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1. Isolamento di E. coli Co-trasformanti <ol><li> Preparare le cellule elettro-competente della appropriata E. coli da trasformare. Trasferimento a 1 ml di terreno LB (Triptone, estratto di lievito, NaCl a 10, 5, e 10 g / L, rispettivamente) una singola colonia del ceppo di scelta, e incubare a 37 ° C in stringono condizioni di 180 giri al minuto. Diluire la precoltura 1: 500 in 25 ml di mezzo fresco LB ed incubare la coltura a 37 ° C. </li><li> A log-fase (0,6 OD) centrifugare la sospensione cellule a 5.000 xg per 20 minuti), e risospendere il pellet in 10%, v / v sterile glicerolo-acqua ghiacciata nella metà del volume cultura originaria. Ripetere questa operazione 4 volte, dimezzando ogni volta il volume risospensione. Infine, risospendere il pellet in glicerolo / acqua e dividere la sospensione cellule in 50 microlitri aliquote. Conservare le aliquote a -80 ° C fino a 6 mesi. </li><li> Sciogliere il plasmide desiderato in acqua sterile integrato con EDTA 0,5 mm. Thaw su ice un&#39;aliquota di cellule elettro-competente e mescolare con una quantità appropriata (2,5-5 ng) di vettore. Versare il composto in una vaschetta 0,1 centimetri adatto per elettroporazione, e applicare 1,8 kV. </li><li> Immediatamente trasferire le cellule elettroporate in 1 ml di terreno SOC (terreno LB integrato con 0,2% w / v di glucosio, 10 mM MgCl 2, 2.5 mM KCl), incubare per 1 ora sotto agitazione, e infine trasferire aliquote di 100 microlitri di piastre di Petri contenenti LB agar con l&#39;antibiotico appropriato. Incubare O / N a 37 ° C. </li><li> Purificare trasformanti strisciando singole colonie su piastre di Petri. </li><li> Preparare le cellule elettro-competente dei trasformanti primari e ripetere i passaggi da 1.1 a 1.5 per trasformare con ulteriori plasmidi. </li><li> Preparare glicerolo scorte di co-trasformanti. Trasferimento di una singola colonia in LB addizionato con antibiotici appropriati, incubare a 37 ° C sotto agitazione, e al log-fase (0,6 OD) centrifugare la sospensione cellule a 5.000 xg per 20 min. Resuspend il pellet in LB-antibiotici mezzo contenente 15% v / v glicerolo. Distribuire in aliquote e conservare a - 80 ° C. </li></ol> 2. Co-espressione di α e ε subunità E. coli DNA polimerasi III <ol><li> Trasferire con un&#39;ansa sterile una piccola quantità di glicerolo magazzino ceppo appropriata (ad esempio, TOP10 / PGOOD-ε243 e TOP10 / PGOOD-ε243-θ / pBADα1160, vedi Figura 1) in una capsula di Petri contenente terreno LB e antibiotici. Lasciate che le cellule di sospensione secca sulla piastra di Petri, e strisciare le cellule gocciolina. Incubare a 37 ° CO / N. </li><li> Trasferimento, utilizzando uno stuzzicadenti sterili, una singola colonia di 1 ml di LB-antibiotici medie. Incubare 8 ore a 37 ° C. Diluire la precoltura 1: 500 in mezzo fresco e incubare a 30 ° C per 15 h. </li><li> Aggiungi IPTG, arabinosio, o arabinosio e IPTG, 1 mm ciascuna. Incubare a 30 ° C per 2,5 ore. Raccogliere le cellule, e memorizzare i pellet a -20 ° C. </li><li> Pellets Scongelare su ghiaccio e risospendere in tampone di lisi (50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, ditiotreitolo 2,5 mm, 1 mm phenylmethylsulfonyl fluoruro (PMSF), pH 8). Omogeneizzare delicatamente la sospensione cellule con un vasaio vetro freddo. </li><li> Sonicare a 15 W per 15 secondi, seguito da 15 intervallo di raffreddamento sec (4 cicli). </li><li> Centrifugare gli estratti proteici totali a 10.000 xg per 20 minuti, a 4 ° C per recuperare la frazione solubile. </li><li> Analizzare mediante SDS-PAGE (12,5% acrilammide) aliquote di ogni estratto proteina solubile. A tale scopo, trasferire 20 microlitri di ogni estratto proteina solubile in provette Eppendorf contenenti 60 ml di H 2 O e 20 microlitri di tampone di caricamento (250 mM Tris-HCl pH 6,8, 10 mM β-mercaptoetanolo, 10% (w / v) SDS, 50% (v / v) di glicerolo, 0,25% (w / v) di blu di bromofenolo) e bollire per 5 min. Carico 18 microlitri di ciascun campione ed eseguire l&#39;elettroforesi a 140 V per circa 1,5 ore. </li></ol> 3. Co-espressione di α subunità di Deinococradiodurans CUS DNA polimerasi III e ε subunità di E. coli DNA polimerasi III <ol><li> Preparare una precoltura