Wir danken Dr. Dr. Michael Cashel (National Institutes of Health, Bethesda, MA, USA) für die Zusendung Bakterienstämme, Plasmide und notwendigen Protokolle. Wir danken Dr. John Glover (Department of Biochemistry, University of Toronto) für die Nutzung des SpecraMax Plattenlesegerät. Großbritannien ist der Empfänger einer Nationalen Sciences and Engineering Research Council of Canada (NSERC) Postgraduate-Stipendium, ein kanadischer Institutes of Health Research Strategic Training Program in die Strukturbiologie von Membranproteinen Verbunden mit Krankheit, und der University of Toronto öffnen Fellowship. Diese Arbeit wurde durch einen Zuschuss von der Canadian Institutes of Health Research (MOP-67210) bis WAH unterstützt.

1) Reagenzien und Geräte <ol><li> Zuerst bereiten Sie die folgenden drei Lösungen: 1 ml Lösung A, die aus 8 mM L-Lysin, 100 mM Natrium-2 wird - (N-Morpholino) ethansulfonsäure (MES) pH 6,5, 0,2 mM Nukleotid, wo die Nukleotid entweder: Guanosin-diphosphat (GDP), Guanosin-Triphosphat (GTP), Guanosin-5&#39;-diphosphat, 3&#39;-diphosphat (ppGpp) oder Guanosin-5&#39;-Triphosphat, 3&#39;-diphosphat (pppGpp), 0,1 mM Pyridoxal-5&#39;-Phosphat- (PLP) und 1 mM β-Mercaptoethanol / (β-ME). 1 ml Lösung B, die von 100 mM Natrium-MES pH 6,5, 0,1 mM PLP, 1 mM β-ME und 50 nM LdcI besteht. LdcI wurde gereinigt, wie in Snider et al. 6 und Kanjee et al. 14. 1 ml Lösung C, die identisch mit Lösung B ist aber nicht enthalten LdcI. </li><li> Bereiten Sie 100 mL der Stop-Lösung, bestehend aus 1 M Natriumcarbonat (10,6 g/100 ml) und 50 ul Aliquot in jedes Well einer 96-Well-Polystyrol-Platte mit einem Mehrkanal-Pipette. Add 30 ul Wasser auf die Platte und dann die Abdeckung. Beachten Sie, dass die Summe der Menge Wasser zugegeben und das Volumen der Probe während der Enzymreaktion extrahiert müssen 50 ul gleich. In diesem Fall werden 20 ul der Enzymreaktion Probe entfernt werden. </li><li> Bereiten Sie 5 ml TNBS Lösung bei 10 mM durch Verdünnung 294 ul von 5% (w / v) TNBS Lager mit 4706 ul Wasser, in Alufolie wickeln und halten auf dem Eis. </li><li> Äquilibrieren der Eppendorf Thermostat und bei 37 ° C. Der Thermostat und verfügt über 24 Brunnen in 6 Spalten. Siehe Abbildung 2 für eine schematische. Label 1,5 ml Eppendorf-Röhrchen unter Angabe der Identität des Nukleotids, die getestet (eine pro Spalte) werden: GTP (Spalte A), BIP (Spalte B), ppGpp (Spalte C), pppGpp (Spalte D), keine Nukleotid (Spalte E ) und Protein-Proben (Spalte F). </li><li> Rack mehrere Kisten mit 200 ul Tipps, so dass jeder abwechselnd hintereinander ausgelassen wird somit insgesamt vier Reihen von Spitzen. Dies ist notwendig für die Nutzung der Multi-Kanal-Pipette mit dem Plus-ThermoStat heatblock während der Enzym-Assay. </li><li> Äquilibrieren Digitale Heatblock (VWR) bei 42 ° C. </li><li> Legen Sie eine 96-well 2,0 ml Polypropylen-Platte auf Eis zu kühlen. </li></ol> 2) LdcI Assay <ol><li> Aliquot 50 ul Lösung A mit dem entsprechenden Nukleotid bis 1,5 mL Eppendorf-Röhrchen in jedem der fünf Säulen des Eppendorf Thermostat. </li><li> Add 330 ul Lösung B auf drei Röhren in Spalte F und fügen 330 ul Lösung C (die nicht-Protein-control), um die endgültige Rohr in Spalte F. </li><li> Äquilibrieren Sie die Lösungen bei 37 ° C für fünf Minuten. Tipp: nach 5 Minuten, schneiden Sie die Kappen der Röhren in der Mitte des Heiz-block, sie stören die Mehrkanalpipette zum nächsten verwendet werden zu verhindern. </li><li> Mit einer 5-50 ul VWR Mehrkanal-Pipette 50 ul aus den Rohren in Spalte F in jedem der Rohre in den Spalten AE. Starten Sie den Timer, sobald das erste Rohr vermischt wird. </li><li> Bei 2, entfernen 4 und 6 Minuten 20 ul der Probe mit dem Mehrkanal-Pipette und fügen Sie die Stop-Lösung. </li><li> Hinweis: Proben in Stop-Lösung kann in Plastikfolie abgedeckt und bei -20 ° C für die nachfolgende Verarbeitung. Die Platten sollten bei Raumtemperatur vor der Reaktion mit TNBS aufgetaut werden. </li></ol> 3) TNBS Reaction und Farbentwicklung <ol><li> Add 50 ul 10 mM TNBS Lösung für die Stop-Lösung mit dem Mehrkanal-Pipette und dann Inkubieren bei 42 ° C für 6 Minuten. Die Lösung wird wiederum ein dunkles gelb / orange Farbe wie die TNBS reagiert mit dem Lysin und Cadaverin. </li><li> Nach 