We wish to acknowledge the partners in the Helmholtz-Alberta Initiative, the Helmholtz Association and the University of Alberta, for the support resulting from participation in this collaboration. Research funding is provided by the Helmholtz Association's Initiative and Networking Fund, the participating Helmholtz Centers and by the Government of Alberta through Alberta Environment's ecoTrust program.
Dr. Tanushree Ghosh is gratefully acknowledged for her critical inputs at a number of crucial stages.

NOTA: Eseguire i protocolli sperimentali nell&#39;ordine descritto di seguito. Il protocollo di coltura batterica è discusso nella sezione 1 (vedi anche figura 1). Sezione 2 descrive il protocollo per arricchire il terreno di coltura utilizzando additivi esterni. La sezione 3 descrive i protocolli per la microscopia multi-mode. I pesi di tutti i singoli componenti possono essere misurati con una bilancia analitica. Volume di ogni soluzione può essere misurata utilizzando un cilindro tarato. NOTA: i protocolli di biosicurezza adeguato devono essere seguite per le sezioni 1 - 2. Consultare l&#39;ufficio di sicurezza istituzionale per i dettagli. 1. coltura batterica <ol><li> Batteri Cultura - Agar piastra Media Preparazione <ol><li> Assemblare attrezzature e gli ingredienti, come piastre di Petri, fiasco, Tris-base, HCl, agar, Millipore acqua, pH-metro etc.Sterilize tutti i contenitori in autoclave a 121 ° C prima dell&#39;uso. </li><li> Preparare 1 L di 0,13 soluzione acquosa M di Tris-buffer mescolando 15.75 g Tris-base con 1 L di acqua Millipore. Per abbassare il pH della soluzione originale (pH 10,4) aggiungere 2.800 ml di HCl (concentrazione 50%). Controllare continuamente con un pH-metro per impostare pH = 9. </li><li> Dividere la soluzione tampone 1 L in due parti: <ol><li> Prendere 800 ml di questa soluzione. Dividerlo equamente in due parti di 400 ml ciascuna. Sciogliere 8 g (NH 4) 2 SO 4 con una soluzione e 16 g estratto di lievito per l&#39;altra soluzione. </li><li> Dai rimanenti (200 ml) soluzione e dividerla nuovamente in due parti di 100 ml ciascuna. Mescolare 2 g (NH 4) 2 SO 4 a uno. Aggiungere 4 g di estratto di lievito e 4 g di agar all&#39;altro. </li></ol></li><li> Autoclave le 4 soluzioni separatamente, dopo aver terminato i rispettivi palloni in foglio di alluminio e attaccare nastri autoclave. NOTA: Se viene utilizzato un apparecchio da banco autoclave, il volume deve essere impostato a 500 ml (temperatura e pressione automatcamente specificato in funzione del volume). </li><li> Dopo averli tolti dal autoclave, impostare i due 400 ml di soluzioni da parte per passo 1.3.1 (qui di seguito). Mescolare le due soluzioni 100 ml di una soluzione di 200 ml. Versare il composto in 10 - 12 capsule di Petri. </li></ol></li><li> Batteri Cultura - piastra di agar Preparazione del campione <ol><li> Rimuovere il magazzino batterica dal congelatore (-80 ° C) e lasciarlo scongelare. Dopo lo scongelamento correttamente, posizionare il magazzino batterica e la piastra di agar all&#39;interno di una cappa di biosicurezza. </li><li> Selezionare la micropipetta di piccola dimensione disponibili (0,5-10 ml è una buona scelta) ad infestare la punta con il brodo pasteurii Sporosarcina. Streak una piastra di agar con la punta micropipetta. Posizionare la piastra di agar striato all&#39;interno di un incubatore non agitazione a 31 ° C per 48 ore. </li><li> Dopo 48 ore, rimuovere la piastra dal termostato di esaminare visivamente l&#39;esistenza di singole colonie. Se non ci sono singole colonie, poi posto in tegli incubatrice per un altro 24 ore. </li><li> Ripetere il processo