Ringraziamo il Dr. Dr. Michael Cashel (National Institutes of Health, Bethesda, MA, USA) per averci inviato ceppi batterici, plasmidi e protocolli necessari. Ringraziamo il Dr. John Glover (Dipartimento di Biochimica, Università di Toronto) per l&#39;utilizzo del lettore di piastre SpecraMax. Regno Unito è il destinatario di una Nazionale di Scienze e Ingegneria Research Council del Canada (NSERC) Borsa di studio post-laurea, un Canadian Institutes of Research Program Formazione Salute strategica nella biologia strutturale delle proteine ​​di membrana collegato a malattie, e una borsa di studio dell&#39;Università di Toronto Open. Questo lavoro è stato sostenuto da un finanziamento della Canadian Institutes of Health Research (MOP-67210) per WAH.

1) Reagenti ed apparecchiature <ol><li> In primo luogo, preparare le seguenti tre soluzioni: 1 ml di soluzione A, che si compone di 8 mM L-lisina, 100 mM di sodio 2 - (N-Morpholino) ethanesulphonic acido (MES) pH 6,5, 0,2 mM nucleotide, in cui il nucleotide è o: guanosina difosfato (GDP), guanosina trifosfato (GTP), guanosina 5&#39;-difosfato, 3&#39;-difosfato (ppGpp), o guanosina 5&#39;-trifosfato, 3&#39;-difosfato (pppGpp), 0,1 mM piridossal 5&#39;-fosfato (PLP), e 1 mM β-mercaptoetanolo / (β-ME). 1 ml di soluzione B che consiste in 100 mM di sodio MES pH 6,5, 0,1 mM PLP, 1 mM β-ME, e 50 nM LdcI. LdcI è stato purificato come descritto nella Snider et al. 6 e Kanjee et al. 14. 1 ml di soluzione C che è identico a B Solution, ma non contiene LdcI. </li><li> Preparare 100 mL di soluzione bloccante costituita da carbonato di sodio 1 M (10,6 g/100 ml) e un&#39;aliquota 50 microlitri in ciascun pozzetto di una piastra a 96 pozzetti in polistirene con una pipetta multicanale. Aggiungere 30 ml di acqua al piatto e poi coprire. Si noti che la somma del volume di acqua aggiunta e il volume di campione estratti durante la reazione enzimatica deve essere uguale a 50 microlitri. In questo caso, 20 l di campione reazione enzimatica saranno rimossi. </li><li> Preparare 5 ml di soluzione TNBS a 10 mm diluendo 294 ml di 5% (w / v) magazzino TNBS con 4706 microlitri di acqua, avvolgetela in un foglio di alluminio e tenere in ghiaccio. </li><li> Equilibrare la ThermoStat Eppendorf Plus. a 37 ° C. Plus ThermoStat ha 24 pozzi in 6 colonne. Vedere la Figura 2 per schematico. Etichetta 1,5 ml provette Eppendorf indicando l&#39;identità del nucleotide che saranno testati (uno per colonna): GTP (colonna A), il PIL (colonna B), ppGpp (colonna C), pppGpp (colonna D), non nucleotide (colonna E ), e campioni di proteine ​​(colonna F). </li><li> Scatole rack diverse punte di 200 microlitri in modo tale che ogni riga si alternano viene omesso per un totale di quattro file di punte. Ciò è necessario per l&#39;utilizzo della pipetta multicanale con il termostato e heatblock durante il dosaggio degli enzimi. </li><li> Equilibrare Digital Heatblock (VWR) a 42 ° C. </li><li> Posizionare un 96-piastra ben 2,0 ml in polipropilene sul ghiaccio per raffreddare. </li></ol> 2) analisi LdcI <ol><li> Aliquota 50 ml di soluzione A con il nucleotide adeguate per 1,5 ml provette Eppendorf in ognuna delle cinque colonne del ThermoStat Eppendorf. </li><li> Aggiungere 330 ml di soluzione B a tre tubi in colonna F e aggiungere 330 ml di soluzione C (la no-proteina di controllo) al tubo finale nella colonna F. </li><li> Equilibrare le soluzioni a 37 ° C per cinque minuti. Suggerimento: dopo 5 minuti, tagliare i tappi dei tubi al centro del riscaldamento blocco per impedire loro di interferire con la pipetta multicanale da utilizzare prossimo. </li><li> Utilizzando un microlitri 5-50 VWR multicanale pipetta, trasferire 50 microlitri dai tubi nella colonna F in ciascuno dei tubi in colonne AE. Avviare il timer non appena il primo tubo è misto. </li><li> A 2, 4, e 6 minuti togliere 20 l di campione con il multi-canale pipetta e aggiungere alla soluzione di stop. </li><li> Nota: i campioni in soluzione di arresto può essere coperto in involucro di plastica e congelato a -20 ° C per l&#39;elaborazione successiva. Le piastre devono essere scongelati a temperatura ambiente prima reazione con TNBS. </li></ol> 3) TNBS di reazione e di sviluppo del colore <ol><li> Aggiungere 50 ml di soluzione di mm 10 TNBS alla soluzione di smettere di usare il multi-canale pipetta e poi incubare a 42 ° C per 6 minuti. La soluzione diventerà di un colore giallo scuro / arancione come TNBS reagisce con la lisina e la cadaverina. </li><li> Dopo 6 minuti, raffreddare la piastra su ghiaccio per rallentare la reazione. </li><li> Rimuovere 100 l di campione mediante una VWR 20-200 microlitri pipetta multicanale e posto nel piatto 2 ml pozzo profondo che è stato raffreddato in ghiaccio. Trasferimento di ghiaccio secchiello per il fumehood. </li><li> Aggiungere 500 microlitri toluene in ogni pozzetto utilizzando una HandyStep (Brand) pipettatore ripetere e 12,5 mL puntale (PLASTIBRAND). </li><li> Togliere ogni eccesso toluene utilizzando un kimwipe e coprire il piatto con le strisce di nastro da imballaggio. Assicurati di premere con decisione per ottenere una buona tenuta. Coprire la piastra a 96 pozzetti con un coperchio piatto e poi agitare energicamente per 1 minuto e 30 secondi. </li><li> Lasciare le soluzioni per risolvere per 5 minuti. Il TNP-cadaverina è ora in fase superiore toluene, mentre il TNP-lisina rimane solubile in acqua (vedi Figura 1). </li><li> Prelevare 200 ml di toluene con il 20-200 microlitri multicanale pipetta in una piastra da 96 pozzetti per la lettura. Nota: assicuratevi di controllare che ogni campione viene rimosso è chiaro e non ha alcun fine della fase acquosa. Nota: consigli per l&#39;uso barriera per evitare danni alla pipetta multicanale dal toluene. </li><li> Leggere l&#39;assorbanza a 340 nm in un lettore di piastre