6 Minuten, kühlen die Platte auf Eis zu einer Verlangsamung der Reaktion. </li><li> Nehmen Sie 100 ul der Probe mit einem VWR 20-200 ul Mehrkanalpipette und Platz in der 2 mL Deepwellplatte, dass auf Eis gekühlt wurde. Transfer-Eis-Eimer zum Abzug durchzuführen. </li><li> Add 500 mL Toluol in jede Vertiefung mit einem HandyStep (Brand) zu wiederholen Pipette und 12,5 ml Pipettenspitze (Plastibrand). </li><li> Wischen Sie überschüssiges Toluol mit einem Kimwipe und die Platte abgedeckt mit Streifen von Packband. Achten Sie darauf, fest andrücken, um eine gute Abdichtung zu erreichen. Decken Sie die 96-Well-Platte mit einem flachen Deckel und dann kräftig schütteln für 1 Minute und 30 Sekunden. </li><li> Verlassen Sie die Lösungen für 5 Minuten ruhen lassen. Die TNP-Cadaverin ist nun in der oberen Toluolphase, während der TNP-Lysin bleibt wasserlöslich (siehe Abbildung 1). </li><li> Nehmen Sie 200 ul von Toluol mit dem 20-200 ul Mehrkanalpipette in eine 96-Well-Platte für das Lesen. Hinweis: Achten Sie darauf, um zu überprüfen, dass jede Probe entfernt ist klar und hat keine der unteren wässrigen Phase. Anmerkung: Mit Barriere Tipps, um Schäden an Mehrkanal-Pipette durch das Toluol zu verhindern. </li><li> Extinktion bei 340 nm in einem SpectraMax 340 Platten-Lesegerät. </li><li> Zur Reinigung der Quarzplatte, spülen Sie das Toluol mit Wasser und legen Sie die Quarzplatte in einer großenGlasschale im Abzug. Pour über 100 ml einer 7:3-Mischung von 70% (v / v) Salpetersäure: 95% (v / v) Ethanol und decken die Quarzplatte mit einer zweiten Glasschale. Achtung: Die Reaktion ist stark exotherm und manchmal explosive tragen angemessenen Schutz und sicherzustellen, dass die Abzugshaube Schärpe um den unteren 6 &quot;eingestellt ist nach dem Abkühlen auswaschen Salpetersäure mit Wasser und dann 95% Ethanol.. </li></ol> 4) Repräsentative Ergebnisse 1) Cadaverin Standardkurve (Abbildung 3) Der Test wurde durchgeführt, wie beschrieben, aber ohne L-Lysin oder LdcI und stattdessen verschiedene Konzentrationen von Cadaverin wurden verwendet, um empirisch die lineare Bereich des Assays. Der Assay wurde linear bis zu einer OD 340 von 0,25, entsprechend ~ 22 nmol Cadaverin (Abbildung 3). 2) Die Aktivität der LdcI (Abbildung 4) Die Aktivität von LdcI allein bestimmt wurde auf 153,5 (± 18,1) nmol Cadaverin min-1 pg LdcI-1 bei pH 6,5 werden. Die Aktivität von LdcI ist unberührt in Gegenwart von 100 uM GTP oder GDP aber stark gehemmt wird (&gt; 10-fach) in Gegenwart von pppGpp und ppGpp (Abbildung 4). <img src="/files/ftp_upload/2094/2094fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Schematische Darstellung der LdcI Reaktion. Die Decarboxylierung von L-Lysin zu CO 2 und Cadaverin zu erzeugen, ist ebenso wie die anschließende Umsetzung mit TNBS bei hohen pH-Wert auf N, N&#39;-bistrinitrophenylcadaverine (TNP-Cadaverin) und N, N erzeugen gezeigt &#39;-bistrinitrophenyllysine (TNP-Lysin). Basierend auf Phan et al.11 <img src="/files/ftp_upload/2094/2094fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Einrichten von Eppendorf Thermostat. Das Layout der Proben in der 24-Well-Eppendorf Thermostat ist zusammen mit Beschreibung der einzelnen Schritte des Assays (angezeigt in Fettdruck) gezeigt. Die Pfeile zeigen die Übertragung von Reaktionslösungen nach dem Protokoll. Das Entfernen der Reaktionslösung auf die Stop-Lösung in der 96-Well-Platte ist ebenfalls angegeben. <img src="/files/ftp_upload/2094/2094fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Cadaverin Standardkurve. Ein Plot der Absorption bei 340 nm gegen nmol Cadaverin wird angezeigt. Fehlerbalken stellen die Standardabweichung von mindestens drei unabhängigen Messungen. Die Trendlinie wird auch gezeigt, und hat einen R2 von 0,996. <img src="/files/ftp_upload/2094/2094fig4.jpg" alt="Abbildung 4" /> Abbildung 4. LdcI Testergebnisse. Eine Darstellung der Rate von LdcI Aktivität (in nmol Cadaverin produziert min-1 pg-1 LdcI) wird in Gegenwart von 100 uM GTP, GDP, pppGpp, ppGpp und in Abwesenheit von Nukleotid gezeigt. Die strengen Antwort Nukleotide (p) ppGpp der Lage sind, signifikant zu inhibieren LdcI Aktivität. Die Fehlerbalken stellen die Standardabweichung von mindestens sechs unabhängigen Messungen.