fino a quando vengono rilevati singole colonie. Non superare i 7 giorni di prova. NOTA: Se singole colonie non appaiono anche dopo una settimana, poi si conclude che le misure non sono state seguite correttamente e l&#39;intero processo deve essere ripetuto dal punto 1. </li></ol></li><li> Batteri Cultura - campione finale Preparazione <ol><li> Mescolare le due soluzioni 400 ml (tampone Tris + (NH 4) 2 SO 4 e tampone Tris + estratto di lievito (1.5.1) insieme per ottenere una soluzione di 800 ml. Trasferire 125 ml di questa soluzione in un pallone. </li><li> Eseguire un esame visivo della superficie della piastra di agar per identificare le regioni ad alta concentrazione di singole colonie. Delicatamente spingere e rompere una delle colonie con una punta micropipetta. </li><li> Immergere la stessa punta micropipetta nel pallone 125 ml e mescolare accuratamente per garantire che il numero sufficiente di cellule per la moltiplicazione robusta vengono trasferite. Porre il pallone in un incubatore agitazione a 150 rpm, 30 ° C per 2 - 3 giorni. Dopo 2 - 3 giorni, togliere il pallone dal termostato. </li></ol></li><li> Batteri Cultura - Finale Cell Count <ol><li> Eseguire diluizioni seriali della soluzione di coltura non diluito con PBS per ottenere una diluizione di almeno dieci milioni (10 -7) garantire appaiono singole colonie numerabili. Disegnare sette linee equidistanti parallele su una delle agar-piastre. <ol><li> Eseguire questa disegnando linee in grassetto sulla superficie inferiore del piatto Petri, abbastanza importante per essere visibile dall&#39;alto. Goccia 3 piccole gocce di soluzione non diluita in un segmento. Aggiungere 1 ml di soluzione non diluita a 9 ml di tampone fosfato (PBS) per ottenere una diluizione 1:10. </li></ol></li><li> Prendere una piccola aliquota (~ 0,1 ml) di questa nuova soluzione diluita con una pipetta e rilasciare 3 più piccole gocce sul segmento successivo. Trasferire la nuova soluzione diluita in un pallone e ulteriormente diluita dieci volte (10x) byaggiungendo PBS. Questo fa scendere la diluizione di 10 -2 o 1: 100. <ol><li> Utilizzare questo 1: soluzione 100 nel segmento successivo. Ripetere questo si elabora con piccoli volumi di soluzioni diluite appena trasferendoli successivamente a nuovi flaconi e diluendo continuamente dieci volte (10x) in tandem con PBS per ottenere campioni sempre più diluite da 10 -3 o 1: 1.000 fino fino a 10 -7 o 1:10 milioni nell&#39;ultimo segmento. </li></ol></li><li> Eseguire Colony-Forming count Unit (CFU) piastra per contare il numero di cellule presenti nella piastra di agar dopo incubazione la piastra per 1 - 2 giorni a 31 ° C. Questo dà una misura quantitativa della carica batterica nel campione non diluito. NOTA: Il valore CFU viene misurata in base alla capacità del sistema di dare origine a colonie nelle condizioni specifiche di mezzo nutriente, temperatura e tempo supponendo che ogni colonia è separata e fondata da una singola cellula microbica vitali. </li><li> Focale piastre di Petri con autosigillante pellicola e conservare rimanendo elementi in frigorifero per un utilizzo futuro. </li></ol></li></ol> 2. Il protocollo per l&#39;arricchimento di nutrienti per accelerare Precipitazioni <ol><li> Transfer 9 ml della coltura liquida preparata (cellule + medium) in diverse provette per centrifuga sterilizzati, ciascuno di 10 ml di volume. </li><li> Preparare una soluzione stock di arricchimento esterno costituito da quattro componenti nelle seguenti concentrazioni in mezzo fresco 100 ml: Urea: 2 g / L, cloruro di ammonio: 1 g / L, bicarbonato di sodio: 212 mg / L, cloruro di calcio: 280 