Gli autori desiderano ringraziare Jen Sheen del Massachusetts General Hospital per la generosa donazione di piantine di Nicotiana benthamiana. Jen Bush ci ha aiutato molto nella consulenza in coltivazione di piante e la creazione di una zona di camera di crescita. Tom Ferrante dell&#39;Istituto Wyss offerto aiuto cruciale con microscopia confocale. Gli autori desiderano soprattutto ringraziare Don Ingber del Children Hospital di Boston e l&#39;Istituto Wyss per la generosa donazione di una pistola Gene e relative forniture. Il finanziamento per questo progetto è stato fornito attraverso un accordo di cooperazione con il Dipartimento dell&#39;Energia Advanced Research Projects Agency (ARPA-E Award # DE-000079) per la PAS, JCW, e MdM, e attraverso Chimerion Biotechnology, Inc. per MJV.

1. Progettazione di sequenze alternativa-splicing per multipla Organelle Targeting <ol><li> Determinare i siti per l&#39;espressione finale di proteine. Per questo lavoro, l&#39;interesse è in mira la cloroplasti, perossisomi, e citosol. </li><li> Utilizzare la letteratura per identificare proteine ​​e DNA sequenze note di indirizzare le proteine ​​per organelli di interesse. In questo caso un cloroplasto di targeting sequenza di Arabidopsis thaliana L-isoaspartate metiltransferasi 6,15, e un perossisomi mira sequenza di Arabidopsis thaliana transthyretin come S-allantoina sintasi 16,17 sono stati determinati. </li><li> Identificare un regime-splicing alternativo che dovrebbe indirizzare le proteine ​​di interesse per gli organelli corretti. Per questo lavoro, splice siti come osservato in un sistema di Arabidopsis naturale 6. La sequenza di mRNA è stata ridisegnata lasciando i siti di splicing naturali, ma le sequenze sono stati collocati codifica peroxis-cloroplasto oome-targeting tag all&#39;interno introni alternativamente-splicing (Figura 1). </li></ol> 2. Assemblea Gibson di parti di DNA Vedi anche figura 2. Il metodo di assemblaggio Gibson dipende dall&#39;azione di numerosi enzimi in un mix pre-made a 1) parzialmente digerire le estremità del DNA a doppio filamento di rendere filamenti singoli e 2) ricottura paia di basi gratuiti della vicina pezzi di DNA. Questo permette la clonazione di frammenti di DNA senza le limitazioni dei siti di restrizione. <ol><li> Scegliere un ordine per gli elementi del costrutto di DNA plasmidico. Come esempio TriTag-1 ha elementi di: a) un vettore plasmidico (pori, contenente un costrutto GFP noto a lavorare in piante, un marcatore di resistenza alla kanamicina, e origini di replicazione di E. coli e Agrobacterium tumefaciens host), b) una sequenza promotore, (P ENTCUP2, mostrato come costitutivo in piante), C) un cloroplasto rivolti sequenza (CTS), c) una sequenza di targeting perossisomi (PTS), e d) una serie di siti di splicing come descritto in precedenza 6. </li><li> Progettare la base-per-base costrutto con il software di progettazione in silico. Scimmia è un pacchetto freeware open source utile per questo lavoro. Nota: TriTag-1, ha l&#39;ordine: plasmide vettore, promoter, ATG, sito di splice donatore, cloroplasti sequenza bersaglio, sito donatore di splicing, luogo neutro, accettore sito di splice, perossisomi sequenza bersaglio, accettore sito di splice, GFP contenute nel vettore plasmidico . <ol><li> Progettare il silico costrutto di DNA in