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

Эта работа была поддержана Советом по естественным наукам и инженерным исследованиям Канады.

1. Получение генов интереса <ol><li> Дизайн синтетических генов. <ol><li> Оптимизируйте последовательность генов для экспрессии E. coli . </li><li> Удалите любые проблемные сайты рестрикции из последовательности (AvrII, NdeI, EcoRI и XbaI). </li><li> Включить сайты рестрикции для клонирования; Реком?...

<ol>
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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sialic+acid+and+N-acyl+sialic+acid+analog+production+by+fermentation+of+metabolically+and+genetically+engineered+Escherichia+coli.">Sialic acid and N-acyl sialic acid analog production by fermentation of metabolically and genetically engineered Escherichia coli.</a> Org. Biomol. Chem. 5 (12), 1903-1909 (2007).</li><li>Horsman, M. E., Lundgren, B. R., Boddy, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Acetylneuraminic+Acid+Production+in+Escherichia+coli+Lacking+N-Acetylglucosamine+Catabolic+Machinery.">N-Acetylneuraminic Acid Production in Escherichia coli Lacking N-Acetylglucosamine Catabolic Machinery.</a> Chem. Eng. 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. 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(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>

Совместная экспрессия множественных генов приобретает все большее значение, особенно при характеристике и восстановлении сложных многогенных метаболических путей 3 , 4 , 5 . Система pMGX делает мультигенную совместную экспрессию в рутин...

Совместная экспрессия нескольких белков становится все более существенной, особенно в приложениях для синтетической биологии, где должны быть выражены несколько функциональных модулей 1 ; При изучении белково-белковых комплексов, где экспрессия и функция часто требуют совместного экспрессии 2 , 3 ; И в характеристике и использовании путей биосинтеза, где каждый ген в пути должен быть выражен 4 , 5 , 6 , 7 , 8 . Был разработан ряд систем для совместной экспрессии, особенно в организме хозяина Escherichia coli , рабочей лошади для экспрессии лабораторного рекомбинантного белка 9 . Например, несколько плазмид с различными выбираемыми маркерами могут быть использованы для экспрессии отдельных белков с использованием богатства различных экс<html> Векторы растяжения 10 , 11 . Одиночные плазмидные системы для множественной экспрессии белка использовали либо множественные промоторы для контроля экспрессии каждого гена 10 , 12 ; Синтетические опероны, где множественные гены кодируются на одном транскрипте 2 , 13 ; Или, в некоторых случаях, один ген, кодирующий полипептид, который в конечном счете протеолитически обработан, давая желаемые представляющие интерес белки 14 . <img alt="Рисунок 1" class="xfigimg" src="/files/ftp_upload/55187/55187fig1.jpg" /> Рисунок 1: рабочий процесс pMGX, показывающий построение полицистронного вектора. Система pMGX обеспечивает гибкую и легкую в использовании стратегию для создания синтетических оперонов под контролем индуцибельного промотора T7.E.com/files/ftp_upload/55187/55187fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Пожалуйста, нажмите здесь, чтобы просмотреть увеличенную версию этого рисунка. В этой рукописи описывается использование высокоэффективной системы для построения мультигенных синтетических оперонов под контролем индуцируемой РНК-полимеразы Т7 ( фиг. 1 ). Эта система позволяет одновременно размножать гены из одной плазмиды. Он основан на плазмидной системе, первоначально называемой pKH22, которая была успешно использована для ряда различных приложений 6 , 7 , 8 . Здесь этот набор плазмид расширяется до четырех связанных векторов: pMGX-A, экспрессирующего вектора, лишенного любых C- или N-концевых тегов, и маркера устойчивости к ампициллину; PMGX-hisA, вектор экспрессии, кодирующий N-концевую гексагистидиновую метку и маркер устойчивости к ампициллину; PMGX-K, вектор экспрессии lПодбирая любые C- или N-концевые метки и маркер устойчивости к канамицину; И pMGX-hisK, вектор экспрессии, кодирующий N-концевую гексагистидиновую метку и маркер устойчивости к канамицину. В этом исследовании продемонстрирован способ генерации полицистронного вектора, содержащего пять генов с использованием системы pMGX, в частности pMGX-A, наряду с успешным продуцированием каждого отдельного белка в 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

Совместная экспрессия нескольких белков приобретает все большее значение для синтетической биологии, изучает белково-белковые комплексы и характеризует и использует пути биосинтеза. В этой рукописи описывается использование высокоэффективной системы для построения мультигенных синтетических оперонов под контролем индуцируемой РНК-полимеразы Т7. Эта система позволяет одновременно размножать гены из одной плазмиды. Здесь набор из четырех связанных векторов, pMGX-A, pMGX-hisA, pMGX-K и pMGX-hisK, с маркером (A и K), устойчивым к ампициллину или канамицину, и либо обладающим, либо отсутствующим N-концевым гексагистидином Тег (его). Подробные протоколы для построения синтетических оперонов с использованием этой векторной системы предоставляются вместе с соответствующими данными, показывая, что система на основе pMGX, содержащая пять генов, может быть легко сконструирована и использована для получения всех пяти кодированных белков в Escherichia coli . Эта системаEm и протокол позволяют исследователям регулярно выражать сложные многокомпонентные модули и пути в E. coli .

В этом исследовании целью было совместное экспрессирование пяти белков из одной плазмиды. Исправленные пять кодонов синтетические фрагменты гена, кодирующие либо N-, либо C-концевые гексагистидиновые метки, были куплены коммерчески. Синтетические гены амплифицирова?...

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

В этом исследовании описаны способы опосредованной T7 совместной экспрессии нескольких генов из одной плазмиды в Escherichia coli с использованием плазмидной системы pMGX.

Индуцируемая T7-РНК-полимераза-опосредованная мультигенная экспрессия, 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

Сопоставление взаимодействия РНК-РНК в глобальном масштабе с использованием биотинилированного псоралена

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 ...

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

Кинетика синтеза Отставание-нити ДНК<em&gt; In Vitro</em&gt; С помощью бактериофага Т7 белков репликации

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

Управляемая Expression ионного канала через Индуцибельные временная трансфекция

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

Учредительные и индуцибельной систем для генетических в естественных условиях изменения мыши гепатоцитов, используя инъекции Вену гидродинамических хвост

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

Полуколичественного обнаружение РНК зависимой РНК-полимеразы активности человека теломеразы транскриптазы белка

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

РНК Pull-down процедура для определения целей РНК РНК, длиной некодирующих

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

Изучения корня микрофлора: извлечение данных бактериальных сообщества из почвы, ризосфере и корень 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

ТРИФОСФАТЫ руководство РНК клонирования млекопитающих систем

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

Сохранение биологических культур и измерения экспрессии генов в nerii тля: Non модель системы для взаимодействия растений насекомое

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

Ингибирование белков индуцибельной и обратимым Доминант отрицательной (DN)

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