Per CM Margaret Smith per aver gentilmente fornire il clone integrasi Un sostegno finanziario a partire da: Genome Canada / Genome Prairie NSERC Discovery e borse di studio progetti strategici

1. Produzione di Cultura <ol><li> Preparare liquidi sterili: TY 10 (5 g / l triptone, 3 g / l di estratto di lievito, 0,44 g / l di cloruro di calcio diidrato), e LB 11 (10 g / l triptone, 5 g / l di estratto di lievito, 5 g / cloruro di sodio, pH 7). </li><li> Inoculare da una singola colonia: SmUW227 (S. meliloti LP-ceppo: verranno descritte altrove, ceppo dettagli costruttivi disponibili su richiesta) () in 5 ml di TY media con 50 ug / ml spectinomicina. Inoculare le seguenti ceppi in 5 ml di liquidi LB, che integra con l&#39;antibiotico data: E. coli DH5a 12 contenente pJC2, il plasmide di espressione integrasi, con 10 pg / ml di tetraciclina; E. coli MT616 13, il mobilizzatore, con 10 pg / ml cloramfenicolo, e E. coli DH5a contenente pJH110, la cassetta plasmide donatore, con 5 pg / ml di gentamicina. </li><li> Incubare E. ceppi di E. notte a 37 &amp;deg; C con costante agitazione. Incubare S. meliloti deformazione a 30 ° C per due giorni con agitazione. È possibile ottenere una S. meliloti cultura per tutta la notte con un inoculo più grande (1:500 sottocultura di una cultura satura). </li></ol> 2. Cultura preparazione e miscelazione <ol><li> Lavare 1,5 ml di coltura di ciascun ceppo raccogliendo il pellet cellulare mediante centrifugazione a 17.000 xg per 30 secondi e risospeso in 1 ml di NaCl sterile 0,85%; ripetere una volta. </li><li> Risospendere il pellet in 100 microlitri di sterile allo 0,85% NaCl. </li><li> Individualmente individuare 10 pl della cultura lavato per ogni ceppo su una pianura piastra di agar TY. Questi punti sono punti di controllo. </li><li> Aggiungere 40 pl di ciascun ceppo esclusa la E. coli DH5a 12 contenente pJC2 in una provetta sterile, mescolare, e spot 120 microlitri di questa miscela in una pianura piastra di agar TY. Questo punto è il no-integrasi controllo negativo. </li><li> Aggiungere 40 pl di ciascun ceppo in un t sterileUbe, mix, e spot 160 microlitri di questa miscela in una pianura piastra di agar TY. Questo luogo è il punto di accoppiamento IMCE. </li><li> Lasciare punti asciugare in una cappa a flusso laminare. </li><li> Piastre di tenuta e incubare a 30 ° C per una notte. </li></ol> 3. Isolamento di Trans-integrants <ol><li> Punti di controllo Streak e spot IMCE su piastre di agar TY integrato con 200 mg / ml di streptomicina (SmUW227 si basa su Rm1021 che trasporta una mutazione conferisce resistenza di alto livello alla streptomicina 14) e 30 mg / ml di gentamicina. </li><li> Per il calcolo del rendimento IMCE, risospendere circa ¼ del punto di accoppiamento IMCE in 500 microlitri sterile NaCl allo 0,85%. Effettuare diluizioni seriali di 10 volte a 10 -7. Piatto diluizioni 10 -2 a 10 -5 su piastre di agar TY integrati con 200 mg / ml di streptomicina e 30 mg / ml di gentamicina, di selezionare per trans integranti. Piatto diluizioni 10 -3 through 10 -7 su piastre di agar TY integrati con 200 mg / ml di streptomicina, per selezionare sia per i trans integranti e dei destinatari potenziali, cioè dei destinatari totali. </li><li> Per l&#39;isolamento di markerless trans integrants risospendere circa un quarto del punto di accoppiamento IMCE in 500 pl di sterile 0,85% NaCl. Effettuare diluizioni seriali di 10 volte a 10 -6. Diluizioni tavola 10 -4 a 10 -6 su quattro piastre di agar TY integrati con 200 mg / ml di streptomicina e 200 mg / ml X-Gluc. </li><li> Incubare le piastre a 30 ° C per 3 giorni. </li><li> Visualizza RFP trans integrants sotto la luce verde (525 nm) e attraverso un filtro rosso (&gt; 610 nm) 15. </li><li> Calcolare l&#39;efficienza IMCE