modo tale che vi sia una sovrapposizione di 50 bp tra ogni pezzo al momento dell&#39;ordine primer per PCR. È possibile utilizzare la sovrapposizione 50 bp come primer: 25 bp ciascuna direzione. <ol><li> Essere sicuri di tenere traccia della provenienza di ciascuno dei pezzi di sequenza. E &#39;come: un plasmide di essere coltivate e miniprepped; una sequenza lineare che può essere utilizzato come modello PCR; o una sequenza che dovrà esseresintetizzato in commercio? Diverse aziende offrono servizi di sintesi del DNA a basso costo per frammenti &lt;500 bp. In questo caso, il TriTag sé è 437 bp quindi era vantaggioso per ordinare commercialmente. </li><li> I migliori risultati arriveranno se tutti i frammenti siano PCR-amplificati e purificati dalla loro DNA template. Il marcatore di resistenza è un buon posto per dividere questo grande DNA in due modelli più piccoli che sono più facilmente PCR-amplificato. </li><li> Di restrizione restrizione enzima DpnI taglia il DNA metilato. Se il modello di PCR era una miniprep da E. coli, trattamento DpnI rimuoverà il contaminante DNA stampo. </li></ol></li></ol></li><li> Purificare tutte le sequenze di DNA separatamente ed eluire in acqua, TE o tampone EB. <ol><li> parte PORE vettore 1, ~ 3000 bp (frecce verdi). PCR amplificato e DpnI trattata. </li><li> PORE vettore parte 2, ~ 3000 bp (frecce nere). PCR amplificato e DpnI trattata. </li><li> TriTag inserto, 437 bp (frecce blu). Ordinato in commercio. Resuspend in DDH 2 O. </li><li> P ENTCUP promotore, ~ 750 bp (frecce arancioni). PCR-amplificato e DpnI trattata. </li></ol></li><li> Misurare le concentrazioni di ciascuno dei pezzi di DNA. Obiettivo per 150 ng / ml dei pezzi di vettore. </li><li> Prelevare un &#39;aliquota del mix di montaggio Gibson fuori dal freezer e scongelare sul ghiaccio. Questa è disponibile in commercio, ma anche di semplice realizzazione in laboratorio 12-14. <ol><li> Ci sono due passi per questa ricetta: a 5x master mix viscoso e 15 reazione dimensioni aliquote di 1.33x microlitri. Il master mix 5x contiene: 3 ml di 1 M Tris-HCl pH 7.5, 300 ml 1 M MgCl 2, 600 microlitri 10 mM di ciascun dNTP, 300 ml 1 M DTT, 1,5 g di PEG-8000, 20 mg di NAD, e DDH 2 O a 6 ml. Preparare 320 microlitri aliquote (18) e congelare tutti tranne uno a -20 ° C. La soluzione sarà molto viscoso. Etichettare questi &quot;tampone isoterma 5X&quot; </li><li> Per quello rimanente (320 ml), aggiungere 1,2 microlitri T5 Exonuclease, 20 microlitri Phusion Alto-Fidelity DNA polimerasi, 160 microlitri Taq DNA Ligase e 700 ml DDH 2 O. Preparare 15 aliquote microlitri (~ 80) sul ghiaccio in provette per PCR e conservare a -20 ° C. </li></ol></li><li> Determinare il volume di quantità equimolari di ciascuno dei frammenti. Ci sono alcuni calcolatori online, tra cui Gibthon.org. Ci dovrebbe essere un totale di 5 ml di tutti i pezzi di DNA. Aggiungerli al 15 microlitri Gibson mix aliquota. Se questo richiede volumi troppo piccolo per pipetta, diluire i frammenti di DNA e / o utilizzare più di una aliquota. <ol><li> Non dimenticate di preparare un controllo del DNA vettore da solo (ad esempio, la parte di DNA che contiene il marcatore di resistenza agli antibiotici) e portare a volume con acqua. </li></ol></li><li> Incubare le provette in un termociclatore a 50-55 ° C per 30-60 minuti. Sostituire