Name: inducible t7 rna polymerase-mediated multigene expression system, pmgx
Uploaded: 2017-06-27T13:00:00
Description: Watch this Scientific Journal Video about Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX at JoVE.com

Questo lavoro è stato sostenuto dal Consiglio di Ricerca delle Scienze Naturali e Ingegneria del Canada.

1. Ottenere Geni di Interesse <ol><li> Generi sintetici di progettazione. <ol><li> Ottimizzare una sequenza genica per l&#39;espressione di E. coli . </li><li> Rimuovere tutti i siti di restrizione problematica dalla sequenza (AvrII, NdeI, EcoRI e XbaI). </li><li> Incorporare siti di restrizione per la clonazione; Si raccomanda un sito 5&#39;-NdeI e un sito 3&#39;-EcoRI. Altri siti possono essere usati, se necessario; Si riferisce alla regione multicloning del plasmide selezionato ( <strong c...

<ol>
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Commun. 203 (10), 1326-1335 (2016).</li><li>Romier, C., 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=Co-expression+of+protein+complexes+in+prokaryotic+and+eukaryotic+hosts:+experimental+procedures,+database+tracking+and+case+studies.">Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies.</a> Acta Crystallogr. D, Biol. Crystallogr. 62 (Pt 10), 1232-1242 (2006).</li><li>Tolia, N. H., Joshua-Tor, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+protein+coexpression+in+Escherichia+coli.">Strategies for protein coexpression in Escherichia coli.</a> Nat. Methods. 3 (1), 55-64 (2006).</li><li>Chanda, P. K., Edris, W. A., Kennedy, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+set+of+ligation-independent+expression+vectors+for+co-expression+of+proteins+in+Escherichia+coli.">A set of ligation-independent expression vectors for co-expression of proteins in Escherichia coli.</a> Protein Expr. Purif. 47 (1), 217-224 (2006).</li><li>Kim, K. -. 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=Two-promoter+vector+is+highly+efficient+for+overproduction+of+protein+complexes.">Two-promoter vector is highly efficient for overproduction of protein complexes.</a> Protein Sci. 13 (6), 1698-1703 (2004).</li><li>Tan, S., Kern, R. C., Selleck, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pST44+polycistronic+expression+system+for+producing+protein+complexes+in+Escherichia+coli.">The pST44 polycistronic expression system for producing protein complexes in Escherichia coli.</a> Protein Expr. Purif. 40 (2), 385-395 (2005).</li><li>Chen, X., Pham, E., Truong, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TEV+protease-facilitated+stoichiometric+delivery+of+multiple+genes+using+a+single+expression+vector.">TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector.</a> Protein Sci. 19 (12), 2379-2388 (2010).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction:+basic+protocol+plus+troubleshooting+and+optimization+strategies.">Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.</a> J. Vis. Exp. (63), e3998 (2012).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. -. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> J. Vis. Exp. (62), (2012).</li><li>Sukumaran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+determination+of+nucleic+acids+and+proteins+using+the+micro-volume+BioSpec-nano-spectrophotometer.">Concentration determination of nucleic acids and proteins using the micro-volume BioSpec-nano-spectrophotometer.</a> J. Vis. Exp. (48), (2011).</li><li>JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. Molecular Cloning. J. Vis. Exp Available from: <a target="_blank" href="https://www.jove.com/science-education/5074/molecular-cloning">https://www.jove.com/science-education/5074/molecular-cloning</a> (2016)</li><li>JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. The Western Blot. J. Vis. Exp Available from: <a target="_blank" href="https://www.jove.com/science-education/5065/the-western-blot">https://www.jove.com/science-education/5065/the-western-blot</a> (2016)</li><li>Laible, M., Boonrod, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homemade+site+directed+mutagenesis+of+whole+plasmids.">Homemade site directed mutagenesis of whole plasmids.</a> J. Vis. Exp. (27), (2009).</li></ol>

La coespressione di geni multipli è sempre più essenziale, in particolare per caratterizzare e ricostituire complessi percorsi metabolici multigene 3 , 4 , 5 . Il sistema pMGX rende coespressione multigene in routine E. coli 6 , 7 , 8 e accessibile a diversi ricercatori. In questo studio sono state dimostrate che cinque prot...