per cento come CFU di trans-integrants CFU rispetto al totale dei destinatari. Circa la metà di trans-integrants sono sottoposti vero scambio cassette che li rende bianchi e spectinomicina sensibile. </li><li> Per trovare i markerless trans integrants (wqui il vettore donatore non contiene Gm r come in pJH110) schermo per una colonia bianco (un periodo di incubazione prolungato, 1-2 giorni supplementari a temperatura ambiente, aumenterà la differenziazione delle colonie tramite X-Gluc, ciò è necessario in S. meliloti SmUW227), confermare la sensibilità del spectinomicina della colonia mediante screening su una piastra di agar TY senza antibiotici e una piastra di agar TY contenente 100 ug / ml spectinomicina. </li></ol> 4. Risultati rappresentativi Dopo tre giorni di incubazione su TY integrato con streptomicina e gentamicina le strisce di controllo non dovrebbe avere alcuna crescita. La striscia IMCE dovrebbe avere crescita confluente sulla striscia testa e molte colonie sulla seconda striscia, come mostrato nella Figura 2. L&#39;efficienza di trans-integrazione, espressa come percentuale di trans-integrants ai destinatari totale, dovrebbe essere nell&#39;intervallo di 0,5%. Circa la metà di trans-integrants sarà spectinomicina sensibili e nerodimostrando di avere subito vero e proprio scambio cassetta. Trans-integrants contenenti il donatore tester rfp cassetta dalla pJH110 dovrebbe visualizzare distinguibile fluorescenza RFP se visto sotto la luce verde (525 nm) e attraverso un filtro rosso (&gt; 610 nm) 15. <img src="/files/ftp_upload/3698/3698fig1.jpg" alt="Figura 1"/> . Figura 1 illustrazione miscela coniugazione: Con l&#39;aiuto della espressione dei geni di trasferimento di pRK600, tutti i plasmidi sono trasferiti in modo casuale da una cella all&#39;altra. Questo trasferimento comporta la creazione di trans-integrants nella miscela, attraverso l&#39;acquisizione LP-ceppo dei due plasmidi necessari per IMCE, l&#39;integrasi (int) pJC2 plasmide helper e la pJH110 plasmide donatore. La cassetta del donatore non replicante plasmide donatore (pJH110) viene scambiata attraverso ΦC31 integrasi con i marcatori del LP-cassetta sul cromosoma, con conseguente perdita dei marcatori LP (SpR e uidA) e il mantenimento della cassetta donatore (RFP e Gm r) nel risultante trans-integrante. <img src="/files/ftp_upload/3698/3698fig2.jpg" alt="Figura 2"/> Figura 2. A:. Macchia piastra di accoppiamento in modo non selettivo piastra TY mostrando miscele di cellule secche sulla superficie agar B: punti di accoppiamento seminata su streptomicina, gentamicina-X-Gluc piatto, dall&#39;alto a sinistra in senso orario alcun controllo integrasi, IMCE in S. meliloti, E. coli DH5a contenente il controllo pJC2, E. coli DH5a contenente pJH110 controllo, E. coli MT616 controllo e S. . meliloti UW227 di controllo C: 10 -2 diluizione di risospensione accoppiamento punto sul TY streptomicina-X-agar Gluc D:. diluizione 10 -2 di risospensione accoppiamento punto sul TY streptomicina-gentamicina-X-Gluc agar (colonie blu sono ricombinanti singoli, colonie bianche sono stati sottoposti a vere. scambio di cassette) E: 10 -2 diluizione di risospensione punto di accoppiamento sulla streptomicina TY-X-agar Gluc che mostra la mancanza di fluorescenza F:. diluizione 10 -2 di risospensione accoppiamento punto sul TY streptomicina-gentamicina-X-agar Gluc che mostra due livelli di fluorescenza (più brillanti colonie corrispondono a colonie blu e hanno una maggiore espressione rfp presumibilmente a causa di promotore leggere, anche se dalla sequenza vettoriale, dove le colonie che hanno subito true-cassette contengono RFP scambio solo con il suo promotore immediata senza read-through da parte del promotore lac in il vettore, che è assente.).