su ghiaccio e trasformare in E. coli attraverso le cellule chimicamente competenti shock termico. Non utilizzare cellule elettrocompetenti. La concentrazione di sale e la concentrazione di DNA relativamente bassapuò causare le cellule a arco. <ol><li> Applicare tutte le 20 microlitri di una preparazione commerciale di 200 microlitri di calcio cloruro competente E. coli. Incubare 30 min in ghiaccio e shock termico 60 sec a 42 ° C </li><li> Ritorno al ghiaccio 2 min, applicare 750 microlitri SOC o terreno LB e recuperare agitazione in una provetta 30 minuti a 37 ° C. Piatto il composto e diluizioni adeguate LB + Kan (o altro antibiotico appropriato). Questo può tradursi in background maggiore (e un numero maggiore di eventi di ricombinazione non-target) rispetto ai tradizionali clonazione a base di legatura, ma vale la pena di schermo di circa 10 colonie. </li></ol></li><li> Conferma clone tramite sequenza e memorizzare una aliquota a -80 ° C </li></ol> 3. Biolistic trasfezione di tabacco con il Gene Gun Questa è una tecnica che si instaura in JoVE 18,19. Passaggi chiave e le differenze sono descritte di seguito. <ol><li> Crescere 50-100 ml E. coli o 200 ml Agrobacterium. Maxi-prep cultura. Una concentrazione di circa 1.500 ng / ml è necessaria per continuare. </li><li> Preparare gene proiettili di pistola come descritto in precedenza. Caricarli nella Gene Gun. </li><li> Per la trasfezione di Nicotiana benthamiana tabacco foglia tessuto epidermico, scegliere un grande foglia vicino alla base di un impianto di 3 mesi. Tagliare con cautela e metterlo lato inferiore su tovaglioli di carta bagnati in un piatto di 15 centimetri Petri. </li><li> Impostare la pressione Lui per il Gene Gun a 200-250 psi. </li><li> Orientare con attenzione la pistola genica tra nervature sul lato inferiore di una foglia, circa 3-5 cm sopra di esso. Rilasciare la sicurezza e fornire le particelle all&#39;anta. Ogni proiettile può essere utilizzato per due volte nella stessa posizione sulla foglia. Se esplode foglia, scartano e avviare una nuova foglia-c&#39;è qualche variazione in foglia tenacità a causa di condizioni di crescita, come l&#39;età, l&#39;umidità, ecc Per una foglia di 12 cm, si aspettano per adattarsi circa 6 colpi. </li><li> Conservare la foglia, coperto con la parte superiore di unCapsula di Petri per 2-3 giorni in condizioni di scarsa luminosità in panchina. </li><li> Esaminare la foglia in un microscopio dissezione dotato di fluorescenza UV, e la ricerca di singole cellule che esprimono GFP. Sezionare 5-10 mm sezioni di foglia. </li><li> Immergere la foglia in un vetrino da microscopio ben profondità sotto ~ 200 microlitri 0,1% Triton X-100. Il detersivo aiuta a prevenire la formazione di bolle d&#39;aria sulla superficie, e la profonda microscopio ben vetrino consente una grande area di esposizione del tessuto. </li></ol> 4. Microscopia confocale di Tissue trasfezionati Queste istruzioni variano per ogni strumento, quindi è essenziale per ottenere adeguatamente formati. <ol><li> Per gli esperimenti di rivelazione di fluorescenza, impostare il laser di eccitazione a 489 nm, e impostare i rivelatori fotomoltiplicatori a 500-569 nm per GFP fluorescenza e 630-700 nm per la clorofilla auto-fluorescenza. Celle di immagine utilizzando un obiettivo 40x immersione in acqua. </li></ol>

