Authors thank Mr. Kotaro Okubo for the kind assistance with SEM imaging. This work was supported by the Japan Society for the Promotion of Science Grant-in-Aid for Basic Research (B) (20310069), Grant-in-Aid for Research Activity Start-up (25880021), and by research grants from the Kurata Memorial Hitachi Science and Technology Foundation and the Nippon Sheet Glass Foundation for Materials Science and Engineering.

1. Preparación de TiO2 presenta el revestimiento de cristal Coverslip <ol><li> Número los cubreobjetos utilizando una punta de trazar diamante. Esto no sólo ayuda a mantener un registro de cada cubreobjetos, sino también para asegurar que el lado correcto de la muestra quede hacia arriba. Limpie los cubreobjetos, primero bajo el chorro de ddH2O, a continuación, sumergiéndolas en una solución de pirañas (H 2 SO 4: H 2 O 2 = 4: 1). Después de 10 minutos, enjuague los cubreobjetos a fondo, 8 veces en ddH2O Seque el cubreobjetos bajo flujo de N2. </li><li> Conjunto TiO 2 de destino en el sistema de pulverización catódica de radiofrecuencia (RF). Coloque el cubreobjetos sobre un soporte de muestras del equipo de pulverización catódica con una cinta de poliamida. Colocar el soporte de muestra en la cámara de pulverización catódica. Evacuar la cámara hasta que la presión alcanza 2,0 × 10 -4 Pa. </li><li> Introducir gas Ar en la cámara y ajustar la presión de deposición a 4,0 mTorr. Mientras se mantiene el obturador cerrado, aumentar gradualmentela potencia de RF a 70 W. </li><li> Abra el obturador y pulverización catódica durante 15 min para obtener una película con un espesor de 120-150 nm de (Figura 1: Paso A). Tasa de crecimiento de la película tiene que ser derivado para cada máquina. </li></ol> 2. Recubrimiento de superficie con Film celular repelente <ol><li> Hidrofilizar la superficie de TiO 2 por tratamiento de la muestra con plasma de O2, siguiendo las instrucciones proporcionadas por el fabricante del reactor de plasma. Tratamos a la muestra durante 5 min a 200 W con O 2 flujo de 100 sccm. Sumergir la muestra en ddH2O y confirme que la superficie es superhidrófila. Seque la superficie a fondo en corriente de N2. </li><li> Preparar octadecyltrichlorosilane 1 mM solución (OTS) mediante la adición de 39,6 OTS mu l a 100 ml de tolueno. Sumergir la muestra en la solución durante 1 hora a RT. Llevar a cabo este paso dentro de una bolsa de N 2 guante -filled (Figura 1: Paso B). </li><li> Para eliminar las moléculas adsorbidas, sonicate la muestra en tolueno, acetona, etanol, y ddH 2 O durante 5 min cada uno, en ese orden. Enjuagar la muestra en cuatro ocasiones en fresco ddH2O y seque la superficie bajo flujo de N2. La superficie debe ser hidrófobo con un ángulo de contacto de 100-110ºC. </li></ol> 3. Ex-Situ Patrones de superficie <ol><li> Trabajando en una campana de flujo laminar, dibujar varias marcas de arañazos con una punta de trazar diamante en la superficie. Las marcas ayudan a mantener un registro de las regiones procesados ​​y también con lo que los microscopios en el foco. Esterilizar el TiO recubierto-OTS 2 mediante la inmersión de la muestra en etanol 70% durante 5 min. Luego enjuague la muestra dos veces en esterilizada ddH2O </li><li> Colocar la muestra en una placa de 35 mm, y añadir 2 ml de medio de crecimiento PC12 (ver Paso 4.2). Incubar durante más de 3 horas en una incubadora de CO 2 (37 ° C). Este procedimiento está destinado a dejar albúminas de suero absorben sobre la superficie. Albúminas adsorbidos inhiben la posterior adsorción de otra proteins y células (Figura 1: la etapa c). </li><li> Mientras espera, configurar el microscopio de fluorescencia invertida. <ol><li> Encienda la lámpara de arco, inserte el cubo de filtro UV, y establecer la lente objetivo de 20X. </li><li> Medir la intensidad de la luz I (W cm -2) utilizando un medidor de UV de intensidad, y calcular el tiempo de irradiación t (en segundos) para una dosis de d (en J cm -2) como: t = d / I. Por ejemplo, para irradiar a una dosis de 200 J cm -2 usando una fuente de luz de 600 mW cm -2, se requiere 333 seg de la irradiación. </li><li> Use un micrómetro y cerrar el diafragma de campo para establecer el tamaño de la región a ser irradiada, por ejemplo, 200 micras. </li></ol></li><li> Después de la incubación de 3 horas, suplementar el medio con 200 l de 3,0 mg ml -1 de colágeno tipo IV (Col-IV; finales de concentración de 300 mg ml -1). </li><li> Transferir el plato de 35 mm a la platina del microscopio. Encuentra el arañazo marca, foco del microscopio sobre la superficie de la muestra, y de