<ol>
	<li>Gale, E. F., Epps, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+the+pH+of+the+medium+during+growth+on+the+enzymic+activities+of+bacteria+(Escherichia+coli+and+Micrococcus+lysodeikticus)+and+the+biological+significance+of+the+changes+produced.">The effect of the pH of the medium during growth on the enzymic activities of bacteria (Escherichia coli and Micrococcus lysodeikticus) and the biological significance of the changes produced.</a> Biochem J. 36, 600-618 (1942).</li><li>Foster, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+acid+resistance:+tales+of+an+amateur+acidophile.">Escherichia coli acid resistance: tales of an amateur acidophile.</a> Nat Rev Microbiol. 2, 898-907 (2004).</li><li>Castanie-Cornet, M. P., Penfound, T. A., Smith, D., Elliott, J. F., Foster, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+acid+resistance+in+Escherichia+coli.">Control of acid resistance in Escherichia coli.</a> J Bacteriol. 181, 3525-3535 (1999).</li><li>Iyer, R., Williams, C., Miller, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine-agmatine+antiporter+in+extreme+acid+resistance+in+Escherichia+coli.">Arginine-agmatine antiporter in extreme acid resistance in Escherichia coli.</a> J Bacteriol. 185, 6556-6561 (2003).</li><li>Sabo, D. L., Boeker, E. A., Byers, B., Waron, H., Fischer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+physical+properties+of+inducible+Escherichia+coli+lysine+decarboxylase.">Purification and physical properties of inducible Escherichia coli lysine decarboxylase.</a> Biochemistry. 13, 662-670 (1974).</li><li>Snider, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+a+distinctive+complex+between+the+inducible+bacterial+lysine+decarboxylase+and+a+novel+AAA++ATPase.">Formation of a distinctive complex between the inducible bacterial lysine decarboxylase and a novel AAA+ ATPase.</a> J Biol Chem. 281, 1532-1546 (2006).</li><li>Kashiwagi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coexistence+of+the+genes+for+putrescine+transport+protein+and+ornithine+decarboxylase+at+16+min+on+Escherichia+coli+chromosome.">Coexistence of the genes for putrescine transport protein and ornithine decarboxylase at 16 min on Escherichia coli chromosome.</a> J Biol Chem. 266, 20922-20927 (1991).</li><li>Schneider, G., Kack, H., Lindqvist, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+manifold+of+vitamin+B6+dependent+enzymes.">The manifold of vitamin B6 dependent enzymes.</a> Structure. 8, 1-6 (2000).</li><li>Cashel, M., Gentry, D. R., Hernandez, V. J., Vinella, D., Curtiss, R., Neidhardt, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Escherichia coli and Salmonella : cellular and molecular biology. , 1458-1496 (1996).</li><li>Magnusson, L. U., Farewell, A., Nystrom, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ppGpp:+a+global+regulator+in+Escherichia+coli.">ppGpp: a global regulator in Escherichia coli.</a> Trends in Microbiology. 13, 236-242 (2005).</li><li>Phan, A. P., Ngo, T. T., Lenhoff, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+assay+for+lysine+decarboxylase.">Spectrophotometric assay for lysine decarboxylase.</a> Anal Biochem. 120, 193-197 (1982).</li><li>Park, Y. K., Bearson, B., Bang, S. H., Bang, I. S., Foster, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+pH+crisis,+lysine+decarboxylase+and+the+acid+tolerance+response+of+Salmonella+typhimurium.">Internal pH crisis, lysine decarboxylase and the acid tolerance response of Salmonella typhimurium.</a> Mol Microbiol. 20, 605-611 (1996).</li><li>Merrell, D. S., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cadA+gene+of+Vibrio+cholerae+is+induced+during+infection+and+plays+a+role+in+acid+tolerance.">The cadA gene of Vibrio cholerae is induced during infection and plays a role in acid tolerance.</a> Mol Microbiol. 34, 836-849 (1999).</li><li>Kanjee, U., Gutsche, I., Alexopoulos, E., Zhao, B., Bakkouri, M. E. l., Thibault, G., Liu, K., Ramachandran, S., Snider, J., Pai, E. F., Houry, W. A., A, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linkage+between+the+Bacterial+Acid+Stress+and+Stringent+Responses+Revealed+by+the+Structure+of+the+Inducible+Lysine+Decarboxylase.">Linkage between the Bacterial Acid Stress and Stringent Responses Revealed by the Structure of the Inducible Lysine Decarboxylase.</a> EMBO Journal. 30, 931-944 (2011).</li></ol>

