Ringraziamo il Department of Homeland Security degli Stati Uniti per la fornitura di una parte del finanziamento.

1. Ceppi batterici e DNA Template Preparazione <ol><li> Crescere ceppi batterici di E. coli O157: H7, non-O157, e di altre specie patogene, quali Salmonella e Shigella, a 37 ° C durante la notte con agitazione. </li><li> Estrarre il DNA da colture batteriche utilizzando l&#39;apposito kit (vedi tabella dei materiali) e seguendo le istruzioni del produttore. </li><li> Misurare la densità ottica (OD 260) per determinare le concentrazioni di DNA utilizzando uno spettrofotometro. </li></ol> 2. Primer and Probe Design per il saggio qPCR <ol><li> E. coli O157: H7 sono le sequenze da GenBank numero di accesso AE00517411. Primer design e la sonda utilizzando il software appropriato per primer e la progettazione della sonda per real-time PCR (vedi tabella materiali per i dettagli). Le sequenze dei primer e la sonda sono i seguenti: Z3276-Forward (F) 5&#39;-TATTCCGCGATGCTTGTTTTT-3 &#39; Z3276-retromarcia (R) 5&#39;-ATTATCTCACCAGCAAACTGGCGG-3 &#39; Z3276-Probe, FAM-CCGCAATCTTTCC-MGBNFQ </li><li> Ricostituire la polvere di primer con acqua, ottenendo una concentrazione di 10 pM come soluzione lavoro primer. </li><li> Diluire le sonde a 10 pM come soluzioni di lavoro della sonda. Sonde marcate variano in potenza con lotti. Pertanto, titolare ogni partita di sonde marcate prima di utilizzare per ottenere prestazioni ottimali qPCR. </li></ol> 3. Impostazione delle qPCR Assay Condizioni <ol><li> Preparare miscela di reazione, che consiste di 25,0 ml di 2x real-time PCR Master Mix, 200 nM di primer forward, 200 nM di primer reverse, e 100 nM di sonda. </li><li> Aggiungere 5 ml di DNA campione (100 pg) e un volume adeguato di acqua fino a raggiungere un volume finale di 50 microlitri. </li><li> Per il controllo nontemplate, usare 5 ml di acqua per sostituire il campione di DNA. </li><li> Impostare le condizioni qPCR come segue: 95 ° C per 10 min per l&#39;attivazione di TaqMan, seguita da 40 cicli di 95 ° C per 10 sec per denaturazione e 60 &amp;# 176; C per 1 minuto per la ricottura / estensione. </li></ol> 4. qPCR Test di sensibilità <ol><li> Effettuare una diluizione di 10 volte in serie da una cultura di fase mid-esponenziale (OD 600 = 0.5; circa 1.5 x 10 8 UFC / ml mediante placcatura). </li><li> Aggiungere 100 ml di diluizioni cellulari su una piastra da 96 pozzetti in triplicato. </li><li> Centrifugare la piastra a 2.500 xg per 10 min. </li><li> Aggiungere 50 ml di soluzione di estrazione del DNA a ciascun bene. </li><li> Pellet cellulari Risospendere con una pipetta multicanale. </li><li> Sigillare la piastra con una pellicola, la piastra bollire a bagnomaria per 10 minuti e si centrifuga a 2500 xg per 2 min. </li></ol> 5. Preparazione Miscele vivi e morti cellulari per il trattamento PMA <ol><li> Crescere 10 ml di E. coli O157: H7 a 37 ° C a metà fase esponenziale e dividere la cultura in due aliquote. </li><li> Bollire una aliquota di cellule per 10 minuti in un bagno d&#39;acqua per cellule morte e lasciare l&#39;altra aliquota di cellule vive. </li><li> Verificare l&#39;re sono cellule vive della aliquota calore ucciso da placcatura le cellule su piastre di agar LB. </li><li> 2 ml di soluzione le cellule vive e uccisi col calore e regolare la concentrazione di 8 x 10 6 UFC / ml di terreno LB. Creare quattro serie di diluizioni cellulari che vanno da 8 x 10 0 a 8 x 10 6 UFC / ml. </li><li> Utilizzare le prime due serie di diluizioni cellulari per cellule vive trattate con PMA o non trattati. </li><li> Per la terza e quarta serie di diluizioni cellulari, aggiungere 8 x 10 6 cellule morte per ogni diluizione cella (8 x 10 0 - 8 x 10 6) per fare miscele di cellule vive e morte. </li><li> Trattare la terza serie di diluizione con PMA e utilizzare la quarta serie di diluizione per il controllo. </li></ol> 6. Trattando le cellule con PMA <ol><li> Sciogliere PMA in dimetilsolfossido (o acqua) per fare magazzino 10 mM soluzione e conservati a -20 ° C al buio. </li><li> Aliquota 400 ml di cellule vive, cellule morte, e la miscela di cellule vive e morte intre microtubi separati. </li><li> Aggiungere 2,0 ml di PMA 10 mM per ogni aliquota di cellule ad una concentrazione finale di 50 mM. </li><li> Incubare i campioni a temperatura ambiente per 5 minuti al buio, agitando delicatamente alcune volte, 3 secondi ogni volta. </li><li> Aggiungere 100 ml di campioni su una piastra a 96 pozzetti. </li><li> Sigillare la piastra con una pellicola ottica e mettere il piatto su ghiaccio. </li><li> Impostare la piastra di 20 centimetri da una W sorgente alogena 650 ed esporre per 2 minuti per l&#39;esposizione alla luce. </li><li> Centrifugare la piastra a 2.500 xg per 10 min, scartare il surnatante delicatamente e accuratamente drenare la piastra su un foglio di carta assorbente. </li><li> Pellet cellulari Risospendere in 50 ml di soluzione di estrazione del DNA da pipettando su e giù per venti volte con un multi-canale Pipetman. </li><li> Sigillare la piastra con un film, bollire per 10 minuti in un bagno d&#39;acqua, e centrifugare a 2500 xg per 2 min. Il surnatante nella piastra è il DNA da cellule trattate con PMA e pronto per qPCR. </li><li> Eseguire qPCR come descriletto sopra, con 5 ml di DNA. </li></ol>