mg / L. Misurare attentamente tutti gli ingredienti con una bilancia analitica e mescolare tutto, ma l&#39;urea con terreno fresco in un bicchiere e posto in autoclave (121 ° C, 15 psi, 15 min). <ol><li> Dopo autoclave, mescolare la quantità necessaria di urea (2 mg) con 1 ml di mezzo fresco e passare attraverso una siringa con filtro a siringa da 0,22 micron per completare il processo di arricchimento. </li><li> Aggiungere 1ml di questo terreno di arricchimento con additivi per le provette da centrifuga sterili contenenti 9 ml di coltura liquida preparata (cellule + media) (vedi punto 2.1). NOTA: Di tutti questi componenti, urea essendo degradabile a temperature elevate, non può essere sterilizzato in autoclave. Quindi, dopo gli altri componenti sono stati autoclave (121 ° C, 15 psi, 15 min), l&#39;urea viene aggiunto per ultimo attraverso un filtro a siringa da 0,22 micron. </li></ol></li><li> Vortex ogni provetta accuratamente con un vortex meccanico. Posizionare i liquidi arricchiti (cellule + additivi medie +) in un incubatore non agitazione a 30 ° C. Monitorare tutte le unità regolarmente per l&#39;inizio di precipitazioni. Utilizzando un microscopio ottico, iniziare osservazioni al microscopio volta insorgenza di precipitazione viene rilevata ad occhio nudo. Questo è di solito tra 30 - 36 ore dopo l&#39;inizio degli esperimenti. </li></ol> 3. Protocollo per Multi-mode Microscopia <ol><li> Trasferire un piccolo volume (~ 1 ml) di fluido ad un alto magnificatione del sistema di microscopia coprioggetti-camerata per eseguire le osservazioni iniziali con modalità campo chiaro. Per cominciare, avviare tutte le osservazioni con l&#39;ingrandimento più basso (4X). che fornisce un&#39;ampia area di copertura e informazioni quindi globale sulla distribuzione di precipitati. </li><li> Selezionare aree particolari con volumi significativi di precipitazioni e zoom (20X, 40X, 60X) aumentando progressivamente l&#39;ingrandimento. NOTA: Dal momento che è impossibile risolvere correttamente le cellule dalla sostanza extracellulare polimerico (EPS) in campo chiaro (a causa di molto vicino indici di rifrazione), attivare la modalità a contrasto di fase in caso di necessità. In questo modo, solo i contorni delle membrane cellulari (essendo una fase differente rispetto alla cella bulk) sono visibili, permettendo così differenziazione visiva. </li><li> Infine, macchiare alcune camere con Live macchia / Morto che tinge selettivamente le componenti in tensione, come verde, mentre la colorazione dei componenti morti in rosso. Fare riferimento a protocolli standard 28 &lt;/ Sup&gt;. </li><li> Attivare la modalità fluorescente di distinguere correttamente tra le regioni rosse e verdi. Red (cubo filtro rosso con eccitazione lunghezza d&#39;onda di 540-580 nm, dicroico specchio di 595 nm e Barriera filtro di 600-660 nm) e passaggio lungo verde (GFP cubo filtro verde con eccitazione lunghezza d&#39;onda di 440-480 nm, dicroico specchio 505 filtro nm e Barriera di 510 cubi nm) di filtro sono utilizzati per facilitare il microscopio a fluorescenza. </li><li> Una volta che sono stati raccolti tutti i dati multi-mode, selezionare le migliori immagini e sovrapposizione manualmente (individuale campo chiaro, contrasto di fase e le immagini fluorescenti) per ottenere il miglior contrasto possibile e chiarezza. </li><li> Eseguire SEM sui campioni solidi secchi di capire strutture cristalline. Questa è una procedura ben consolidata e standard. 22 Eseguire XPS per identificare le firme chimiche dei gruppi funzionali (carbonati). Questa è una procedura ben consolidata e standard pure. 23 </li></ol>