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In questo studio, strategie semplici sono descritti per la localizzazione di una singola proteina transgenica a più compartimenti cellulari nelle piante. L&#39;obiettivo era di progettare costrutto che esprimesse un singolo gene in più di un organello in Nicotiana benthamiana. Le strategie includono progettazione razionale di base di GFP-costrutti di DNA, il montaggio Gibson, la consegna dei plasmidi a celle foglia con il Gene Gun, e l&#39;osservazione dei risultati con microscopia confocale. <p class="jo...

Questo lavoro è un progetto di ingegneria / biologia sintetica metabolica in cui le cellule vegetali sono progettati per esprimere una proteina reporter in diversi organelli ma solo con un singolo costrutto di DNA. Un approccio per proteine ​​bersaglio a più di una posizione comporta clonazione più copie genetiche, ciascuna contenente un diverso peptide localizzazione. Ogni copia deve essere introdotto da successive ritrasformazione, o, in alternativa, da backcrossing singole trasformazioni 1. Ciò comporta la clonazione supplementare, ed è limitata da un tag di localizzazione per ogni terminale. Un altro modo per localizzare una proteina a più riprese è attraverso splicing alternativo 2-5. RNA è trascritto da un singolo gene, ma diverse copie della trascrizione sono trattati in modo diverso, spesso in più di un modo per cella. Ciò può comportare più di un RNA messaggero nella cella trascritto da un singolo gene. Questi diversi messengeRNA R possono codificare per diverse isoforme della stessa proteina, o nel caso di un frameshift, una proteina diversa. Sebbene splicing alternativo è stato descritto in letteratura per molti anni, i meccanismi di azione e donatore conservati e splice siti recettoriali sono solo stati chiariti più recente 6. Poiché questi siti vengono meglio descritti, si aprono opportunità per l&#39;ingegneria. Gli ingegneri metaboliche delle piante si trovano ad affrontare una sfida quando esprime una proteina in più organelli. Per una proteina che è da localizzare a più organello, il tecnico deve clonare il gene stesso più volte, ciascuno con una sequenza segnale separato dirigendolo al organello di interesse. Per un singolo gene in tre organelli, questo è semplicemente tre geni. Ma per una via metabolica sei gene, questo si espande a 18 geni, un notevole sforzo di clonazione. La combinazione di più sequenze di localizzazione in un unico segno gene alternativa-impiombatoriduce ificantly questo sforzo. Ad esempio, re-engineering fotorespirazione 7,8 e sintesi isoprenoide 9,10 coinvolge sia i cloroplasti e perossisomi. Nel nostro caso abbiamo approfittato dei siti di splicing come osservato in un sistema di Arabidopsis thaliana naturale descritto in precedenza 6. Abbiamo razionalmente riprogettato la sequenza di mRNA di lasciare i siti di splicing naturali solo, ma messo sequenze che codificano tag-perossisomi rivolti-cloroplasto o all&#39;interno introni alternativamente-splicing (Figura 1). La proteina espressa può o non può avere un tag, a seconda che il pre-mRNA che codifica è stato escisso come un introne (Figure 1g e 1h). Per ulteriori informazioni sulla progettazione dei costrutti presentati in questo lavoro, vedere l&#39;articolo compagna 11. Perché questo è ancora uno sforzo significativo clonazione, il montaggio Gibson, un nuovo metodo di clonazione costrutti di DNA, è used in costruzione. Il metodo Gibson può essere utilizzato per qualsiasi sequenza, indipendentemente siti di restrizione (Figura 2) 12-14. Il mix specifico di enzimi permette un solo passaggio, assemblaggio isotermica. In questo metodo, più parti a doppio filamento lineare di DNA sono progettati in modo tale che essi hanno sequenze sovrapposte di ~ 50 bp. L&#39;assemblaggio miscela enzimatica Gibson digerisce parzialmente le parti di DNA lineari, esponendo singoli filamenti di sequenze omologhe. Questi parziali sequenze a singolo filamento sono ri-ricotti nella miscela di reazione, con un conseguente rapido, one-step, sequenza-indipendente, reazione subcloning privo di legatura. Questo lavoro descrive 1) progettazione razionale costrutti splicing alternativo per l&#39;espressione di cloroplasti delle piante, perossisomi, e citosol, 2) la loro clonazione utilizzando il nuovo metodo privo di legatura di riunione Gibson, 3) la loro consegna nelle cellule delle foglie di tabacco con Gene Gun, e 4) I risultati mostrano il targeting organelli, come osservato con GFPe microscopia confocale.