<ol>
	<li>Barletta, F., 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=Validation+of+five-colony+pool+analysis+using+multiplex+real-time+PCR+for+detection+of+diarrheagenic+Escherichia+coli.">Validation of five-colony pool analysis using multiplex real-time PCR for detection of diarrheagenic Escherichia coli.</a> J. Clin. Microbiol. 47, 1915-1917 (2009).</li><li>Gannon, V. P., King, R. K., Kim, J. Y., Thomas, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+sensitive+method+for+detection+of+Shiga-like+toxin-producing+Escherichia+coli+in+ground+beef+using+the+polymerase+chain+reaction.">Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction.</a> Appl. Environ. 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Microbiol. 35, 1066-1070 (1997).</li><li>Fricker, M., Messelh&#228;u&#223;er, U., Bushch, U., Scherer, S., Ehling-Schulz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+real-time+PCR+assays+for+detection+of+emetic+Bacillus+cereus+strains+in+foods+and+recent+food-borne+outbreaks.">Diagnostic real-time PCR assays for detection of emetic Bacillus cereus strains in foods and recent food-borne outbreaks.</a> Appl. Environ. Microbiol. 73, 1892-1898 (2007).</li><li>Li, B., Chen, J. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+methodology+for+selective+detection+of+viable+Escherichia+coli+O157:H7+cells+by+targeting+Z3276+as+a+genetic+marker.">Real-time PCR methodology for selective detection of viable Escherichia coli O157:H7 cells by targeting Z3276 as a genetic marker.</a> Appl Environ. Microbiol. 78, 5297-5304 (2012).</li><li>Perna, N. T., 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=Genome+sequence+of+enterohaemorrhagic+Escherichia+coli+O157:H7.">Genome sequence of enterohaemorrhagic Escherichia coli O157:H7.</a> Nature. 409, 529-533 (2001).</li><li>Wang, S., Levin, R. 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Un obiettivo primario dello studio comporta l&#39;uso di una ORF Z3276, unico per E. coli O157: H7, come unico biomarcatore per l&#39;analisi qPCR. Allo stato attuale, la maggior parte dei saggi qPCR stanno prendendo di mira geni di virulenza, come stx1, stx2, e dell&#39;EAEA 1,2,5,6 o geni fenotipici condivisi, come uidA 3,4, rfbE e geni FLIC 8,9. Occasionalmente, una specie o ceppo non possono essere definitivamente identificati dal gene virulenza (s), come STX 1e STX 2 geni, come quei geni sono presenti in specie o ceppi 16 e diversi. Utilizzando rfbE e FLIC per geni target, sono necessari entrambi i geni ad essere utilizzati per l&#39;identificazione completa 17. Uso ORF Z3276 come biomarker, questo dosaggio qPCR è stata dimostrata per essere saggio sensibile e specifico (Tabella 1). Questo test è stato sottoposto a test di inclusività ed esclusività severe, tra cui O157: H7 (n = 367), oltre 100 ceppi non-O157, e di altre specie patogene, quali Salmonella e Shigella. Tutti O157: H7 ceppi esaminati sono stati positivamente individuati, e nessun cross-reattività è stato trovato dai ceppi non-O157 (Tabella 1), indicando che ORF Z3276 è un biomarker unico e forte per il rilevamento di E. coli O157: H7. L&#39;altro obiettivo dello studio è quello di individuare selettivamente le cellule vive dalle cellule morte dal trattamento PMA prima dell&#39;estrazione del DNA. PMA può penetrare all&#39;interno delle cellule morte con membrana compromessa e legarsi al DNA, e quindi inibisce l&#39;amplificazione del DNA di cellule morte in PCR 13. Abbiamo modificato la procedura PMA-trattamento, e adattato in un formato piastra a 96 pozzetti per la procedura. La giusta intensità di esposizione alla luce è indispensabile per ottenere un effetto ottimale reticolazione. In precedenza, microtubi sono stati utilizzati in questo passaggio 13-15. Tuttavia, microtubi separati sono difficili da gestire in luce expasso posure, e questi tubi non sono assolutamente trasparente per avere la migliore trasversale piacimento. Utilizzando una piastra a 96 pozzetti, abbiamo migliorato l&#39;efficienza della PMA-trattamento. Questi cambiamenti possono influire sul dosaggio in diversi modi: rendono più facile ottenere un effetto di reticolazione completa e uniforme, soprattutto con numerosi campioni, i campioni trattati possono essere spostati da una piastra all&#39;altra per rendere possibile il rilevamento di un gran numero di campioni, e prevedono questo test PMA-qPCR con potenziale automazione per l&#39;intero processo. Il limite di questo saggio PMA-qPCR è che il trattamento PMA aumenta il costo PCR po e leggermente ridotta la sensibilità del saggio PCR.