La coespressione di molteplici proteine ​​è sempre più essenziale, in particolare nelle applicazioni di biologia sintetica, in cui devono essere espressi più moduli funzionali 1 ; Nello studio dei complessi proteico-proteici, in cui espressione e funzione richiedono spesso co-espressione 2 , 3 ; E nel caratterizzare e sfruttare percorsi biosintetici, in cui ogni gene nel percorso deve essere espresso 4 , 5 , 6 , 7 , 8 . Sono stati sviluppati diversi sistemi per la coespressione, in particolare nell&#39;organismo ospitante Escherichia coli , il cavallo da lavoro per l&#39;espressione di proteine ​​ricombinanti di laboratorio 9 . Ad esempio, plasmidi multipli con differenti marcatori selectable possono essere utilizzati per esprimere singole proteine ​​usando una ricchezza di diversi es<html> Vettori di pressione 10 , 11 . I singoli sistemi plasmidi per l&#39;espressione multipla di proteine ​​hanno utilizzato sia promotori multipli per controllare l&#39;espressione di ciascun gene 10 , 12 ; Operoni sintetici, in cui i geni multipli sono codificati su una singola trascrizione 2 , 13 ; O, in alcuni casi, un singolo gene che codifica un polipeptide che viene ultimamente elaborato in modo proteolitico, fornendo le proteine ​​desiderate di interesse 14 . <img alt="Figura 1" class="xfigimg" src="/files/ftp_upload/55187/55187fig1.jpg" /> Figura 1: flusso di lavoro pMGX che mostra la costruzione di un vettore policistronico. Il sistema pMGX fornisce una strategia flessibile e facile da usare per la costruzione di operoni sintetici sotto il controllo di un promotore T7 induttivo.E.com/files/ftp_upload/55187/55187fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Fare clic qui per visualizzare una versione più grande di questa figura. In questo manoscritto è descritto l&#39;uso di un sistema altamente efficace per la costruzione di operoni sintetici multigene sotto il controllo di una polimerasi RNA T7 indottabile ( figura 1 ). Questo sistema permette che molti geni siano espressi simultaneamente da un plasmide. È basato su un sistema plasmidico, originariamente chiamato pKH22, che è stato utilizzato con successo per una serie di applicazioni diverse 6 , 7 , 8 . Qui, questo set di plasmidi viene ampliato per includere quattro vettori correlati: pMGX-A, un vettore di espressione privo di qualsiasi etichetta C- o N-terminale e con il marker di resistenza dell&#39;ampicillina; PMGX-hisA, un vettore di espressione che codifica un tag di esahistidina N-terminale e con il marker di resistenza dell&#39;ampicillina; PMGX-K, un vettore di espressione lAcking qualsiasi etichetta C- o N-terminale e con il marker di resistenza alla kanamycina; E pMGX-hisK, un vettore di espressione che codifica un tag di hexahistidine N-terminale e con il marker di resistenza alla kanamycina. In questo studio è stato dimostrato il metodo per la generazione di un vettore polistronicico contenente cinque geni usando il sistema pMGX, in particolare pMGX-A, insieme alla produzione di ogni singola proteina in Escherichia coli .

