Sie die Thermocycler-Programm: 1 Zyklus von 60 min bei 37 ° C von 1 Zyklus von 15 min bei 55 ° C gefolgt. Verwenden Proben sofort oder einfrieren und bei -20 ° C. </li></ol> 5. Transformation und Kolonieselektion <ol><li> Bereiten Sie Luria Base (LB) Agar-Petrischalen mit 1 mM Konzentration Ampicillin (Amp) und eine 10 mM Konzentration von Arabinose (ARB). Bereiten Sie mindestens 10 ml 1 M L -Arabinose Stammlösung , da diese Stammlösung wird in späteren Schritten verwendet werden. </li><li> Verwenden Sie einen hocheffizienten (10. August - 10. SEPTEMBER Transformanten / ug) chemisch kompetente E. coli (JM109) Zellen für die Transformation. 9 Für die besten Ergebnisse, 3-5 ul der Ligationsreaktion zu 50 ul kompetente Zellen hinzuzufügen. Wenn chemische kompetente Zellen verwenden, befolgen Sie die Transformationsprotokoll spezifisch für die Zellen verwendet wird. </li><li> Die Hälfte (25 ul) der transformierten Zellen auf vorgewärmte LB (Amp / arb) Agarplatten einer sterilen L-förmigen &quot;Hockeyschläger&quot; mit und Inkubation O / N bei 37 ° C. Aufgrund der schnellen Geschwindigkeit der Zersetzung des Ampicillin, nicht inkubieren nicht über 24 h bei 37 ° C. </li><li> Führen Kolonieselektion basierend auf Farbe durch visuelle Inspektion. Eine erfolgreiche Unterbindung sollte weiß / gelb in sichtbares Licht und fluoresziert grün under Blau (450 nm) oder UV-Licht. Ein erfolgloser Ligatur wird eine Kolonie produzieren, die nach 18 in Farbe unter sichtbarem Licht blau ist - 20 Stunden. </li></ol> 6. Überprüfung und Quantifizierung von Terminator <ol><li> Bereiten Sie 5 ml sterile LB-Brühe Röhrchen mit 1 mM Ampicillin und 10 mM Arabinose. Die Anzahl der Rohre erforderlich ist, ist abhängig von der Anzahl der Terminatoren getestet und Kontrollen (Zellen, die mit Ampicillin-Resistenz, die fluoreszieren, und die Zellen nicht den ungeschnittenen pGR Blau Plasmid enthält) wird. </li><li> Unter sichtbarem Licht wählen Sie eine sterile Schleife mit 2-3 weiß / gelb Kolonien (Schritt 5.4). Lassen Sie die Proben bei 37 ° C und 160 rpm für 16-18 h in Brühen auf einem Schüttler inkubiert. Sie brüten nicht diese Proben für mehr als 24 Stunden. <ol><li> Optional können ein Spektrophotometer optische Zelle Dichte bei 600 nm (OD 600) , um zu bestimmen ausreichende Zellwachstum zu gewährleisten. Im Allgemeinen wird ein OD 600 von ~ 0,8-1,0 nach 16-18 h des Wachstums beobachtet. </li></ol></li><li> Benutzeneine sterile 96-Well-Platte für die verbleibenden Schritte aus. Führen Sie diese Schritte in einer aseptischen Umgebung und in dreifacher Ausfertigung. In 199 ul sterilem LB + Amp / Arabinose Brühe (1 mM Ampicillin / 10 mM Arabinose) in jede Vertiefung. Fügen Sie Platz auf der Platte für drei separate Steuerungen. <ol><li> Verwenden Sie die folgenden drei Steuerelemente: Ein gut (1) mit LB + Amp / Arabinose Brühe nur und ein gut (2) für nicht-transformierten kompetenten Zellen (leer) als Rohling zu dienen. Für die Quantifizierung (3) des Terminators Stärke, ungeschnittene ursprüngliche pGR enthaltenden Zellen oder mit einem Gehalt pGR-Blau mit einer nicht abbrechenden Sequenz ligiert sollte (Referenzsteuerung für die Quantifizierung) einbezogen werden. Siehe Abbildung 1 für Plattenleser Layout. </li></ol></li><li> Pipette 1 ul der Zelllösung aus dem O / N Brühen in einzelne Wells der 96-Well-Platte. </li><li> Dichtungsplatten mit einer atmungsfähigen Deckel und schütteln für 18-20 Stunden bei 37 ° C und 160 Umdrehungen pro Minute. </li><li> Mit Hilfe eines Plattenlesegerät, bestimmen OD 600 durch LesenExtinktion bei 600 nm. Messen Sie GFP - Fluoreszenz wie folgt: Anregung 395 und Emission 509. Measure RFP Fluoreszenz wie folgt: Anregung 575 und Emission 605. Nach 18-20 h, die OD 600 sollte etwa 0,5 betragen. </li><li> Um eine Veränderung der Wachstums normalisieren, verwenden Sie die Gleichung: <img alt="Gleichung 1" src="/files/ftp_upload/54064/54064eq1.jpg" /> sowohl für GFP und RFP. </li><li> Nach der Normalisierung, bestimmen die Gleichung relativ Terminator Stärke unter Verwendung von <img alt="Gleichung 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.