PCR è una tecnica comune per il rilevamento di E. coli O157: H7 da campioni alimentari ed ambientali. Identificazione di biomarcatori specifici per E. coli O157: H7 è uno dei fattori chiave per la rilevazione successo in PCR. I biomarcatori comunemente utilizzati in saggi di PCR per il rilevamento di E. coli O157: H7 includono tossine Shiga (STX e STX 1 2) 1,2, 3,4 uidA, dell&#39;EAEA 5,6, rfbE 7, e FLIC geni 8,9. Questi marcatori possono fornire efficienza variabile per l&#39;identificazione, tuttavia, la maggior parte dei biomarker non possono essere utilizzati come biomarker unico per il rilevamento di E. coli O157: H7. Questa carenza di alimentazione differenziazione dei geni bersaglio richiede biomarker (s) più specifico e più forte per l&#39;identificazione di E. coli O157: H7 10. Numerose sonde per geni o ipotetiche fasi di lettura aperta (ORF) in E. coliO157: H7 11 sono stati progettati per qPCR saggio basato sugli indizi di microarray di genotipizzazione del DNA del nostro laboratorio e di ricerca all&#39;interno del database GenBank del National Center for Biotechnology Information. La sequenza di Z3276 ORF, che è un gene fimbrial 11, è stato selezionato per il saggio qPCR. Successivamente, un saggio qPCR è stato sviluppato attraverso numerose prove sui parametri di PCR come concentrazioni di primer e sonde, nonché le temperature di ricottura e di estensione (dati non mostrati). Dopo i test inclusività di incentivazione e di esclusività su ceppi di riferimento come E. coli O157: H7, non-O157 e Shigella ceppi nel qPCR, la ORF Z3276 è stato identificato come un marcatore genetico specifico e forte per l&#39;identificazione di E. coli O157: H7. Poi sviluppiamo un nuovo metodo qPCR utilizzando questo biomarker unico per l&#39;identificazione di E. coli O157: H7. Un&#39;altra sfida nella rilevazione di E. coli O157: H7 in contaminati foods e l&#39;ambiente è accurata determinazione di cellule vive da campioni. La presenza estesa di DNA da cellule morte e l&#39;incapacità di differenziare redditività nel PCR potrebbe causare letture di falsi positivi, con conseguente sovrastima del numero di cellule vive di rilevamento. Di conseguenza, questo limita l&#39;utilizzo della PCR in un rilevamento accurato di patogeni 12. Un nuovo approccio per differenziare DNA delle cellule morte è stata adottata combinando propidio monoazide (PMA) trattamento con procedura PCR negli ultimi anni 13-15. PMA è in grado di andare all&#39;interno di cellule morte, e intercalare nel DNA quando esposto alla luce, ma non può andare all&#39;interno di cellule vive per reagire con il DNA delle cellule vive. Così, in miscele PMA-trattate di cellule vive e morte, il DNA di cellule morte non può essere amplificato in PCR 13. Abbiamo combinato il trattamento PMA con il saggio qPCR di recente sviluppato per rilevare selettivamente dal vivo E. coli O157: H7 cellule. Inoltre, abbiamo fatto la PMA-qPCR unssay possibile per il rilevamento ad alta velocità.

