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

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.

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.

Konjugative Mating Assays für sequenzspezifische Analyse der Übertragung beteiligten Proteine ​​in bakteriellen Konjugation

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

Forschung

Genetik

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

Ein universelles Protokoll für groß angelegte gRNA Bibliothek Produktion aus jeder DNA-Quelle

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

G2-Seq: Ein hoher Durchsatz Sequenzierung-basierten Methode zur Identifizierung von spät repliziert Regionen des Genoms

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

Eine Hefe 2-Hybrid Bildschirm im Batch Proteininteraktionen vergleichen

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

Biochemie

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Induzierbaren, Zelle Typ-spezifischen Ausdruck in Arabidopsis Thaliana durch LhGR-vermittelten Trans-Aktivierung

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

Schnelle Montage von Multi-Gen-Konstrukten mit modularem Golden Gate Klonen

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

Biotechnik

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the CRISPR-Cas systems and their natural inhibitors-the anti-CRISPR proteins (Acrs)-provides a new tool to design and implement in vivo gene digital circuits. Here, we describe a protocol that follows the idea of the "Design-Build-Test-Learn" biological engineering cycle and makes use of dCas9/dCas12a together with their corresponding Acrs to establish small transcriptional networks, some of which behave like Boolean gates, in Saccharomyces cerevisiae. These results point out the properties of dCas9/dCas12a as transcription factors. In particular, to achieve maximal activation of gene expression, dSpCas9 needs to interact with an engineered scaffold RNA that collects multiple copies of the VP64 activation domain (AD). In contrast, dCas12a shall be fused, at the C terminus, with the strong VP64-p65-Rta (VPR) AD. Furthermore, the activity of both Cas proteins is not enhanced by increasing the amount of sgRNA/crRNA in the cell. This article also explains how to build Boolean gates based on the CRISPR-dCas-Acr interaction. The AcrIIA4 fused hormone-binding domain of the human estrogen receptor is the core of a NOT gate responsive to &#946;-estradiol, whereas AcrVAs synthesized by the inducible GAL1 promoter permits to mimic both YES and NOT gates with galactose as an input. In the latter circuits, AcrVA5, together with dLbCas12a, showed the best logic behavior.

CRISPR-Cas systems and anti-CRISPR proteins were integrated into the scheme of Boolean gates in Saccharomyces cerevisiae. The new small logic circuits showed good performance and deepened the understanding of both dCas9/dCas12a-based transcription factors and the properties of anti-CRISPR proteins.

Digitale Genschaltkreise auf Basis von CRISPR-Cas-Systemen und Anti-CRISPR-Proteinen

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

Maus-Genom-Engineering mit Designer Nukleasen

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

Biologie

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 ...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

Plasmid stammende DNA-Strand Displacement Gatter zum Implementieren Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...

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

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Konjugative Mating Assays für sequenzspezifische Analyse der Übertragung beteiligten Proteine in bakteriellen Konjugation

Ein universelles Protokoll für groß angelegte gRNA Bibliothek Produktion aus jeder DNA-Quelle

G2-Seq: Ein hoher Durchsatz Sequenzierung-basierten Methode zur Identifizierung von spät repliziert Regionen des Genoms

Eine Hefe 2-Hybrid Bildschirm im Batch Proteininteraktionen vergleichen

Induzierbaren, Zelle Typ-spezifischen Ausdruck in Arabidopsis Thaliana durch LhGR-vermittelten Trans-Aktivierung

Schnelle Montage von Multi-Gen-Konstrukten mit modularem Golden Gate Klonen

Digitale Genschaltkreise auf Basis von CRISPR-Cas-Systemen und Anti-CRISPR-Proteinen

Maus-Genom-Engineering mit Designer Nukleasen

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

Plasmid stammende DNA-Strand Displacement Gatter zum Implementieren Chemical Reaction Networks

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

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Konjugative Mating Assays für sequenzspezifische Analyse der Übertragung beteiligten Proteine ​​in bakteriellen Konjugation

Ein universelles Protokoll für groß angelegte gRNA Bibliothek Produktion aus jeder DNA-Quelle

G2-Seq: Ein hoher Durchsatz Sequenzierung-basierten Methode zur Identifizierung von spät repliziert Regionen des Genoms

Eine Hefe 2-Hybrid Bildschirm im Batch Proteininteraktionen vergleichen

Induzierbaren, Zelle Typ-spezifischen Ausdruck in Arabidopsis Thaliana durch LhGR-vermittelten Trans-Aktivierung

Schnelle Montage von Multi-Gen-Konstrukten mit modularem Golden Gate Klonen

Digitale Genschaltkreise auf Basis von CRISPR-Cas-Systemen und Anti-CRISPR-Proteinen

Maus-Genom-Engineering mit Designer Nukleasen

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

Plasmid stammende DNA-Strand Displacement Gatter zum Implementieren Chemical Reaction Networks

Konjugative Mating Assays für sequenzspezifische Analyse der Übertragung beteiligten Proteine in bakteriellen Konjugation

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