<table border="0" cellpadding="0" cellspacing="0" width="1224">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Enzymes</td>
			<td style="width:312px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alkaline Phosphatase, Calf Intestinal (CIP)</td>
			<td style="width:312px;">New England Biolabs</td>
			<td style="width:139px;">M0290S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AvrII</td>
			<td style="width:312px;">New England Biolabs</td>
			<td style="width:139px;">R0174S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EcoRI</td>
			<td style="width:312px;">New England Biolabs</td>
			<td style="width:139px;">R0101S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NdeI</td>
			<td style="width:312px;">New England Biolabs</td>
			<td style="width:139px;">R0111S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XbaI</td>
			<td style="width:312px;">New England Biolabs</td>
			<td style="width:139px;">R0145S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Herculase II Fusion DNA Polymerase</td>
			<td style="width:312px;">Agilent Technologies</td>
			<td style="width:139px;">600677</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA Ligase</td>
			<td style="width:312px;">New England Biolabs</td>
			<td style="width:139px;">M0202S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Name</td>
			<td style="width:312px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Reagents</td>
			<td style="width:312px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 kb DNA ladder</td>
			<td style="width:312px;">New England Biolabs</td>
			<td style="width:139px;">N3232L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-20% Mini-PROTEAN TGX Stain-Free Protein Gels</td>
			<td style="width:312px;">Bio-Rad</td>
			<td style="width:139px;">456-8095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 x TAE</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">BP1332-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">BP1423-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">BP160-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicilin</td>
			<td style="width:312px;">Sigma-Alrich</td>
			<td style="width:139px;">A9518-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BL21 (DE3) chemically comeptent cells</td>
			<td></td>
			<td style="width:139px;"></td>
			<td style="width:448px;">Comeptent cell prepared in house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-PER Bacterial Protein Extraction Reagent</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td>PI78243</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dNTP mix</td>
			<td style="width:312px;">Agilent Technologies</td>
			<td style="width:139px;"></td>
			<td>Supplied with polymerase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel Extraction Kit</td>
			<td style="width:312px;">Omega</td>
			<td style="width:139px;">D2500-02</td>
			<td>E.Z.N.A Gel Extraction, supplied by VWR Cat 3: CA101318-972</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">BP381-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">His Tag Antibody [HRP], mAb, Mouse</td>
			<td style="width:312px;">GenScript</td>
			<td style="width:139px;">A00612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immobilon Western Chemiluminescent HRP Substrate</td>
			<td style="width:312px;">EMD Millipore</td>
			<td style="width:139px;">WBKLS0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IPTG</td>
			<td style="width:312px;">Sigma-Alrich</td>
			<td style="width:139px;">15502-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LB</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">BP1426-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">A411-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteurized instant skim milk powder</td>
			<td style="width:312px;">Local grocery store</td>
			<td style="width:139px;"></td>
			<td>No-name grocery store milk is adequate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Nitrocellulose membrane</td>
			<td style="width:312px;">Amersham Protran (GE Healthcare Life Sciences)</td>
			<td style="width:139px;">10600007</td>
			<td>Membrane PT 0.45 &#181;m 200 mm X 4 m, supplied by VWR Cat #: CA10061-086</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid DNA Isolation Kit</td>
			<td style="width:312px;">Omega</td>
			<td style="width:139px;">D6943-02</td>
			<td>E.Z.N.A Plasmid DNA MiniKit I, supplied by VWR Cat #: CA101318-898</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMGX</td>
			<td style="width:312px;">Boddy Lab</td>
			<td style="width:139px;"></td>
			<td>Request from the Boddy Lab Contact cboddy@uottawa.ca</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primers</td>
			<td style="width:312px;">Intergrated DNA Technologies</td>
			<td style="width:139px;"></td>
			<td>Design primers as needed for desired gene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synthetic Gene</td>
			<td style="width:312px;">Life Technologies</td>
			<td style="width:139px;"></td>
			<td>Design and optimize as needed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thick Blot Filter Paper</td>
			<td style="width:312px;">Bio-Rad</td>
			<td style="width:139px;">1703932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris base</td>
			<td style="width:312px;">BioShop</td>
			<td style="width:139px;">TRS001.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td style="width:312px;">Sigma-Alrich</td>
			<td style="width:139px;">P9416-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XL1-Blue chemically competent cells</td>
			<td></td>
			<td style="width:139px;"></td>
			<td style="width:448px;">Comeptent cell prepared in house</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Name</td>
			<td style="width:312px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Equipment</td>
			<td style="width:312px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BioSpectrometer</td>
			<td style="width:312px;">Eppendorf</td>
			<td style="width:139px;">RK-83600-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel box - PAGE</td>
			<td style="width:312px;">Bio-Rad</td>
			<td style="width:139px;">1658005</td>
			<td>Mini-PROTEIN Tetra Vertical Electrophoresis Cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel Imager</td>
			<td style="width:312px;">Alpha Innotech</td>
			<td style="width:139px;"></td>
			<td>AlphaImager EC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator-oven</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">11-690-650D</td>
			<td>Isotemp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator-shaker</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">SHKE6000-7</td>
			<td>MaxQ 6000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Personna Razors</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">S04615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Power Pack</td>
			<td style="width:312px;">Bio-Rad</td>
			<td style="width:139px;">S65533Q</td>
			<td>FB300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transilluminator</td>
			<td style="width:312px;">VWR International</td>
			<td style="width:139px;">M-10E,6W</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermocylcer</td>
			<td style="width:312px;">Eppendorf</td>
			<td>Z316091</td>
			<td>Mastercycler Personal, supplied by Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV Face-Shield</td>
			<td style="width:312px;"></td>
			<td style="width:139px;">18-999-4542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waterbath</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">15-460-2SQ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Western Transfer Apparatus</td>
			<td style="width:312px;">Bio-Rad</td>
			<td style="width:139px;">1703935</td>
			<td>Mini-Trans Blot Cell</td>
		</tr>
	</tbody>
</table>