Der wichtigste Schritt in diesem Protokoll ist die richtige Oligonukleotid-Design bis zur Bestellung vor. Die Oligonukleotide müssen die entsprechenden klebrigen Enden sowohl an die 5&#39;-Enden der oberen und unteren hinzugefügt Stränge, um sicherzustellen, dass GGA Einbau möglich ist. Darüber hinaus ist es wichtig, die Ausrichtung von links mit Blick auf Terminatoren (Terminatoren, die Transkription auf dem Untertrum stoppen), die der rechten Verblendung (endet Transkription auf dem oberen Strang) Terminatoren we...

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.

Dieses Protokoll wird produzieren Zellen pGR-Blau mit einem Terminator ligiert zwischen GFP und RFP mit Golden Gate Assembly (Abbildung 2) enthält. Positive Kolonien ligierte Insert enthält, kann auf der Grundlage Farbe ausgewählt werden. Im sichtbaren Licht werden positive Kolonien weiß / gelb und Fehlalarme werden blaue Kolonien nach 18-20 h Inkubation bei 37 ° C (Abbildung 3) zu erzeugen. <p class="jove_content" fo:keep-together.within-page="...

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

Schnelle Überprüfung von Terminatoren Mit PGR-Blau Plasmide und Golden Gate Assembly

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

Serie: 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.

Purifying Plasmid-DNA aus Bakterien-Kolonien mit dem 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 ...

Forschung

Biologie

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.

Transformation von Plasmid-DNA in E. coli mit dem Hitzeschock-Methode

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.

In Vitro Nuclear Assembly mit Fraktionierte Xenopus Egg Auszüge

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.

Montage-, Lade-und Alignment einer analytischen Ultrazentrifuge Messkammerdirektanschluss

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.

Primäre Kultur und Plasmid Elektroporation der Murine Corti-Organ.

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.

Rapid PCR Thermocycling mit Microscale thermische Konvektion

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.

High-throughput Hefeplasmid Überexpression Bildschirm

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

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.

Erzeugung von Plasmid-Vektoren, die FLAG-markierten Proteine ​​Nach der Verordnung der Menschen Elongation Factor-1α Promoter Mit Gibson Assembly

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.

Filtration Isolierung von Nukleinsäuren: Eine einfache und schnelle DNA-Extraktionsmethode

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

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI), a protocol based on the insertion of restriction enzyme sites into synonym DNA sequences and functional plasmid recovery. This protocol is a fast and low-cost method for fusing protein-coding genes.

CAPRRESI: Chimären Assembly durch Plasmid Recovery und Restriktion Enzym Site Insertion

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the ...