SpectraMax 340. </li><li> Per pulire la piastra di quarzo, sciacquare il toluene con acqua e posizionare la piastra di quarzo in un grandevassoio di vetro della cappa. Versare sopra 100 ml di una miscela 7:03 del 70% (v / v) di acido nitrico: 95% (v / v) di etanolo e coprire la piastra di quarzo con un vassoio di vetro secondo. Attenzione: la reazione è altamente esotermica protezione contro l&#39;usura e, a volte esplosiva e accertarsi che l&#39;anta cappa è impostato per abbassare il &quot;livello 6 Dopo il raffreddamento, l&#39;acido nitrico lavare con acqua e poi etanolo al 95%.. </li></ol> 4) Rappresentante Risultati 1) cadaverina curva standard (Figura 3) Il test è stato eseguito come descritto ma senza L-lisina o LdcI e le concentrazioni invece diverse cadaverina sono stati utilizzati per determinare empiricamente la gamma lineare del test. Il test è stato lineare a un OD 340 di 0,25, corrispondente a ~ 22 nmoli di cadaverina (Figura 3). 2) Attività di LdcI (Figura 4) L&#39;attività di LdcI solo è stato determinato in 153,5 (± 18.1) nmoli cadaverina min-1 mg LdcI-1 a pH 6,5. L&#39;attività di LdcI non è influenzato in presenza di 100 mM GTP o PIL, ma è fortemente inibita (&gt; 10 volte) in presenza di pppGpp e ppGpp (Figura 4). <img src="/files/ftp_upload/2094/2094fig1.jpg" alt="Figura 1" /> Figura 1. Schema della reazione LdcI. La reazione di decarbossilazione della L-lisina per generare CO 2 e cadaverina è mostrata così come la reazione successiva con TNBS a pH elevato per generare N, N-bistrinitrophenylcadaverine (TNP-cadaverina) e N, N &#39;-bistrinitrophenyllysine (TNP-lisina). Sulla base di Phan et al.11 <img src="/files/ftp_upload/2094/2094fig2.jpg" alt="Figura 2" /> Figura 2. Impostazione del termostato Eppendorf. La disposizione dei campioni nei 24 pozzetti ThermoStat Eppendorf è indicato insieme con la descrizione delle fasi del test (indicata in grassetto). Le frecce indicano il trasferimento di soluzioni di reazione secondo il protocollo. Rimozione della soluzione di reazione alla soluzione fermare nella piastra a 96 pozzetti è anche indicato. <img src="/files/ftp_upload/2094/2094fig3.jpg" alt="Figura 3" /> Figura 3. Cadaverina curva standard. Un grafico dell&#39;assorbanza a 340 nm contro nmoli di cadaverina è mostrato. Barre di errore rappresentano la deviazione standard di almeno tre misurazioni indipendenti. La retta di regressione è anche mostrato e ha una R2 di 0,996. <img src="/files/ftp_upload/2094/2094fig4.jpg" alt="Figura 4" /> Figura 4. Risultati del dosaggio LdcI. Un grafico del tasso di attività LdcI (in nmoli cadaverina prodotta min-1-1 mg LdcI) è indicato in presenza di mM GTP 100, PIL, pppGpp, ppGpp e in assenza di nucleotide. I nucleotidi risposta stringenti (p) ppGpp sono in grado di inibire in modo significativo l&#39;attività LdcI. Le barre di errore rappresentano la deviazione standard di almeno sei misurazioni indipendenti.