inducible t7 rna polymerase-mediated multigene expression system, pmgx

La coespressione di molteplici proteine ​​è sempre più essenziale per la biologia sintetica, studiando complessi proteico-proteici e caratterizzando e sfruttando percorsi biosintetici. In questo manoscritto è descritto l&#39;uso di un sistema altamente efficace per la costruzione di operoni sintetici multigene sotto il controllo di una polimerasi RNA T7 indottabile. Questo sistema permette che molti geni siano espressi simultaneamente da un plasmide. In questo caso, un insieme di quattro vettori correlati, pMGX-A, pMGX-hisA, pMGX-K e pMGX-hisK, con il marker selettivo di resistenza a ampicillina o kanamycina (A e K) e posseggono o mancano di una esahistidina N-terminale (Il suo) sono divulgati. Sono forniti protocolli dettagliati per la costruzione di operoni sintetici che utilizzano questo sistema vettoriale, insieme ai dati corrispondenti, che dimostrano che un sistema basato su pMGX contenente cinque geni può essere facilmente costruito e utilizzato per produrre tutte e cinque le proteine ​​codificate in Escherichia coli . Questo sistemaEm e il protocollo consentono ai ricercatori di esprimere in modo frequente moduli multipli complessi e percorsi in E. coli .

In questo studio, l&#39;obiettivo era quello di co-esprimere cinque proteine ​​da un unico plasmide. I frammenti di gene sintetici ottimizzati a cinque codoni che codificano i tag di esahistidine di N- o C-terminali sono stati acquistati in commercio. I geni sintetici sono stati amplificati mediante PCR e clonati individualmente in un vettore PCR-blunt e sequenziati. Per generare il plasmide policistronico, i cinque geni di interesse sono stati prima clonati in un idoneo plasmide pMG...

Watch this Scientific Journal Video about Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX at JoVE.com

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Questo studio descrive metodi per la coespressione mediata da T7 di geni multipli da un singolo plasmide in Escherichia coli usando il sistema plasmid pMGX.