<table border="0" cellpadding="0" cellspacing="0" width="693">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oligonucleotide primers</td>
			<td style="width:191px;">IDT</td>
			<td style="width:119px;">(custom)</td>
			<td style="width:167px;">Design specifically for construct</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GeneBlocks</td>
			<td style="width:191px;">IDT</td>
			<td style="width:119px;">(custom)</td>
			<td>500 bp oligonucleotides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ApE software</td>
			<td style="width:191px;">U Utah</td>
			<td style="width:119px;">(download)</td>
			<td>http://biologylabs.utah.edu/jorgensen/wayned/ape/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MinElute kit</td>
			<td style="width:191px;">Qiagen</td>
			<td style="width:119px;">28004</td>
			<td>Used to purify PCR products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QIAprep spin miniprep kit</td>
			<td style="width:191px;">Qiagen</td>
			<td style="width:119px;">27104</td>
			<td>Used to prepare cloning-appropriate amounts of plasmid</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phusion Master Mix with GC Buffer</td>
			<td style="width:191px;">NEB</td>
			<td style="width:119px;">M0532S</td>
			<td>Used to PCR-amplify gene of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gibson assembly reaction mix</td>
			<td style="width:191px;">NEB</td>
			<td style="width:119px;">E2611L</td>
			<td>Master mix of the following 9 ingredients</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 M Tris-HCl pH7.5</td>
			<td style="width:191px;">Teknova</td>
			<td style="width:119px;">T1075</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 M MgCl2</td>
			<td style="width:191px;">G Biosiences</td>
			<td style="width:119px;">82023-086</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dNTP mix</td>
			<td style="width:191px;">Fermentas</td>
			<td style="width:119px;">R0192</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 M DTT</td>
			<td style="width:191px;">Fermentas</td>
			<td style="width:119px;">R0861</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PEG-8000</td>
			<td style="width:191px;">Affymetrix</td>
			<td style="width:119px;">19966</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NAD</td>
			<td style="width:191px;">Applichem</td>
			<td style="width:119px;">A1124,0005</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T5 exonuclease</td>
			<td style="width:191px;">Epicentre</td>
			<td style="width:119px;">T5E4111K</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Phusion polymerase</td>
			<td style="width:191px;">NEB</td>
			<td style="width:119px;">F530S</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Taq DNA ligase</td>
			<td style="width:191px;">NEB</td>
			<td style="width:119px;">M0208L</td>
			<td>Gibson assembly mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gibthon.org</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>Website to simplify calculations</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Plasmid PLUS Maxi kit</td>
			<td style="width:191px;">Qiagen</td>
			<td style="width:119px;">12963</td>
			<td>Used to prepare DNA for gene gun bullets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gene Gun system</td>
			<td style="width:191px;">BioRad</td>
			<td style="width:119px;">165-2451</td>
			<td>Includes all parts necessary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nicotania benthamiana</td>
			<td style="width:191px;">(n/a)</td>
			<td style="width:119px;">(n/a)</td>
			<td>Gift of Jen Sheen, MGH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Confocal microscope</td>
			<td style="width:191px;">Leica</td>
			<td style="width:119px;">SP5 X MP</td>
			<td>Imaging of resultant cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Deep well slides</td>
			<td style="width:191px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">71561-01</td>
			<td>Used for confocal imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">A Plasmid Editor (ApE) </td>
			<td style="width:191px;">University of Utah </td>
			<td style="width:119px;"></td>
			<td>http://biologylabs.utah.edu/jorgensen/wayned/ape</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gibthon:Ligation calculator</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>http://django.gibthon.org/tools/ligcalc/</td>
		</tr>
	</tbody>
</table>