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Non ci sono conflitti di interesse dichiarati.

La tecnica IMCE consente l&#39;integrazione efficace di una singola cassetta DNA attB affiancato nella LP-locus di un ceppo precedentemente progettato. Una volta che il costrutto desiderato viene clonato al posto di RFP per creare la cassetta donatore, la tecnica non richiede successiva purificazione del DNA e trasformazione, che rende molto robusto. È fondamentale che controlli crescita appropriati sono inclusi, per essere certi della resistenza agli antibiotici è dovuta alla creazione di trans-inte...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Streptomicina </td><td> Bioshop Canada Inc. </td><td> STP101 </td><td></td></tr><tr><td> Spectinomicina </td><td> Bioshop Canada Inc. </td><td> <a href="https://ssl34.alentus.com/bioshopcanada/detail.asp?Pin=SPE201">SPE201</a> </td><td></td></tr><tr><td> Gentamicina </td><td> Bioshop Canada Inc. </td><td> GTA202 </td><td></td></tr><tr><td> Choramphenicol </td><td> Bioshop Canada Inc. </td><td> CLR201 </td><td></td></tr><tr><td> Tetracycline </td><td> Bioshop Canada Inc. </td><td> TET701 </td><td></td></tr><tr><td> Kanamicina </td><td> Bioshop Canada Inc. </td><td> KAN201 </td><td></td></tr><tr><td> Batteriologica agar grade </td><td&gt; Bioshop Canada Inc. </td><td> AGR001 </td><td></td></tr><tr><td> Triptone </td><td> Bioshop Canada Inc. </td><td> TRP402 </td><td></td></tr><tr><td> Estratto di lievito </td><td> Bioshop Canada Inc. </td><td> YEX401 </td><td></td></tr><tr><td> Cloruro di sodio </td><td> Bioshop Canada Inc. </td><td> SOD001 </td><td></td></tr><tr><td> Cloruro di calcio </td><td> Bioshop Canada Inc. </td><td> CCL444 </td><td></td></tr><tr><td> X-Gluc </td><td> Oro Biotechnology Inc. </td><td> G1281C1 </td><td></td></tr><tr><td> MT616 ceppo E. coli </td><td> Disponibile su richiesta </td><td></td><td> Utilizzato anche al di fuori del nostro laboratorio </td></tr><tr><td> PJC2 ceppo E. coli </td><td> In casa, disponibile su richiesta </td><td></td><td></td></tr><tr><td> PJH110 ceppo E. coli </td><td> In casa, a disposizione dal richt </td><td></td><td></td></tr><tr><td> SmUW227 ceppo </td><td> In casa, disponibile su richiesta </td><td></td><td></td></tr></tbody></table>

site-specific bacterial chromosome engineering: &#934;c31 integrase mediated cassette exchange (imce)

Il cromosoma batterico può essere utilizzato per mantenere stabilmente il DNA estraneo nel mega-base gamma 1. Integrazione nel cromosoma elude questioni quali la replicazione del plasmide, la stabilità plasmide, incompatibilità plasmide, e il plasmide varianza numero di copie. Questo metodo utilizza il site-specific integrasi dal fago Streptomyces (Φ) C31 2,3. Il ΦC31 integrasi catalizza una ricombinazione diretto tra due specifici siti del DNA: ATTB e attP (34 e 39 bp, rispettivamente) 4. Tale ricombinazione è stabile e non ripristinare 5. Una &quot;pista&quot; (LP) costituito da una sequenza spectinomicina gene di resistenza, aadA (SPR), e la E. coli ß-glucuronidasi gene (uidA) affiancato da siti attP è stato integrato nei cromosomi di Sinorhizobium meliloti, Ochrobactrum anthropi e tumefaciens in una regione intergenica, l&#39;ampC locus, e il locus teta, rispettivamente. S. meliloti viene utilizzato in questo protocollo. Vettori contenenti siti donatori mobilizzabile attB fiancheggiano una stuffer rosso proteina fluorescente (RFP) gene e un gene di resistenza agli antibiotici sono stati costruiti. In questo esempio la gentamicina pJH110 resistente plasmide viene utilizzato. Il gene RFP 6 può essere sostituito con un costrutto desiderata con Sph I e Pst I. In alternativa, un costrutto sintetico fiancheggiata da siti attB possono essere subclonato in un vettore mobilizzabile come pK19mob 7. L&#39;espressione del gene ΦC31 integrasi (clonato da pHS62 8) è guidata dal promotore lac, su una vasta gamma mobilizzabile ospite plasmide pRK7813 9. Un protocollo accoppiamento tetraparental viene utilizzato per trasferire la cassetta nel ceppo donatore LP sostituendo i marcatori nella sequenza LP con la cassetta donatore. Queste cellule sono trans-integrants. Trans-integrants sono formate con una efficienza tipica di 0,5%. Trans-integrants trovano tipicamente entro i primi 500-1.000 colonie schermati dalla sensibilità agli antibiotici o blu-nero screening utilizzando 5-bromo-4-cloro-3-indolil-beta-D-glucuronico (X-Gluc). Questo protocollo contiene le procedure di accoppiamento e di selezione per la creazione e isolare trans integranti.