Inducibile T7 RNA Polymerase-mediato sistema di espressione multigene, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

The overall goal of this procedure is to construct multigene synthetic operons under the control of an inducible T7 RNA polymerase for expressing multiple proteins simultaneously. This method can help researchers in synthetic biology and biochemistry, enabling them to build multi-protein pathways or study protein complexes that are dependent on co-expression multiple approaches. The main advantage of this technique is that it allows multiple genes to expressed simultaneously in Escherichia coli from a single plasma.Demonstrating the procedure from my lab, will be Mohamed Hassan, a PhD student and Angela Thompson, a Co-op student. After obtaining and amplifying genes of interest according to the text protocol, prepare digestion reactions for the first gene of interest and the pMGX factor. Using a 40 microliter reaction containing 0.5 to 1.5 micrograms of DNA into the indel enzyme.Incubate the reactions at 37 degree Celsius for one hour. Then add EcoRI and allow the digest to proceed for an additional hour. NdeI is added prior to EcoRI to ensure that it digests effectively.NdeI is sensitive to the length in double-stranded DNA on either side of the restriction site. Adding it first ensures that it cuts the full-length circular plasma. Load the digestion reactions on to a 0.7%agarose gel and electrophores the samples at 100 volts for 55 minutes.Then, with a clean scalpel or razor blade, excise the insert and vector bands and place the excise gel segments into 1.5 milliliter tubes. Use a gel extraction kit according to the text protocol to purify the DNA. Set up a 20 microliter ligation reaction containing 0.15 to 0.5 micrograms of vector DNA and appropriate amount of insert depending on the size of the gene and one microliter of T4 DNA ligase.Under aseptic conditions, thaw on ice 100 microliter aliquots of chemically competent XL1-Blue E.coli cells for five minutes. And then add five microliters of the ligation reactions containing a gene of interest to the cells. Incubate the transformations on ice for 30 minutes.Next, heat shock the cells for 45 seconds in a 42 degree Celsius water bath. Then, place the transformations on ice and then add 200 microliters of cold LB medium. Leave the cells to incubate for two minutes.Incubate the cells at 37 degree Celsius in 220 rotations per minute for one hour and spread 100 microliters onto LB agar plates containing an appropriate selectable marker. To screen colonies for possible transformants, compare the number of colonies on the negative control versus the ligation plates. A colony count ratio of one to two is desired.Select four to eight individual colonies from each ligation reaction to inoculate 4 milliliters of LB with the appropriate antibiotic. Incubate the cultures at 37 degree Celsius and 220 rotations per minute overnight. With the plasmid DNA isolation kit, purify the plasmid DNA according to the text protocol and set up 20 microliter digestion reactions with 150 to 500 nanograms of DNA using one microliter each of NdeI and EcoRI.Incubate the digestion reactions at 37 degrees Celsius for two hours. To insert gene two into the pMGX vector containing gene one, setup a 40-microliter digestion reaction containing 0.5 to 1.5 micrograms of vector DNA containing the first gene of interest and one microliter of AVR2. Incubate the reaction at 37 degrees Celsius for 1.5 hours before adding 1.5 microliters of Calf-intestinal phosphatase.Incubate for an additional 30 minutes. Next, set up a 40 microliter digestion reaction containing 0.5 to 1.5 micrograms of the pMGX vector containing the second gene of interest. Using one microliter of AVR2 and xpol.Incubate the reaction at 37 degree Celsius for two hours. Electrophoris the restriction digests on 0.7 agarose gel. And use a clean scalpel eraser to excise the insert and vector bands as demonstrated earlier.After extracting the plasmid DNA and quantifying the sample according to the text protocol, set up a ligation reaction using a 3:1 insert to vector ratio of gene 2 and pMGX-yfg1. Include a negative control using pMGX-yfg1 only. Transform five microliters of each reaction into XL1-Blue cells as demonstrated earlier.Then compare the colony count on the negative control and ligation plates. If there are a large number of colonies on the negative control plate, review the CIP treatment. Select four to eight individual colonies from the ligation reaction and use each colony to inoculate four microliter of LB plus the appropriate antibiotic.Grow at 37 degrees Celsius and 220 rotations per minute overnight. After isolating and quantifying the plasmid DNA as before, screen for effective insertion of the second gene by setting up a digestion reaction containing 150 to 500 nanograms of DNA and one microliter of EcoRI. Incubate the reactions at 37 degrees Celsius for two hours.EcoRI is recommended for screening purposes but in some cases, EcoRI may generate two or more inserts that are similar in size which are difficult to distinguish on a gel. To ensure success, alternative restriction digest enzyme such as NdeI should be used. Separate the digestion reactions on an agarose gel, looking for a band that corresponds to the size of the inserted gene 2.Clone additional genes into the plasmid according to the text protocol. To generate the polycistronic plasmid, the five genes were first cloned into a suitable plasmid, pMGX A.This figure shows pCR-Blunt-yfg1 and pCR-Blunt-yfg2 in the pMGX A plasmid as well as pMGX A digested with indel and EcoRI. To clone the first two genes into pMGX A, the plasmid was digested with indel and EcoRI.After cloning, pMGX-yfg1 and pMGX-yfg2 where digested with AVR2 and xpol to confirm that successful clones were obtained. This figure shows the pMGXyfg1 vector digested with AVR2 and the pMGX-yfg2 vector digested with AVR2 and xpol to isolate gene of interest two. The resulting insert was cloned pMGX-yfg1 to create the pMGX-yfg1 two vector.As seen here, digestion of pMGX-yfg1 two with EcoRI confirmed the successful cloning of gene interest number two into pMGX-yfg1. This gel image demonstrates the difference between digestive plasmids that either had the insert in the correct orientation or in a reverse orientation. An insert in the reverse orientation would two EcoRI sites in close proximity making the resulting digested 50 base pair fragments undetectable on the gel and the backbone larger.Finally, after generating a five-gene polycistronic expression vector, a western blot of the expressed proteins confirms that all five proteins were expressed from a single vector. Note that the protein produced by yfg5 is the same size as yfg1 and is not separated on the gel. Once mastered, this technique can be completed in three or four days for every gene inserted into pMGX backbone if it is done correctly.While attempting this procedure, it's important to remember to treat and linearize vector with the phosphatase such as Calf-intestinal phosphatase to dephosphorylate the five vector. Following this procedure, further experiments such as in vivo multigene expression can be performed in order to characterize and harness biosynthetic pathways for the production natural products or to study protein-protein complexes. This technique paved the way for researchers in the synthetic biology field to produce complex carbohydrates and genetically engineered E.Coli.After watching this video, you should have a good understanding of how to implement the multigene expressions system in E.coli.