In der Lysin-Decarboxylase-Test ist TNBS mit dem primären Aminen von L-Lysin und Cadaverin reagierte auf TNP-Lysin und TNP-Cadaverin Addukte (Abbildung 1). Durch die Anwesenheit der Carbonsäuregruppe auf TNP-Lysin, bleibt dieses Addukt in Wasser löslich ist, während der TNP-Cadaverin, ohne die Carbonsäuregruppe, ist in der Lage Aufteilung in Toluol 11. Diese Art von Test kann im weiteren Sinne auf andere Arten von Aminosäuren, wo der Verlust einer Carbonsäuregruppe tritt bei der Reaktion genutzt werden...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2,4,6-trinitrobenzensulfonic acid </td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P2297</td>
<td>&nbsp;</td>
<tr>
<td>0.1 mM pyridoxal 5'-phosphate (PLP)</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>P9255</td>
<td>&nbsp;</td>
<tr>
<td>Cadaverine</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>D22606</td>
<td>&nbsp;</td>
<tr>
<td>96 well polystyrene plates</td>
<td>&nbsp;</td>
<td>Sarstedt</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>96-well quartz plate</td>
<td>&nbsp;</td>
<td>Hellma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>VWR Digital Heatblock</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ThermoStat Plus</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>2.0 mL 96-well polypropylene plates</td>
<td>&nbsp;</td>
<td>Axygen</td>
<td>P-DW-20-C</td>
<td>&nbsp;</td>
<tr>
<td>Handy Step Repeat Pipettor</td>
<td>&nbsp;</td>
<td>Brand</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>12.5 mL Repeat Pipettor Tips</td>
<td>&nbsp;</td>
<td>Brand (Plastibrand)</td>
<td>702378</td>
<td>&nbsp;</td>
<tr>
<td>SpectraMax 340PC Plate Reader</td>
<td>&nbsp;</td>
<td>SpectraMax</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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an assay for measuring the activity of escherichia coli inducible lysine decarboxyase