Watch this Scientific Journal Video about Site-specific Bacterial Chromosome Engineering: Î¦C31 Integrase Mediated Cassette Exchange IMCE at JoVE.com

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

Un metodo rapido ed efficace per integrare DNA estraneo di interesse in pre-fatti ceppi accettori, chiamati ceppi pista di atterraggio, è descritta. Il metodo permette integrazione sito-specifica di una cassetta DNA nel locus ingegnerizzato atterraggio di un dato ceppo, tramite coniugazione e l&#39;espressione del ΦC31 integrasi.

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 ...

site-specific-bacterial-chromosome-engineering-c31-integrase-mediated

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Bioengineering

15.5K Views.  University of Waterloo. Un metodo rapido ed efficace per integrare DNA estraneo di interesse in pre-fatti ceppi accettori, chiamati ceppi pista di atterraggio, è descritta. Il metodo permette integrazione sito-specifica di una cassetta DNA nel locus ingegnerizzato atterraggio di un dato ceppo, tramite coniugazione e l'espressione del ΦC31 integrasi.

Video: Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Procedura per l&#39;Ingegneria del polmone

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

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Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

Volatili che caratterizzano batterica secondaria utilizzando Electrospray ionizzazione Spettrometria di Massa (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

Alta efficienza, sito-specifica trasfezione di cellule aderenti con siRNA mediante matrici microelettrodi (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capillare Forza litografia per Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

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 ...

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 ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

Produzione robusta fibra naturale preforme Utilizzando batterica cellulosa come Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea (ureolysis) in naturally occurring environments through secretion of an enzyme urease. This process of ureolysis, through a chain of chemical reactions, leads to the formation of calcium carbonate precipitates. This is known as Microbiologically Induced Calcite Precipitation (MICP). The proper culture protocols for MICP are detailed here. Finally, visualization experiments under different modes of microscopy were performed to understand various aspects of the precipitation process. Techniques like optical microscopy, Scanning Electron Microscopy (SEM) and X-Ray Photo-electron Spectroscopy (XPS)&#160;were employed to chemically characterize the end-product. Further, the ability of these precipitates to clog pores inside a natural porous medium was demonstrated through a qualitative experiment where sponge bars were used to mimic a pore-network with a range of length scales. A sponge bar dipped in the culture medium containing the bacterial cells hardens due to the clogging of its pores resulting from the continuous process of chemical precipitation. This hardened sponge bar exhibits superior strength when compared to a control sponge bar which becomes compressed and squeezed under the action of an applied external load, while the hardened bar is able to support the same weight with little deformation.

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 ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Ingegneria dei tessuti da parte intrinseca Vascolarizzazione in un<em&gt; In Vivo</em&gt; Camera ingegneria dei tessuti

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

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

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Volatili che caratterizzano batterica secondaria utilizzando Electrospray ionizzazione Spettrometria di Massa (SESI-MS)

Alta efficienza, sito-specifica trasfezione di cellule aderenti con siRNA mediante matrici microelettrodi (MEA)

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