<table border="0" cellpadding="0" cellspacing="0" width="901">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">AB 7900HT Fast Real-time PCR system</td>
			<td style="width:164px;">ABI</td>
			<td style="width:171px;"></td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">2 x Universal master mix</td>
			<td style="width:164px;">ABI</td>
			<td align="right" style="width:171px;">4304437</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">PrepMan Ultra</td>
			<td style="width:164px;">ABI</td>
			<td align="right" style="width:171px;">4318930</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">Primer Express</td>
			<td style="width:164px;">ABI</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="58" rowspan="2" style="height:58px;width:249px;">Probes</td>
			<td rowspan="2" style="width:164px;">ABI</td>
			<td>Top of Form</td>
			<td rowspan="2"></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">4316033 Bottom of Form</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">PMA</td>
			<td style="width:164px;">Biodium</td>
			<td align="right" style="width:171px;">40013</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="87" rowspan="3" style="height:87px;width:249px;">Puregen cell and tissue kit</td>
			<td rowspan="3" style="width:164px;">Qiagene</td>
			<td>Top of Form</td>
			<td rowspan="3"></td>
		</tr>
		<tr height="29">
			<td align="right" height="29" style="height:29px;">158622</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;">Bottom of Form</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">DU530 spectrophotometer</td>
			<td style="width:164px;">Beckman</td>
			<td style="width:171px;"></td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">Spectrophotometer</td>
			<td style="width:164px;">NanoDrop Technology</td>
			<td style="width:171px;">&#160;ND-1000</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">650 w halogen light source</td>
			<td style="width:164px;">Britek</td>
			<td style="width:171px;">H8061S</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">Otptical Adhesive film</td>
			<td>ABI</td>
			<td align="right">4311971</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:249px;">Optical 96-well reaction plate</td>
			<td>ABI</td>
			<td align="right">4316813</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of live escherichia coli o157:h7 cells by pma-qpcr

Una cornice unica di lettura aperta (ORF) Z3276 è stato identificato come un marcatore genetico specifico per E. coli O157: H7. Un saggio qPCR stato sviluppato per il rilevamento di E. coli O157: H7 di mira ORF Z3276. Con questo test, possiamo individuare a partire da un paio di copie del genoma del DNA di E. coli O157: H7. La sensibilità e specificità del test sono state confermate dal test di convalida intensive con un gran numero di E. coli O157: H7 (n = 369) e ceppi non-O157 (n = 112). Inoltre, abbiamo procedura di propidio monoazide (PMA) in combinazione con il protocollo qPCR di nuova concezione per la rilevazione selettiva delle cellule vive da cellule morte. Amplificazione del DNA da cellule morte PMA-trattati è stata quasi completamente inibita in contrasto con l&#39;amplificazione praticamente inalterati di DNA da cellule vive PMA-trattati. Inoltre, il protocollo è stato modificato e adattato ad un formato piastra a 96 pozzetti per un facile trasporto e coerente di un gran numero di campioni. Til suo metodo dovrebbe avere un impatto sulla microbiologico accurata e il monitoraggio epidemiologico della sicurezza alimentare e di origine ambientale.