Research

JoVE Journal

Genetics

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Mappatura delle interazioni RNA-RNA globalmente utilizzando Psoralen biotinilato

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Ricerca

Genetica

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Cinetica di Lagging filamento sintesi del DNA<em&gt; In Vitro</em&gt; Dai replica proteine ​​batteriofago T7

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

Controllable Ion Channel Expression through Inducible Transient Transfection

Controllabile Espressione Ion Canale attraverso inducibile trasfezione transiente

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Sistemi costitutive e inducibile per la modificazione genetica In Vivo degli epatociti Mouse usando l'iniezione della vena della coda idrodinamica

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Rilevamento semi-quantitativa dell'attività della RNA polimerasi RNA-dipendente della proteina di Human Telomerase Reverse Transcriptase

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

Procedura di RNA Pull-down per identificare gli obiettivi di RNA di un RNA lunghi Non codificanti

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

Esplorare il microbioma radice: estrazione dei dati di comunità batteriche dal suolo e la rizosfera radice Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

CRISPR Guide RNA Cloning for Mammalian Systems

CRISPR guida RNA clonazione per sistemi mammiferi

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative ...

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Mantenimento di colture biologiche e misurazione dell'espressione genica in Aphis nerii: un sistema Non-modello per le interazioni pianta-insetto

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms ...

Inducible and Reversible Dominant-negative (DN) Protein Inhibition

Inducible and Reversible Dominant-negative DN Protein Inhibition

Inibizione della proteina viscoelastica e reversibile dominante-negativo (DN)

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based ...