<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>

Nessun conflitto di interessi dichiarati.

Nel test lisina decarbossilasi, TNBS viene fatto reagire con le ammine primarie di L-lisina e cadaverina per formare TNP-lisina e TNP-cadaverina addotti (Figura 1). A causa della presenza del gruppo carbossilico sul TNP-lisina, questo addotto rimane solubile in acqua, mentre il TNP-cadaverina, manca il gruppo carbossilico, è in grado di partizionamento in toluene 11. Questo tipo di analisi può essere utilizzata più ampiamente su altri tipi di aminoacidi in cui la perdita di un gruppo carbossilico si verifi...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<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>
		</table>
		</div>

an assay for measuring the activity of escherichia coli inducible lysine decarboxyase

Escherichia coli è un batterio intestinale che è in grado di crescere in un ampio intervallo di valori di pH (pH 5 - 9) 1 e, incredibilmente, è in grado di sopravvivere acido estrema sottolinea anche il passaggio attraverso lo stomaco dei mammiferi in cui il pH può scendere al livello più basso come pH 1 - 2 2. Per consentire ad una vasta gamma di sopravvivenza a pH acido, E. coli possiede quattro diversi decarboxylases aminoacido inducibile che decarbossilano loro substrato amino acidi in un protone-dipendente aumentando così il pH interno. Il decarboxylases includono il acido glutammico decarboxylases Gada e GadB 3, l&#39;arginina decarbossilasi Adia 4, la lisina decarbossilasi LdcI 5, 6 e l&#39;ornitina decarbossilasi SpeF 7. Tutti questi enzimi utilizzano piridossale-5&#39;-fosfato come co-fattore 8 e funzione con membrana interna substrato-prodotto antiporters che rimuovono i prodotti decarbossilazione al supporto esterno in cambio di substrato fresco 2. Nel caso di LdcI, la lisina-cadaverina antiporter si chiama Cadb. Recentemente, abbiamo determinato la struttura a raggi X di cristallo di LdcI a 2.0 Å, e abbiamo scoperto un romanzo piccole molecole legato LdcI il severo guanosina regolatore risposta 5&#39;-difosfato, 3&#39;-difosfato (ppGpp) 14. La risposta rigorosa verifica quando le cellule in crescita esponenziale esperienza privazione nutritiva o uno di una serie di sollecitazioni di altri 9. Di conseguenza, le cellule producono ppGpp che porta ad una cascata di segnali si conclude con il passaggio da una crescita esponenziale alla crescita fase stazionaria 10. Abbiamo dimostrato che ppGpp è un inibitore specifico della LdcI 14. Qui descriviamo il test lisina decarbossilasi, modificato dal test sviluppato da Phan et al. 11, che abbiamo usato per determinare l&#39;attività di LdcI e l&#39;effetto di pppGpp / ppGpp su tale attività. La reazione di decarbossilazione LdcI rimuove l&#39;α-carbossi gruppo di L-lisina e produce anidride carbonica e la cadaverina poliammine (1,5-diaminopentane) 5. L-lisina e cadaverina si può reagire con 2,4,6-trinitrobenzensulfonic acido (TNBS) a pH elevato per generare N, N-bistrinitrophenylcadaverine (TNP-cadaverina) e N, N&#39;-bistrinitrophenyllysine (TNP-lisina), rispettivamente 11. Il TNP-cadaverina può essere separato dal TNP-lisina in quanto il primo è solubile in solventi organici come il toluene, mentre la seconda non lo è (vedi figura 1). Il range lineare del test è stato determinato empiricamente utilizzando purificata cadaverina.

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

L&#39;attività della decarbossilasi inducibile lisina è monitorato facendo reagire il substrato L-lisina e il prodotto cadaverina con 2,4,6-trinitrobenzensulfonic acido per formare addotti che hanno solubilità differenziale in toluene.

Un test per misurare l&#39;attività di Escherichia coli Inducibile Lisina 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 Views.  University of Toronto. L'attività della decarbossilasi inducibile lisina è monitorato facendo reagire il substrato L-lisina e il prodotto cadaverina con 2,4,6-trinitrobenzensulfonic acido per formare addotti che hanno solubilità differenziale in toluene.

Video: 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.

Misurare la rigidezza flessionale di cellule batteriche Utilizzando una trappola ottica

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

Ricerca

Biologia

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.

Throughput Screening elevati di attività Endoglucanase fungine 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.

Applicazione di MassSQUIRM per le misure quantitative delle attività demetilasi lisina

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 Protocollo per la preparazione di elettrocompetenti<em&gt; Escherichia coli</em&gt; E<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.

Protocolli per l&#39;implementazione di un<em&gt; Escherichia coli</em&gt; Sulla base TX-TL cellulare-Free Expression System per la biologia sintetica

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

Rilevamento dal vivo<em&gt; Escherichia coli</em&gt; O157: H7 Celle di 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-base Espressione Proiezione di proteine ​​di membrana 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.

I benefici multiforme Protein Co-espressione 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.

L&#39;incorporazione specifici residuo di non canonici amminoacidi in proteine ​​modello utilizzando un<em&gt; Escherichia coli</em&gt; Trascrizione-traduzione sistema non cellulare

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.

Misurazione dell'attività enzimatica della lattasi in laboratorio

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