transient gene expression in tobacco using gibson assembly and the gene gun

Al fine di indirizzare una singola proteina a più organelli subcellulari, piante tipicamente duplicare i geni rilevanti, ed esprimono ogni gene separatamente utilizzando complesse strategie di regolamentazione, tra cui promotori differenziali e / o sequenze di segnali. Ingegneri metaboliche e biologi sintetici interessato a colpire enzimi per un particolare organello trovano ad affrontare una sfida: per una proteina che è da localizzare a più di un organello, il tecnico deve clonare il gene stesso più volte. Si presenta una soluzione a questa strategia: sfruttando splicing alternativo dell&#39;mRNA. Questa tecnologia sfrutta cloroplasto stabilito e dei perossisomi mira sequenze e li combina in un unico mRNA che viene alternativamente impiombata. Alcune varianti di splicing vengono inviati al cloroplasto, alcuni al perossisoma, e alcuni al citosol. Qui il sistema è progettato per multipli organello targeting con splicing alternativo. In questo lavoro, GFP si aspettava di essere espressed nei cloroplasti, citosol, e perossisomi da una serie di razionalmente progettati 5 &#39;tag mRNA. Questi tag hanno il potenziale per ridurre la quantità di clonazione necessaria quando i geni eterologhi devono essere espressi in più organelli subcellulari. I costrutti sono stati progettati in opera precedente 11, e sono stati clonati usando Gibson assembly, un metodo di clonazione indipendente legatura che non richiede enzimi di restrizione. I plasmidi risultanti sono stati introdotti in Nicotiana benthamiana celle foglia epidermica con un protocollo Gene Gun modificato. Infine, foglie trasformate sono state osservate con microscopia confocale.

Lo sforzo progettuale è stato il risultato di una pianificazione significativa. Novel di questo progetto è l&#39;uso di splicing alternativo per creare un pre-mRNA che viene tradotto in proteine ​​differenzialmente espressi. Queste proteine ​​sono espresse in diversi organelli, in questo caso il cloroplasti, perossisomi e / o citosol. Abbiamo adattato gene Arabidopsis naturale che viene alternativamente impiombato 6, e collocato conosciuti cloroplasto 6 e perossisomi 17</sup...

Watch this Scientific Journal Video about Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun at JoVE.com

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

Questo lavoro descrive un nuovo metodo per selettivamente organelli subcellulari nelle piante, saggiata usando l&#39;BioRad Gene Gun.

Transient espressione genica in tabacco con all&#39;Assemblea Gibson e Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

transient-gene-expression-tobacco-using-gibson-assembly-gene

Research

JoVE Journal

Environment

20.4K Views.  Harvard University.  Questo lavoro descrive un nuovo metodo per selettivamente organelli subcellulari nelle piante, saggiata usando l'BioRad Gene Gun. 

Video: Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

Determinazione del microbica extracellulare Enzyme Activity in Acque, Suoli e sedimenti utilizzando High Throughput micropiastre saggi

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

Ricerca

Ambiente

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

Trascrizione e metabolita Profiling per la valutazione del Tabacco Albero e pioppo come materia prima per l&#39;industria con sede a Bio

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne illness. Ready-to-eat (RTE) processed foods such as deli meats, cheese, and in some cases fresh produce, have been involved in foodborne disease outbreaks due to contamination with pathogens such as Listeria monocytogenes. With respect to L. monocytogenes, deli slicers are often the main source of cross contamination. The goal of this study was to use a fluorescent compound to simulate bacterial contamination and track this contamination in a retail setting. A mock deli kitchen was designed to simulate the retail environment. Deli meat was inoculated with the fluorescent compound and volunteers were recruited to complete a set of tasks similar to those expected of a food retail employee. The volunteers were instructed to slice, package, and store the meat in a deli refrigerator. The potential cross contamination was tracked in the mock retail environment by swabbing specific areas and measuring the optical density of the swabbed area with a spectrophotometer. The results indicated that the refrigerator (i.e. deli case) grip and various areas on the slicer had the highest risk for cross contamination. The results of this study may be used to develop more focused training material for retail employees. In addition, similar methodologies could also be used to track microbial contamination in food production environments (e.g.&#160;small farms), hospitals, nursing homes, cruise ships, and hotels.