<ol>
	<li>Gibson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+investigation+of+the+Bacillus+pasteurii+group.">An investigation of the Bacillus pasteurii group.</a> Journal of Bacteriology. 28, 491-502 (1934).</li><li>Greenfield, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+and+concentration+of+calcium+and+magnesium+and+precipitation+of+calcium+carbonate+by+a+marine+bacterium.">Metabolism and concentration of calcium and magnesium and precipitation of calcium carbonate by a marine bacterium.</a> Annals of the New York Academy of Sciences. 109, 23-45 (1963).</li><li>Phillips, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+applications+of+ureolytic+biomineralization:+a+review.">Engineered applications of ureolytic biomineralization: a review.</a> Biofouling. 29, 715-733 (2013).</li><li>Dhami, N. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomineralization+of+calcium+carbonates+and+their+engineered+applications:+a+review.">Biomineralization of calcium carbonates and their engineered applications: a review.</a> Frontiers in microbiology. 4, 314 (2013).</li><li>Cuthbert, M. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controls+on+the+rate+of+ureolysis+and+the+morphology+of+carbonate+precipitated+by+S.+Pasteurii+biofilms+and+limits+due+to+bacterial+encapsulation.">Controls on the rate of ureolysis and the morphology of carbonate precipitated by S. Pasteurii biofilms and limits due to bacterial encapsulation.</a> Ecological Engineering. 41, 32-40 (2012).</li><li>Okwadha, G. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimum+conditions+for+microbial+carbonate+precipitation.">Optimum conditions for microbial carbonate precipitation.</a> Chemosphere. 81, 1143-1148 (2010).</li><li>Stocks-Fischer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiological+precipitation+of+CaCO3.">Microbiological precipitation of CaCO3.</a> Soil Biology and Biochemistry. 31, 1563-1571 (1999).</li><li>Lauchnor, E. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterially+induced+calcium+carbonate+precipitation+and+strontium+coprecipitation+in+a+porous+media+flow+system.">Bacterially induced calcium carbonate precipitation and strontium coprecipitation in a porous media flow system.</a> Environmental science &amp; technology. 47, 1557-1564 (2013).</li><li>Al Qabany, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+Affecting+Efficiency+of+Microbially+Induced+Calcite+Precipitation.">Factors Affecting Efficiency of Microbially Induced Calcite Precipitation.</a> Journal of Geotechnical and Geoenvironmental Engineering. 138, 992-1001 (2012).</li><li>Morita, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcite+precipitation+by+marine+bacteria.">Calcite precipitation by marine bacteria.</a> Geomicrobiology Journal. 2, 63-82 (2009).</li><li>Chafetz, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marine+peloids:+A+product+of+bacterially+induced+carbonate+precipitation.">Marine peloids: A product of bacterially induced carbonate precipitation.</a> Journal of Sedimentary Petrology. 56, 812-817 (1986).</li><li>Whiffin, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Microbial CaCO3 precipitation for the production of biocement. , (2004).</li><li>Paassen, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale+up+of+BioGrout:+a+biological+ground+reinforcement+method.">Scale up of BioGrout: a biological ground reinforcement method.</a> Proceedings of the 17th International Conference on Soil Mechanics and Geotechnical Engineering. , 2328-2333 (2009).