Escherichia coli ist ein Bakterium, das in der Lage enterischen wachsender über einen weiten Bereich von pH-Werten (pH 5 bis 9) ist 1 und, unglaublich, ist in der Lage, extreme Säure betont einschließlich Passage durch den Magen von Säugetieren, wo der pH-Wert fallen können, um so niedrig überleben wie pH 1 bis 02 Februar. Um ein so breites Spektrum von sauren pH Überleben, E. coli besitzt vier verschiedenen induzierbaren Aminosäure Decarboxylasen dass decarboxylieren ihrem Substrat Aminosäuren in ein Proton-abhängigen Weise überhöhen dadurch die internen pH-Wert. Die Decarboxylasen gehören Glutaminsäure Decarboxylasen GADA und GadB 3, der Arginin-Decarboxylase Adia 4, die Lysin-Decarboxylase LdcI 5, 6 und die Ornithindecarboxylase SPEF 7. Alle diese Enzyme nutzen Pyridoxal-5&#39;-Phosphat als Co-Faktor 8 und Funktion zusammen mit der inneren Membran-Substrat-Produkt Antiporter dass Decarboxylierung Produkte mit dem externen Medium im Austausch für frisches Substrat 2 zu entfernen. Im Falle von LdcI, wird das Lysin-Cadaverin Antiporter genannt Cadb. Kürzlich haben wir festgestellt, das X-ray Kristallstruktur von LdcI bis 2,0 Å, und wir entdeckten ein neues kleines Molekül gebunden LdcI die strengen Response-Regulator Guanosin-5&#39;-diphosphat, 3&#39;-diphosphat (ppGpp) 14. Die strengen Reaktion tritt ein, wenn exponentiell wachsenden Zellen Nährstoffmangel oder eines einer Reihe von anderen betont 9 Erfahrungspunkte. Als Folge produzieren die Zellen ppGpp, die zu einer Signalkaskade ihren Höhepunkt in der Verlagerung von exponentiellem Wachstum bis zur stationären Phase das Wachstum 10 führt. Wir haben gezeigt, dass ppGpp ein spezifischer Inhibitor der LdcI 14 ist. Hier beschreiben wir die Lysin-Decarboxylase-Test aus dem Test von Phan et al modifiziert. 11, die wir verwendet haben, um die Aktivität von LdcI und die Wirkung der pppGpp / ppGpp diese Tätigkeit zu bestimmen. Die LdcI Decarboxylierung entfernt die α-Carboxygruppe von L-Lysin und produziert Kohlendioxid und das Polyamin Cadaverin (1,5-Diaminopentan) 5. L-Lysin und Cadaverin kann mit 2,4,6-trinitrobenzensulfonic (TNBS) bei hohen pH umgesetzt werden, um N, N&#39;-bistrinitrophenylcadaverine (TNP-Cadaverin) und N, N&#39;-bistrinitrophenyllysine (TNP-Lysin) zu generieren bzw. 11. Die TNP-Cadaverin aus der TNP-Lysin als der ehemalige löslich in organischen Lösungsmitteln wie Toluol, während letzteres nicht (siehe Abbildung 1) getrennt werden. Der lineare Bereich des Assays wurde empirisch ermittelt unter Verwendung von gereinigten Cadaverin.