The overall goal of this study was to demonstrate the potential cross contamination mechanism of foodborne pathogen Listeria monocytogenes in a retail deli setting. This methodology may be applied to a variety of different environments to track pathogen contamination. &#160;

Monitoraggio contaminazione microbica in ambienti di vendita al dettaglio usando fluorescente Powder - Un dettaglio Delicatessen Ambiente Esempio

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne ...

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture organisms, such as hyphae-building fungi and anaerobic bacteria, has hastened the use of recombinant technologies in this field. Plant expression methods are a desirable system for large-scale production of enzymes and other industrially useful proteins. Herein, methods for the transient expression of a fungal endoglucanase, Trichoderma reesei Cel5A, in Nicotiana tabacum are demonstrated. Successful protein expression is shown, monitored by fluorescence using an mCherry-enzyme fusion protein. Additionally, a set of basic tests are used to examine the activity of transiently expressed T. reesei Cel5A, including SDS-PAGE, Western blotting, zymography, as well as&#160;fluorescence and dye-based substrate degradation assays. The system described here can be used to produce an active cellulase in a short time period, so as to assess the potential for further production in plants through constitutive or inducible expression systems.

Expression of Recombinant Cellulase Cel5A from Trichoderma reesei in Tobacco Plants

Tobacco plants were used to produce a fungal cellulase, TrCel5A, via a transient expression system. The expression could be monitored using a fluorescent fusion protein, and the protein activity was characterized post-expression.

Espressione ricombinante Cellulase Cel5A da<em&gt; Trichoderma reesei</em&gt; In piante di tabacco

Cellulose degrading enzymes, cellulases, are targets of both research and industrial interests. The preponderance of these enzymes in difficult-to-culture ...

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.

This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs.

Screening prodotti alimentari per la Classe 1 integroni e Gene cassette

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major ...

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That represents 100 million tons of food being destroyed or diverted to non-human consumption each year. Aflatoxins are powerful carcinogens normally accumulated by the fungi Aspergillus flavus and A. parasiticus in cereals, nuts, root crops and other agricultural products. Silencing of five aflatoxin-synthesis genes by RNA interference (RNAi) in peanut plants was used to control aflatoxin accumulation following inoculation with A. flavus. Previously, no method existed to analyze the effectiveness of RNAi in individual peanut transgenic events, as these usually produce few seeds, and traditional methods of large field experiments under aflatoxin-conducive conditions were not an option. In the field, the probability of finding naturally contaminated seeds is often 1/100 to 1/1,000. In addition, aflatoxin contamination is not uniformly distributed. Our method uses few seeds per transgenic event, with small pieces processed for real-time PCR (RT-PCR) or small RNA sequencing, and for analysis of aflatoxin accumulation by ultra-performance liquid chromatography (UPLC). RNAi-expressing peanut lines 288-72 and 288-74, showed up to 100% reduction (p&#8804;0.01) in aflatoxin B1 and B2 compared to the control that accumulated up to 14,000 ng.g-1 of aflatoxin B1 when inoculated with aflatoxigenic A. flavus. As reference, the maximum total of aflatoxins allowable for human consumption in the United States is 20 ng.g-1. This protocol describes the application of RNAi-mediated control of aflatoxins in transgenic peanut seeds and methods for its evaluation. We believe that its application in breeding of peanut and other crops will bring rapid advancement in this important area of science, medicine and human nutrition, and will significantly contribute to the international effort to control aflatoxins, and potentially other mycotoxins in major food crops.

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

We demonstrate a method for the analysis of aflatoxins and transgene expression in peanut seeds that contain RNA-interference signals for silencing aflatoxin-synthesis genes in the fungus Aspergillus flavus. RNAi-mediated control of mycotoxins in plants has not been reported previously.