della E. coli ceppo TOP10 / pBAD-α Dr / PGOOD-ε243 in 1 ml di terreno LB-ampicillina-tetraciclina e incubare 8 ore a 37 ° C. Diluire 1: 1.000 in terreno fresco e incubare O / N a 37 ° C. </li><li> Diluire 1: 100 in 200 ml di mezzo fresco, incubare a 37 ° C per 3 ore, poi indurre per 3 ore con arabinosio e IPTG, 1 mM ciascuno. Raccogliere le cellule, e conservare il pellet a -20 ° C. </li><li> Risospendere il pellet in 50 mM Tris-HCl pH 8, NaCl 150 mM, EDTA 1 mM, PMSF 1 mM. Mescolare e distruggere le cellule, come descritto in 2.4 e 2.5. </li><li> Centrifugare immediatamente le cellule lisato a 10.000 xg per 20 min. Eliminare il pellet. Filtrare il surnatante con un imbuto Buchner dotato di 3 strati di carta da filtro. Applicare dolce di vuoto. Per evitare eccessiva formazione di schiuma, mantenere la beuta vuoto su ghiaccio durante la filtrazione. </li><li> Determinare la concentrazione di proteine ​​secondo Bradford 14. </li></ol> 4. Gel Filtration Chromatography <ol><li> Equilibrare un 16X70-rivestito di acqua (200) colonna di gel filtrazione con 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8. carico sulla colonna dell&#39;estratto proteina solubile. Per una risoluzione ottimale, utilizzare un ciclo campione di 1 ml. Eseguire la cromatografia a 0.6 ml / min. Mantenere la temperatura della colonna a 4 ° C in tutto. </li><li> Raccogliere 0,9 ml frazioni e analizzare con SDS-PAGE. Trasferire 20 ml di ogni frazione rilevante per provette Eppendorf contenenti 60 ml di H 2 O e 20 microlitri di tampone di caricamento (250 mM Tris-HCl pH 6,8, 10 mM β-mercaptoetanolo, 10% (w / v) di SDS, 50% ( v / v) di glicerolo, 0,25% (w / v) di blu di bromofenolo) e far bollire per 5 min. Carico 18 microlitri di ciascun campione ed eseguire l&#39;elettroforesi a 140 V per circa 1,5 ore. </li><li> Determinare il 3&#39;-5 &#39;esonucleasi e l&#39;attività della DNA polimerasi di ciascuna frazione in 96-cill micropiastre. Analizzare l&#39;attività di esonucleasi 5&#39;-utilizzando timidina monofosfato estere p-nitrofenil (p NP-TMP) come substrato 15. Stima polimerasi DNA usando l&#39;PPX (pirofosfatasi, Purine nucleoside fosforilasi, xantina ossidasi) enzima accoppiato dosaggio 16 e 2- (4-Iodophenyl) -3- (4-nitrofenil) -5-cloro fenil-2H-tetrazolio (INT) come accettore di elettroni 16. </li></ol> 5. Mutazione Analisi delle popolazioni Co-esprimono la wild-type ε subunità di E. coli DNA polimerasi III e mutageni εD12A Variant <ol><li> Trasferire una singola colonia di E. coli TOP10 contenente il pBAD-ε e le pGOOD1-εD12A 11 vettori a 1 ml di LB-antibiotici media. Incubare O / N a 37 ° C. </li><li> Diluire la precoltura 1: 250 in 3 fiasche contenenti mezzo fresco (10 ml), aggiungere le appropriate induttori (arabinosio, IPTG o arabinosio e IPTG, 1 mM ciascuno) e incubare a 37 ° Cper 8 ore. Preparare in culture non-indotta paralleli. </li><li> Raccogliere aliquote di 1 ml, poi diluito 1: 500 in nuove fiasche contenenti mezzo fresco (10 ml) integrato o meno con gli induttori appropriati. Incubare O / N a 37 ° C. </li><li> Ripetere i punti 5.2 e 5.3 e raccogliere aliquote di 1 ml. </li><li> Determinare il numero di generazioni è verificato in ciascuna cultura. Trasferimento su piastre LB, 100 ml di appropriate diluizioni seriali di inoculo e cultura. Incubare O / N a 37 ° C. Contare le colonie su piastre LB, e calcolare il logaritmo del numero di cellule presenti nel inoculo (log I) e nella coltura alla fine della crescita (log C). Per determinare il numero di generazioni, utilizzare la formula: (log C - log I) /0.301. </li><li> Centrifugare a 5.000 xg per 20 min e risospendere le cellule in 1 ml di 50 mM Tris-HCl pH 7,6, NaCl 50 mM. Per permeabilize le celle, aggiungere 2-3 gocce di cloroformio e vortex per 20 sec. </li><li> Determinare il β-Attività glucosidasi di ciascuna aliquota in una micropiastra a 96 pozzetti, utilizzando p -nitrophenyl-β-D-glucopiranoside (PNPGluc) come substrato. Aggiungere a ciascun pozzetto 100 l di cellule permeabilizzate e 100 ml di substrato (16 mg / ml soluzione madre in H 2 O). Fare attenzione ad evitare bolle d&#39;aria nei pozzetti. Leggere l&#39;assorbanza a 420 nm, utilizzando un lettore di micropiastre e il filtro appropriato. </li></ol>