Watch this Scientific Journal Video about An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase at JoVE.com

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Die Aktivität der induzierbaren Lysin-Decarboxylase wird durch Umsetzung des Substrates L-Lysin und das Produkt Cadaverin mit 2,4,6-trinitrobenzensulfonic Säure-Addukte, die unterschiedliche Löslichkeit in Toluol Form überwacht.

Ein Assay zur Messung der Aktivität von Escherichia coli Inducible Lysin Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

an-assay-for-measuring-activity-escherichia-coli-inducible-lysine

Research

JoVE Journal

Biology

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

14.1K Aufrufe.  University of Toronto. Die Aktivität der induzierbaren Lysin-Decarboxylase wird durch Umsetzung des Substrates L-Lysin und das Produkt Cadaverin mit 2,4,6-trinitrobenzensulfonic Säure-Addukte, die unterschiedliche Löslichkeit in Toluol Form überwacht.

Serie: An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

Die Messung der Biegesteifigkeit von Bakterienzellen mit einer optischen Falle

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Forschung

Biologie

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

High Throughput Screening von Fungal Endoglucanaseaktivität in Escherichia coli</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

Die Anwendung des MassSQUIRM für quantitative Messungen von Lysin-Demethylase

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Rapid-Protokoll für die Herstellung von Elektrokompetente<em&gt; Escherichia coli</em&gt; Und<em&gt; Vibrio cholerae</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

Protokolle für die Implementierung einer<em&gt; Escherichia coli</em&gt; Basierend TX-TL Zellfreie Expression System für Synthetische Biologie

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

Erkennung von Live-<em&gt; Escherichia coli</em&gt; O157: H7-Zellen durch PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-basierte Screening Expression von Membranproteinen in<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

Die vielfältigen Vorteile von Protein Co-expression in<em&gt; Escherichia coli</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

Rückstands spezifische Einbau nichtkanonischer Aminosäuren in Modellproteine ​​eine Verwendung<em&gt; Escherichia coli</em&gt; Zellfreie Transkription-Translation-System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 &#176;C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

The enzymatic activity of lactase is essential for the catabolic processing of the disaccharide lactose. Here, the activity of lactase found in dietary supplements is assayed using a colorimetric assay. This provides students with an experimental platform for understanding the activity of lactase and enzyme kinetics.

Messung der Laktase enzymatische Aktivität in das Lehrlabor

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

Ein Assay zur Messung der Aktivität von Escherichia coli Inducible Lysin Decarboxyase

Zusammenfassung

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Zusammenfassung

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