Rilevamento di E. coli O157: H7 da qPCR utilizzando ORF Z3276 Uno dei fattori chiave di questo saggio è la sensibilità e la specificità della sonda Z3276 utilizzato nella qPCR. E &#39;in grado di rilevare a partire da un paio di copie del genoma del DNA di E. coli O157: H7 come mostrato nella Figura 1A. Dei 120 ceppi non-O157 esaminati, compresi i sei principali ceppi non-O157 STEC O104: H4 ceppi, Salmonella, e ceppi Shigella, non cross-reattività è stata trovata, come mostrato nella Tabella 1. Combinazione del trattamento PMA con qPCR Covare E. coli O157: H7 cellule con PMA (50 mm) per 5 min al buio ed esponendo alla luce per 2 min sono stati selezionati per le condizioni di trattamento di PMA. Inoltre, abbiamo modificato procedura di trattamento di PMA. Rispetto ai vari microtubi trasparenti utilizzati nel passaggio di reticolazione, una piastra a 96 pozzetti è risultato essere più efficaceraggiungere l&#39;effetto di reticolazione ottimale e più conveniente di gestire un grande numero di provette per campioni durante il processo di esposizione alla luce. L&#39;amplificazione del DNA da cellule vive e morte PMA trattati in qPCR Gli effetti di inibizione PMA-mediata di amplificazione del DNA da cellule morte in questo saggio qPCR sono stati mostrati in Figura 1 usando il DNA derivato da due 10 diluizioni cellulari vivi o morti seriali. I risultati mostrano che le curve generate dalle cellule vive trattate con PMA (curva rossa) e senza PMA (curva blu) sembrano lineare e quasi uguali tra loro. Differenze di valore CT Lievi stati mostrati tra le cellule vive trattate con PMA e senza PMA (Figura 1A), suggerendo che il trattamento PMA avuto poco effetto sulla amplificazione di DNA delle cellule vive nella qPCR. Ma un 15 - differenza di valore CT (32.000 volte) è stato mostrato tra le cellule morte trattate con PMA e senza PMA, demonstrating che il trattamento PMA efficacemente soppressa amplificazione del DNA dalle cellule morte (Figura 1B). Rilevazione di cellule vive da miscele di cellule vive e morte in qPCR Due serie di diluizioni di cellule vive (8 x 10 0-8 x 10 6 CFU) sono stati trattati con PMA o senza PMA per rilevare le cellule vive da miscele di cellule vive / morte. I risultati qPCR (Figura 2a) ha dimostrato un parallelo inverso andamento progressivo dei valori CT come correlata al numero di cellule vive trattate (barre viola) ai campioni non trattati (barre blu). Le cellule vive trattate prodotto un valori CT leggermente superiori a quelli delle cellule vive non trattate. Un simile inverso andamento progressivo in valori CT è stata osservata con il numero reale di cellule vive nelle miscele di cellule vive / morte trattate con PMA (barre verdi) e nella Figura 2B. Inoltre, questa tendenza al ribasso dei valori CT eraosservata con il numero di cellule vive nelle miscele nonostante la presenza di 106 cellule morte. Questi risultati hanno mostrato che i valori CT della miscela di cellule vive / morte rappresentato il DNA da solo le cellule vive, mentre l&#39;amplificazione del DNA dalle cellule morte era efficacemente soppressa mediante trattamento PMA. Ma, i valori CT di miscele di cellule live / morti trattati con la PMA sono stati fluttuato e non hanno rispecchiato il numero di cellule vive nelle miscele in figura 2B (barre gialle). <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/50967/50967fig1highres.jpg" src="/files/ftp_upload/50967/50967fig1.jpg" /> Figura 1. Dal vivo e le cellule morte trattate con PMA o senza PMA nel saggio qPCR. (A) di serie di 10-volte-diluito in diretta E. coli O157: H7 diluizioni cellulari (8 x 10 0-8 x 10 6 CFU) sono stati trattati con PMA o senza PMA.I valori CT rappresentano i valori medi di CT dei triplicati nel saggio qPCR. (B) Confronto di amplificazione del DNA delle cellule vive e cellule morte trattate con PMA e senza PMA nel confronto q qPCR dosaggio. ΔRn si riferisce a fluorescenza cambiamento di intensità. <a href="https://www.jove.com/files/ftp_upload/50967/50967fig1highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita.</a> <img alt="Figura 2" fo:content-width="5in" fo:src="/files/ftp_upload/50967/50967fig2highres.jpg" src="/files/ftp_upload/50967/50967fig2.jpg" /> Figura 2. Differenziazione delle cellule vive da miscele di cellule live / morti di PMA-qPCR Quattro serie di diluizioni di 10 volte di colture cellulari (8 x 10 0-8 x 10 6 CFU). Stati fatti come indicato. (A) Prima serie di diluizione cellulare è stata trattata con PMA mentre la seconda diluizione cella era o non trattati prima dell&#39;estrazione del DNA.(B) 8 x 10 6 cellule morte sono state mescolate con la terza e quarta serie di diluizioni cellulari per fare due serie di miscele di cellule vive / morte come indicato. Una serie di miscele di cellule è stato trattato con PMA e l&#39;altra serie è stata trattata senza PMA prima dell&#39;estrazione del DNA. Ogni barra rappresenta i valori medi di CT di un esperimento triplice copia ± SD. <table><tbody><tr><td> Organismo </td><td> Sierotipo </td><td> Numero di ceppi testati </td></tr><tr><td rowspan="15"> E. coli </td><td> 2006 spinaci O157: H7 isolati focolai </td><td> 186 </td></tr><tr><td> 2006 Taco Bell O157: H7 isolati </td><td> 58 </td></tr><tr><td> 2006 Taco John O157: H7 isolati </td><td> 11 </td></tr><tr><td> Collezioni di deformazione DMB dagli altri O157: H7 focolai </td><td> 112 </td></tr><tr><td> O104: H4 </td><td> 3 </td></tr><tr><td> O26 </td><td> 18 </td></tr><tr><td> O103 </td><td> 13 </td></tr><tr><td> O111 </td><td> 24 </td></tr><tr><td> O121 </td><td> 2 </td></tr><tr><td> O45 </td><td> 5 </td></tr><tr><td> O145 </td><td> 9 </td></tr><tr><td> Ø113 </td><td> 2 </td></tr><tr><td> O91 </td><td> 1 </td></tr><tr><td> O55 </td><td> 9 </td></tr><tr><td> Altro </td><td> 19 </td></tr><tr><td rowspan="2"> Salmonella </td><td> Typhi </td><td> 1 </td></tr><tr><td> Newport </td><td> 2 </td></tr><tr><td> Shigella dysenteriae </td><td> 6 </td><td> 2 </td></tr><tr><td> Shigella sonnei </td><td> Sconosciuto </td><td> 2 </td></tr><tr><td> Shigella flexneri </td><td> 4 </td><td> 1 </td></tr><tr><td> Shigella boydii </td><td> 1 </td><td> 1 </td></tr><tr><td colspan="2"> Totale </td><td> 481 </td></tr></tbody></table> Tgrado 1. ceppi utilizzati per la validazione in tempo reale PCR per l&#39;identificazione di E. coli O157: H7.