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The authors declare that they have no competing financial interests.

Le proteine ​​possono essere intrinsecamente disordinato, con le regioni la cui struttura terziaria non è limitata a un numero limitato di conformazioni 25. Queste proteine ​​sono disordinati solito incline all&#39;aggregazione 25, e l&#39;isolamento e la caratterizzazione potrebbero rappresentare un compito difficile. La subunità ε di E. DNA polimerasi coli III dispone di due domini distinti 26,27, vale a dire: i) il dominio N-ter, recante la &#39;attività ...

L&#39;introduzione di tecnologie iperespressione impulso sia gli studi biochimici di basso numero di copie enzimi e la produzione industriale di proteine ​​farmacologicamente attive (ad esempio, insulina). Con l&#39;avvento di queste tecnologie, progressi significativi sono stati raggiunti per aumentare la resa e la qualità delle proteine ​​ricombinanti. Inoltre, procariote ed eucariote 1,2 3 sistemi iperespressione sono stati sviluppati nel corso degli anni, offrendo alternative utili al &quot;cavallo di lavoro&quot; di proteine ​​biotecnologie, cioè, Escherichia coli. In particolare, la disponibilità di piattaforme alternative a E. coli ha portato alla produzione di peptidi o proteine ​​ricombinanti recanti modificazioni post-traduzionali. Tuttavia, va detto che E. coli rappresenta ancora l&#39;organismo di riferimento per la produzione di proteine ​​ricombinanti. Ciò è dovuto a diversi fattori, tra cui la più rilevante può essereconsiderato: i) la disponibilità di un certo numero di sistemi iperespressione (vettori di espressione e ceppi) per E. coli 1,2; ii) i tempi breve generazione, e alti rendimenti biomassa, di E. coli in una varietà di media ricchi e sintetici; iii) la manipolazione facile né al biochimico e a livello genetico di questo microrganismo; iv) l&#39;isolamento di ceppi capaci di produzione di proteine ​​tossiche 4; v) la costruzione di ceppi caratterizzati induzione omogenea a livello di popolazione 5,6. Inoltre, è stato recentemente dimostrato che i sistemi di espressione adatti per la produzione in E. coli di proteine ​​post- traslazione modificati può essere concepita e costruita 2. Allo stato attuale, la proteina sovraespressione è principalmente utilizzato per ottenere proteine ​​monomeriche o omo-oligomeri, le cui hypersynthesis può essere eseguita con un singolo gene clonato in un plasmide appropriata. Tuttavia, l&#39;attenzione è stata recentementeversato alla costruzione di E. sistemi di proteine ​​co-espressione coli, sfidando la produzione, in vivo, di complessi etero-oligomerica 2. È interessante notare che i primi esperimenti di proteine ​​co-espressione di fronte all&#39;Assemblea inter-specie di grandi e piccole subunità di carbossilasi cianobatteri ribulosio-1,5-bisfosfato / ossigenasi 7,8, e l&#39;associazione di forme troncate e lungometraggi di HIV-1 trascrittasi inversa 9. Questi studi pionieristici hanno dimostrato che la proteina co-espressione rappresenta una potente alternativa al tradizionale ricostituzione vitro. Inoltre, la proteina co-espressione in E. coli è stato usato per produrre diverse proteine ​​recanti modificazioni