Controllo RNAi mediata di aflatossine in arachidi: metodo per analizzare produzione di micotossine e transgene espressione nel Peanut /<em&gt; Aspergillus</em&gt; Patosistema

The Food and Agriculture Organization of the United Nations estimates that 25% of the food crops in the world are contaminated with aflatoxins. That ...

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly relevant as a model for the studies of osmoregulatory mechanisms because of its extreme tolerance to low salinity. It is also widely used as a model of settling biology, in particular in relation to antifouling research. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae for studies. A protocol for the all-year-round culturing of B. improvisus has been developed and a detailed description of all steps in the production line is outlined (i.e., the establishment of adult cultures on panels, the collection and rearing of barnacle larvae, and the administration of feed for adults and larvae). The description also provides guidance on troubleshooting and discusses critical parameters (e.g., the removal of contamination, the production of high-quality feed, the manpower needed, and the importance of high-quality seawater). Each batch from the culturing system maximally yields roughly 12,000 nauplii and can deliver four batches in a week, so up to almost 50,000 larvae per week can be produced. The method used to culture B. improvisus is, probably, to a large extent also applicable to other marine invertebrates with free-swimminglarvae. Protocols are presented for the dissection of various tissues from adults as well as the production of high-quality RNA for studies on gene expression. It is also described how cultured adults and reared cyprids can be utilized in a wide array of experimental designs for examining gene expression in relation to external factors. The use of cultured barnacles in gene expression is illustrated with studies of possible osmoregulatory roles of Na+/K+ ATPase and aquaporins.

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The barnacle Balanus (Amphibalanus) improvisus is a model for studying osmoregulation and antifouling. However, natural seasonal spawning yields an unpredictable supply of cyprid larvae. Here, a protocol for the all-year-round culturing of B. improvisus is described, including the production of larvae. The use of cultured barnacles in gene expression studies is illustrated.

Il Barnacle Balanus BalanUs come modello Marine - coltura e l'espressione genica

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into the plant&#39;s genome. Here, we present two transformation systems to genetically modify potato (Solanum tuberosum) plants. In A. tumefaciens transformation, leaves are infected, the transformed cells are selected and a new complete transformed plant is regenerated using phytohormones in 18 weeks. In A. rhizogenes transformation, stems are infected by injecting the bacteria with a needle, the new emerged transformed hairy roots are detected using a red fluorescent marker and the non-transformed roots are removed. In 5-6 weeks, the resulting plant is a composite of a wild type shoot with fully developed transformed hairy roots. To increase the biomass, the transformed hairy roots can be excised and self-propagated. We applied both Agrobacterium-mediated transformation methods to obtain roots expressing the GUS reporter gene driven by a suberin biosynthetic gene promoter. The GUS staining procedure is provided and allows the cell localization of the promoter induction. In both methods, the transformed potato roots showed GUS staining in the suberized endodermis and exodermis, and additionally, in A. rhizogenes transformed roots the GUS activity was also detected in the emergence of lateral roots. These results suggest that A. rhizogenes can be a fast alternative tool to study the genes that are expressed&#160;in roots.

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Here, we present two protocols to transform potato plants. The Agrobacterium tumefaciens transformation leads to a complete transgenic plant while the Agrobacterium rhizogenes produces transgenic hairy roots in a wild type shoot that can be self-propagated. We then detect promoter activity by GUS staining in the transformed roots.

Agrobacterium tumefaciens e Agrobacterium rhizogenes-mediata trasformazione di patate e l'attività del promotore di un Gene di suberina da GUS Staining

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression.
The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified.
Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

Secondo i saggi foglia distaccati per semplificare gli studi sull'espressione genica nelle patate durante l'infestazione da mawing Insect Manduca sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

Stima dell'indice dell'area foglia utilizzando tre metodi distinti in stand deciduous puro

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...