</li><li>Cunningham, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbially+enhanced+geologic+containment+of+sequestered+supercritical+CO2.">Microbially enhanced geologic containment of sequestered supercritical CO2.</a> Energy Procedia. 1, 3245-3252 (2009).</li><li>Mitchell, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+enhanced+geologic+sequestration+of+supercritical+CO2.">Biofilm enhanced geologic sequestration of supercritical CO2.</a> International Journal of Greenhouse Gas Control. 3, 90-99 (2009).</li><li>Cunningham, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+the+risk+of+well+bore+leakage+of+CO2+using+engineered+biomineralization+barriers.">Reducing the risk of well bore leakage of CO2 using engineered biomineralization barriers.</a> Energy Procedia. 4, 5178-5185 (2011).</li><li>Jonkers, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+bacteria+as+self-healing+agent+for+the+development+of+sustainable+concrete.">Application of bacteria as self-healing agent for the development of sustainable concrete.</a> Ecological Engineering. 36, 230-235 (2010).</li><li>Tobler, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+Sporosarcina+pasteurii+in+sandstone+and+its+significance+for+subsurface+engineering+technologies.">Transport of Sporosarcina pasteurii in sandstone and its significance for subsurface engineering technologies.</a> Applied Geochemistry. 42, 38-44 (2014).</li><li>Mortensen, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+environmental+factors+on+microbial+induced+calcium+carbonate+precipitation.">Effects of environmental factors on microbial induced calcium carbonate precipitation.</a> Journal of applied microbiology. 111, 338-349 (2011).</li><li>Phillips, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+CO2+leakage+reduction+through+biofilm-induced+calcium+carbonate+precipitation.">Potential CO2 leakage reduction through biofilm-induced calcium carbonate precipitation.</a> Environmental science &amp; technology. 47, 142-149 (2013).</li><li>vander Heide, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> X-ray Photoelectron Spectroscopy: An introduction to Principles and Practices. , (2011).</li><li>Wiley, W. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+an+alkaline+pH+and+ammonia+for+substrate+oxidation+by+Bacillus+pasteurii.">Requirement of an alkaline pH and ammonia for substrate oxidation by Bacillus pasteurii.</a> Journal of Bacteriology. 84, (1962).</li><li>Tagliaferri, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observing+strain+localisation+processes+in+bio-cemented+sand+using+x-ray+imaging.">Observing strain localisation processes in bio-cemented sand using x-ray imaging.</a> Granular Matter. 13, 247-250 (2011).</li><li>Kumar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+confinement+features+can+affect+biofilm+formation.">Microscale confinement features can affect biofilm formation.</a> Microfluidics and Nanofluidics. 14, 895-902 (2012).</li><li>Valiei, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+web+of+streamers:+biofilm+formation+in+a+porous+microfluidic+device.">A web of streamers: biofilm formation in a porous microfluidic device.</a> Lab on a chip. 12, 5133-5137 (2012).</li><li>. LIVE/DEAD Bacterial Viability kit, Two-color bacterial viability assay Available from: <a target="_blank" href="https://tools.lifetechnologies.com/content/sfs/manuals/mp07007.pdf">https://tools.lifetechnologies.com/content/sfs/manuals/mp07007.pdf</a> (2004)</li></ol>