Watch this Scientific Journal Video about Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR at JoVE.com

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

Un test qPCR è stato sviluppato per il rilevamento di Escherichia coli O157: H7 mira un marcatore genetico unico, Z3276. La qPCR è stato combinato con propidio monoazide (PMA) Trattamento per il rilevamento di cellule vive. Questo protocollo è stato modificato e adattato ad un formato piastra a 96 pozzetti per maneggevolezza e coerente di numerosi campioni

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. ...

detection-of-live-escherichia-coli-o157h7-cells-by-pma-qpcr

Research

JoVE Journal

Biology

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

16.4K Views.  Food and Drug Administration.  Un test qPCR è stato sviluppato per il rilevamento di Escherichia coli O157: H7 mira un marcatore genetico unico, Z3276. La qPCR è stato combinato con propidio monoazide (PMA) Trattamento per il rilevamento di cellule vive. Questo protocollo è stato modificato e adattato ad un formato piastra a 96 pozzetti per maneggevolezza e coerente di numerosi campioni 

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

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine 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 acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

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

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility 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 ...

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 ...

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Phenotypes are determined by a complex series of physical (e.g. protein-protein) and functional (e.g. gene-gene or genetic) interactions (GI)1. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

Mappatura batteriche reti funzionali e sulle vie di<em&gt; Escherichia Coli</em&gt; Utilizzando matrici sintetiche genetiche

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identificazione di complessi proteici in<em&gt; Escherichia coli</em&gt; Con sequenziale purificazione per affinità Peptide in combinazione con Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

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 ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Purificazione non-cromatografico di polipeptidi di elastina ricombinante simili e le loro fusioni con Peptidi e Proteine ​​da<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

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 ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

Metodo per l'etichettatura di trascrizioni in cellule individuali Escherichia coli per singola molecola di fluorescenza In Situ gli esperimenti di ibridazione