post-traduzionali 2, per ottenere proteine ​​contenenti amminoacidi innaturali 2, e per aumentare la resa di proteine ​​di membrana sovraespresso 2. Inoltre, il potenziale della proteina co-espressione come uno strumento per conferire a E. coli competerence della secrezione di proteine ​​è sotto inchiesta attiva 2. Due strategie principali di proteine ​​co-espressione in E. coli può essere perseguito: i) l&#39;uso di un singolo plasmide per ospitare diversi geni da sovraespresso; ii) l&#39;uso di più plasmidi in singole cellule co-esprimono proteine ​​bersaglio. Nel primo caso, i criteri per la scelta del plasmide non differiscono da quelle dei tradizionali esperimenti singole proteine ​​iperespressione, anche se particolari plasmidi contenenti tandem elementi promotore / operatore stati costruiti per co-espressione 10. Questo primo approccio è quindi molto semplice. Tuttavia, va detto che l&#39;uso di un singolo plasmide co-esprimere proteine ​​diverse facce due grandi difficoltà: i) la massa molecolare del vettore aumenta con il numero di geni ospitati, limitando il numero di proteine ​​co-espresse; ii) quando più geni clonati sono sotto il controllo di un unico promotore, polarità può decreasall&#39;e l&#39;espressione dei geni distali dal promotore. L&#39;uso di plasmidi doppi o multipli in un&#39;unica E. coli deve compiere la compatibilità dei vettori di scelta, quindi imporre vincoli alle combinazioni ammissibili di plasmidi. Tuttavia, questa seconda strategia co-espressione presenta il vantaggio di contenere la massa molecolare di vettori, e limita la polarità. Recentemente abbiamo costruito un sistema di co-espressione della proteina progettato per facilitare la spola di geni tra plasmidi co-espressione 11. In particolare, abbiamo costruito la serie vettori PGOOD, le caratteristiche rilevanti dei quali sono: i) origine p15A di replica, per garantire la compatibilità dei plasmidi PGOOD con i vettori commerciali che contiene l&#39;origine ColE1 (ad esempio, la serie pBAD 12); ii) una cassetta tetraciclina resistenza; iii) la presenza di lac -derived elementi regolatori, cioè, il promotore-operatore (O 1) Coppia e Laci </em&gt; gene q. Utilizzando un adeguato paio pBAD-PGOOD, siamo stati in grado di iperespressione nucleo catalitico di E. DNA polimerasi coli III, composto da tre differenti subunità, cioè, α (il &#39;polimerasi, ε (3&#39;-5 5&#39;-3)&#39; exonuclease) e θ (stabilizzazione ε) 13. In particolare, abbiamo dimostrato che la co-espressione del complesso αεθ era strettamente dipendente dalla aggiunta alla E. coli terreno di coltura sia di IPTG e arabinosio, innescando sovraespressione da PGOOD e pBAD rispettivamente (Figura 1A). Nella presente relazione, illustriamo come proteine ​​co-espressione può efficacemente risolvere le difficoltà legate alla scarsa solubilità di un complesso proteico subunità. Inoltre, si mostra come nei test di complementazione proteica vivo possono essere eseguiti, e abbiamo finalmente riportiamo l&#39;uso di proteine ​​co-espressione di frequenza sintonizzare mutazione in E. coli. A questo scopo, abbiamo utilizzato le coppie PGOOD-PBAD idonei per illustrare esempi rilevanti di ciascun caso di studio.