The authors have nothing to disclose.

Passi critici: Questo manoscritto descrive in dettaglio i protocolli per la coltura di un campione vitale di S. pasteurii. Una volta che la coltura è stato preparato, deve essere opportunamente arricchito. Questo è un passo chiave fondamentale per il successo dell&#39;esperimento, perché un fallimento per fornire l&#39;ambiente chimico adeguato porta a uno scale temporali molto lunghi di precipitazioni o di una completa mancanza della stessa. S. pasteurii è molto sensibile a divers...

Sporosarcina pasteurii è un batterio gram-positivo in grado di sopravvivere in ambienti fortemente alcaline (pH ~ 10) 1 ed è una delle specie batteriche che possono diventare un agente causativo di un fenomeno chiamato Microbiologicamente Induced Calcite Precipitation (MICP) 2-4. MICP è un processo in cui la precipitazione di carbonato di calcio è indotta da certi microbi in condizioni ambientali idonee. S. pasteurii ha assunto importanza negli ultimi anni a causa della sua identificazione come possibile agente per indurre un significativo volume di MICP in determinate condizioni. Questa possibilità deriva dal fatto che S. pasteurii ha la capacità unica di secernere grandi quantità di ureasi. Questo enzima agisce come un catalizzatore, promuovendo una lisi accelerata di urea (un composto biochimico naturale con fornitura diffusa e abbondante) in presenza di molecole di acqua. Attraverso una cascata di reazioni, questo processo finalely porta alla generazione di ioni carbonato carica negativa. Questi ioni, a sua volta, reagire con ioni metallici positivi come calcio per formare infine precipitati di carbonato di calcio (calcite); da qui l&#39;etichetta MICP 5-9. Il processo di MICP è stato conosciuto e studiato per diversi decenni 10,11. Nel corso degli ultimi anni, MICP è stato studiato per un&#39;ampia gamma di applicazioni tecniche e ambientali, compresa bottom-up costruzione verde 12, il miglioramento delle strutture di grandi dimensioni 13,14 e il sequestro del carbonio e lo stoccaggio 15,16. Ad esempio, Cunnigham 17 et. Al progettato un reattore a flusso temperatura moderata ad alta pressione contenente un nucleo di arenaria Berea. Il reattore è stato inoculato con il batterio S. fridgidimarina e in condizioni di alta pressione carbonica supercritica iniezione anidride, un massiccio accumulo di biomassa all&#39;interno del poro volUmes stato osservato, che ha portato ad una riduzione superiore al 95% in permeabilità. Jonkers e Schlangen 18 hanno studiato l&#39;effetto di alcuni ceppi di batteri speciali sul processo di auto-guarigione in calcestruzzo. acqua esterna trasportata nella rete dei pori che entra attraverso i pori della superficie si prevede di attivare i batteri dormienti che a loro volta aiutano resistenza strutturale tramite MICP. Tobler 19 et al. hanno confrontato l&#39;attività ureolytic di S. pasteurii con una falda indigena ureolytic microcosmo in condizioni che favoriscono MCIP su larga scala e ha scoperto che S. pasteurii ha una capacità costante di migliorare la calcite precipitazioni anche quando le comunità indigene mancava attività ureasica prima. Mortensen 20 et.al hanno studiato gli effetti di fattori esterni come il tipo di suolo, la concentrazione di cloruro di ammonio, salinità, concentrazione di ossigeno e lisi delle cellule su MICP. La loro dimostrazione che il processo di trattamento biologico è molto robusto con respect ad un&#39;ampia variazione nella spazio dei parametri sostanzia l&#39;idoneità di questo processo per varie applicazioni di bonifica su larga scala di cui un processo di arricchimento adeguato per rinforzare i batteri è intrapresa. Phillips 21 et. al progettato esperimenti per studiare i cambiamenti nella permeabilità e la forza di una colonna di sabbia ed un nucleo di arenaria dopo l&#39;iniezione con S. culture pasteurii. Essi hanno scoperto che, mentre la permeabilità diminuita 2 - 4 volte mentre la resistenza alla frattura è aumentato tre volte. S. pasteurii e il suo ruolo nella MICP sono argomenti di ricerca attiva e di diverse questioni relative al meccanismo di precipitazione chimica non sono ancora pienamente compresi. Alla luce di questo, è molto importante avere un set di protocolli standardizzati coerenti con precisione la cultura un magazzino opportunamente arricchito di S. pasteurii per raggiungere MICP. Qui, descriviamo un protocollo rigoroso che assicuri ripetibilità e la riproducibilità. Questo manuscript descrive i protocolli dettagliati per la coltura di S. pasteurii ed opportunamente arricchendo il terreno di coltura per indurre la precipitazione. Il processo è indagato attraverso varie tecniche microscopiche, come ottica e microscopia elettronica a scansione (SEM) e X-Ray Photo-elettrone Spectroscopy (XPS). Il focus del manoscritto è sul processo di MICP. Procedure come SEM e SIMS, essendo protocolli standard consolidati, non sono descritte separatamente.

<table border="0" cellpadding="0" cellspacing="0" width="705">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Petridish</td>
			<td style="width:108px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875712</td>
			<td style="width:255px;">Petridishes being used as Agar plate</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:224px;">Pyrex Flasks</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S63268</td>
			<td style="width:255px;">Corning Erlenmeyer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Tris-Base</td>
			<td style="width:108px;">Promega</td>
			<td style="width:119px;">H5133</td>
			<td style="width:255px;">being used to make Tris-Buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Hydrochloric Acid</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">H9892</td>
			<td style="width:255px;">1.0 N, Bioreagent, suitable for cell culture</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:224px;">Agar Powder</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">A1296</td>
			<td style="width:255px;">microbiology tested, plant cell culture tested, cell culture tested, powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Sulphate</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4418</td>
			<td style="width:255px;">for Molecular Biology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Yeast extract powder</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">51475</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Measuring Cylinder</td>
			<td style="width:108px;">Cole-Parmer</td>
			<td style="width:119px;">CP08559GC</td>
			<td style="width:255px;">Cole-Parmer Class A Graduated Cylinder w/Cal Cert,TC;1000ml,1/Pk</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Analytical Balance</td>
			<td style="width:108px;">OHAUS</td>
			<td style="width:119px;">AX124E</td>
			<td style="width:255px;">being used to measure weight of reagents</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Autoclave</td>
			<td style="width:108px;">Brinkmann</td>
			<td style="width:119px;">58619000</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Autoclave Tape</td>
			<td style="width:108px;">VWR</td>
			<td style="width:119px;">52428864</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Aluminum Foil</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z185140</td>
			<td style="width:255px;">being used to seal the flask before placing it in Autoclave</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Bacterial Stock</td>
			<td style="width:108px;">Cedarlane</td>
			<td style="width:119px;">11859</td>
			<td style="width:255px;">-80&#176;C stock of S. pasteurii, ATCC No. is mentioned against Cat. No.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:224px;">Mline Single-Channel Mechanical Pipettors, Variable Volume</td>
			<td style="width:108px;">Biohit</td>
			<td style="width:119px;">725010</td>
			<td style="width:255px;">Marketed by VWR under catalog number 14005976</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Micropipette Tip</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">212772B</td>
			<td style="width:255px;">Used for scratching Agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Incubator</td>
			<td style="width:108px;">Binder</td>
			<td style="width:119px;">80079098</td>
			<td style="width:255px;">Microbiology Incubator,BF Series</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">Shaking Incubator</td>
			<td style="width:108px;">VWR</td>
			<td style="width:119px;">14004300</td>
			<td style="width:255px;">VWR Signature Benchtop Shaking Incubators</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Saline (PBS)&#160;</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7059</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:224px;">BD Falcon Express Pipet-Aid Pipetting Device</td>
			<td style="width:108px;">BD Biosciences</td>
			<td style="width:119px;">357590</td>
			<td style="width:255px;">Marketed by VWR under catalog number 53106220</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Parafilm</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7793</td>
			<td style="width:255px;">Being used to seal Agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Urea</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">U1250</td>
			<td>Enrichment for nutrient medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Sodium Bicarbonate</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8875</td>
			<td>Enrichment for nutrient medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:224px;">Calcium chloride</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1016</td>
			<td>Enrichment for nutrient medium</td>
		</tr>
	</tbody>
</table>

microbiologically induced calcite precipitation mediated by sporosarcina pasteurii

La particolare batterio sotto inchiesta qui (S. pasteurii) è unico nella sua capacità, nelle giuste condizioni, di indurre l&#39;idrolisi di urea (ureolysis) in ambienti presenti in natura attraverso la secrezione di un ureasi. Questo processo di ureolysis, attraverso una catena di reazioni chimiche, porta alla formazione di precipitati di carbonato di calcio. Questo è noto come Microbiologicamente Induced Calcite Precipitazioni (MICP). I protocolli di coltura adeguate per MICP sono descritti qui. Infine, esperimenti di visualizzazione in diverse modalità di microscopia sono state effettuate per capire i vari aspetti del processo di precipitazione. Tecniche come la microscopia ottica, microscopia elettronica a scansione (SEM) e X-Ray Photo-elettroni Spectroscopy (XPS) sono stati impiegati per caratterizzare chimicamente il prodotto finale. Inoltre, la capacità di questi precipitati ad ostruire i pori all&#39;interno di un mezzo poroso naturale è stato dimostrato attraverso un esperimento qualitativa dove spugnabarre sono stati usati per simulare un poro-rete con una gamma di scale di lunghezza. Una barra spugna imbevuta nel terreno di coltura contenente le cellule batteriche indurisce l&#39;intasamento dei suoi pori risultanti dal processo continuo di precipitazione chimica. Questa barra spugna indurito presenta una resistenza superiore rispetto a un bar spugna controllo che si comprime e spremuto sotto l&#39;azione di un carico esterno applicato, mentre la barra indurito è in grado di sostenere lo stesso peso con poca deformazione.