<table border="0" cellpadding="0" cellspacing="0" width="627">
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			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
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			<td height="21" style="height:21px;width:216px;">Agar</td>
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			<td height="21" style="height:21px;width:216px;">KCl</td>
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			<td height="21" style="height:21px;width:216px;">NaCl</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PNP-gluc</td>
			<td style="width:125px;">Sigma-Aldrich</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pNP-TMP</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">T0251</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tetracyclin</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">87128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trizma base&#160;</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:119px;">T1503</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Tryptone</td>
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			<td style="width:119px;">95039</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Yeast extract</td>
			<td style="width:125px;">Fluka</td>
			<td style="width:119px;">70161</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acrylamide solution 30%</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0158</td>
			<td>For gel electrophoresis</td>
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			<td style="width:119px;">161-0700</td>
			<td>For gel electrophoresis</td>
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			<td height="21" style="height:21px;width:216px;">Glycine</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0718</td>
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			<td height="21" style="height:21px;width:216px;">TEMED</td>
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			<td style="width:119px;">161-0800</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">161-0719</td>
			<td>For gel electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cuvettes 0.1 cm</td>
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			<td style="width:119px;">1652089</td>
			<td>For electroporation</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
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			<td height="21" style="height:21px;width:216px;">Centrifuge 5415R</td>
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			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge Allegra 21R</td>
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			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Chromatography apparatus GradiFrac</td>
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			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Gene Pulser II electroporation</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microplate Reader 550</td>
			<td style="width:125px;">Biorad</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MiniProtean 3 cell</td>
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			<td height="21" style="height:21px;width:216px;">Sonicator 3000</td>
			<td style="width:125px;">Misonix</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

La subunità ε di E. coli DNA polimerasi III è composto da 243 aminoacidi e dispone di scarsa solubilità 17,18, a meno che i residui 187-243 vengono cancellati 17. Tuttavia, abbiamo già dimostrato 11 che il co-espressione di α full-length, ε, e subunità θ produce solubile DNA polimerasi III nucleo catalitico (Figura 1). In particolare, utilizzando il sistema pBAD-PGOOD co-espressione, abbiamo dimostrato che: i) la sovraespressione di α e subunità ε pu...

Watch this Scientific Journal Video about The Multifaceted Benefits of Protein Co-expression in Escherichia coli at JoVE.com

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 ...

the-multifaceted-benefits-of-protein-co-expression-in-escherichia-coli

Research

JoVE Journal

Biology

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

11.8K Views.  University of Bologna. 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. 

Video: The Multifaceted Benefits of Protein Co-expression in Escherichia coli

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 ...

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. ...

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 ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Spazi di giunzione intercellulare di comunicazione: un biomarker funzionale per valutare gli effetti avversi dei tossici e tossine, e benefici per la salute di prodotti naturali

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

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