S. pasteurii essere un alkaliphile 24 possono sopravvivere in condizioni relativamente difficili. Quando il protocollo cultura di cui sopra è seguita, e S. pasteurii è cresciuto all&#39;interno di una camera, i batteri porta alla precipitazione di carbonato di calcio nel tempo (Figura 2A). Figura 2 (b) mostra un contrasto di fase ottica dell&#39;immagine microscopica della popolazione cellulare batterica nel terreno di colt...

Watch this Scientific Journal Video about Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii at JoVE.com

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

Microbiologicamente indotta Calcite Precipitazioni Mediata da<em&gt; Sporosarcina pasteurii</em

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea ...

microbiologically-induced-calcite-precipitation-mediated-sporosarcina

Research

JoVE Journal

Bioengineering

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

21.7K Views.  University of Alberta. Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

Video: Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6. Gold is particularly used because of its low toxicity 7-9. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12.
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15 or nanostars 16. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.

We synthesized star shaped gold nanostars using a silver seed mediated growth method. The diameter of the nanostars ranges from 200 to 300 nm and the number of tips vary from 7 to 10. The nanoparticles have a broad surface plasmon resonance mode centered in the near infrared.

Sintesi Nanostar oro con un metodo d&#39;argento crescita Seed Mediated

The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5. There is a great interest ...

Ricerca

Bioingegneria

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

Site-specific Ingegneria batterica Cromosoma: ΦC31 integrasi Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Raccolta murini macrofagi alveolari e Valutare l&#39;attivazione cellulare indotta da nanoparticelle polianidride

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

Osservazione e quantificazione della compattazione del gel di fibrina mediata da fibroblasti

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.&#160;low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

Investigare la separazione tridimensionale del flusso indotta da un modello di polipo piega vocale

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

Sintesi di una proteina artificiale mediata da inteina Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Utilizzando impedenza Cell-substrato e imaging cellulare dal vivo per misurare i cambiamenti in tempo reale in Cellular Adesione e De-adesione indotta da Matrix Modifica

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of exosomes to determine if they carry disease discriminatory biomarkers. For performing exosomal analysis, it is necessary to develop a method for extracting and analyzing exosomes from target biofluids without damaging the internal content.
Electric field-induced release and measurement (EFIRM) is a method for specifically extracting exosomes from biofluids, unloading their cargo, and testing their internal RNA/protein content. Using an anti-human CD63 specific antibody magnetic microparticle, exosomes are first precipitated from biofluids. Following extraction, low-voltage electric cyclic square waves (CSW) are applied to disrupt the vesicular membrane and cause cargo unloading. The content of the exosome is hybridized to DNA primers or antibodies immobilized on an electrode surface for quantification of molecular content.
The EFIRM method is advantageous for extraction of exosomes and unloading cargo for analysis without lysis buffer. This method is capable of performing specific detection of both RNA and protein biomarker targets in the exosome. EFIRM extracts exosomes specifically based on their surface markers as opposed to size-based techniques.
Transmission electron microscopy (TEM) and assay demonstrate the functionality of the method for exosome capture and analysis. The EFIRM method was applied to exosomal analysis of 9 mice injected with human lung cancer H640 cells (a cell line transfected to express the exosome marker human CD63-GFP) in order to test their exosome profile against 11 mice receiving saline controls. Elevated levels of exosomal biomarkers (reference gene GAPDH and protein surface marker human CD63-GFP) were found for the H640 injected mice in both serum and saliva samples. Furthermore, saliva and serum samples were demonstrated to have linearity (R = 0.79). These results are suggestive for the viability of salivary exosome biomarkers for detection of distal diseases.

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

Rilevazione di Exosomal Biomarker da campo elettrico indotto rilascio e di misura (Efirm)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formazione Light-mediata ed il modello di idrogel per colture cellulari

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...