irradiar luz UV a una dosis de 200 J cm -2 (Figura 1: Paso D). El área de la irradiación UV puede ser alterado ya sea ajustando la abertura del diafragma de campo o mediante la sustitución del diafragma de campo con una máscara de metal de la geometría arbitral. </li><li> Sustituya el medio con un medio de crecimiento fresco (sin Col-IV) y luego colocar la muestra de nuevo en la incubadora. </li></ol> 4. Cultivo Celular <ol><li> Cultivo de rutina de las células PC12 se lleva a cabo en un plato de plástico recubierta con colágeno. <ol><li> Para cubrir una de 60 mm placa de cultivo de tejidos con Col-IV, mojar primero la superficie con ddH2O y aspirar todo ddH 2 O. </li><li> Preparar 300 mg ml -1 Col-IV diluyendo la solución original (3 mg ml -1) 10x con ddH 2 O. </li><li> Añadir 200 l de 300 mg ml -1 Col-IV por 60-mm plato. Deje que la solución se extendió a través de la superficie. </li><li> T secoque plato en una campana de flujo laminar durante aproximadamente 1 hora. </li><li> Enjuague la superficie dos veces con 4 ml de solución salina tamponada con fosfato de Dulbecco (D-PBS). Cuando no utilice el plato recubierto de inmediato, enjuague el plato una vez con 4 ml de ddH 2 O. Después de aspirar ddH2O, almacenar el plato en la incubadora. Trate de no mantenerla almacenada durante más de dos semanas. </li></ol></li><li> Las células PC12 se cultivan en un medio de crecimiento que consiste en: Medio (bajo nivel de glucosa) de Eagle modificado por Dulbecco suero solución de penicilina-estreptomicina + 10% de suero bovino fetal al 5% + caballo + 1%. Se incuban las células en una incubadora de CO 2 (37 ° C, 5% de CO 2), y reemplazar la mitad del medio cada dos días. Células Passage antes de llegar a la confluencia. <ol><li> Aspirar el medio de crecimiento, y añadir 2 ml de PBS + (D-PBS + 10 mg ml -1 albúmina de suero bovino (BSA) + EDTA 10 mM) precalentado a 37 ° C. Incubar durante 5 minutos a 37 ° C. Golpee suavemente el plato de separar todo ceLLS. </li><li> Recoger las células en un tubo cónico de 15 ml. Enjuague el plato con 3 ml de fresco D-PBS, precalentada a 37 ° C. </li><li> Centrifugar el tubo a 150 × g durante 4 min. </li><li> Aspirar el sobrenadante, y se añade 1 ml de medio de crecimiento. </li><li> Para el cultivo de rutina, dividir las células 1: 3 a 1: 5. </li></ol></li><li> Para placa células en la OTS / TiO2 muestra modificada UV, cuente densidad celular y añadir 3,0 × 10 5 células en una placa de 35 mm (Figura 1: Paso E). Incubar el plato en el incubador humidificado (37 ° C, 5% de CO 2) durante 1-2 días. Tanto el factor de crecimiento ingenua y el nervio (NGF), las células PC12 -differentiated se pueden utilizar para los experimentos de modelado. Para NGF diferenciación, 100 ng ml -1 de 7S-NGF se añade a la de crecimiento medio varios días antes en placas las células en la muestra. NGF-diferenciación parece aumentar la adhesividad de las células PC12 y hace que el manejo más fácil, especialmente en el in-situ e patronesxperimentos. </li></ol> 5. In-Situ Patrones de superficie <ol><li> Después de 1-2 días de cultivo en la superficie ex-situ modificado, confirman que las células se adhieran y crezcan sólo en la región UV-irradiado. Configure el microscopio, como se describe en el Paso 3.4. </li><li> Transferir la muestra a un nuevo 35-mm placa de cultivo tisular que contiene el medio de crecimiento, 100 ng ml -1 NGF (en el caso de la utilización de células diferenciadas-NGF), y 100 mg ml -1 Col-IV. </li><li> Coloque el plato en la platina del microscopio. Encontrar la posición adecuada, e irradiar la luz UV a una dosis de 200 J cm -2 (Figura 1: Paso F, G). La región permisiva recién creado se indica con un asterisco en la Figura 1: Paso G. </li><li> Trata de completar el procesamiento de una sola muestra dentro de los 30 min. Después de la irradiación, la transferencia de la muestra de nuevo en el medio sin Col-IV. </li><li> Tenga mucho cuidado al realizar el plato con cucélulas ltured y la transferencia de la muestra de plato a plato para evitar desprendimiento de las células estampadas. </li></ol><img alt="Figura 1" src="/files/ftp_upload/53045/53045fig1highres.jpg" width="700" /> Figura 1. Esquema del proceso general. Véase el texto para más detalles. <a href="https://www.jove.com/files/ftp_upload/53045/53045fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

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The authors have nothing to disclose.

En nuestro protocolo actual, TiO2 película fue formada por pulverización catódica RF-magnetrón. Estamos a favor de este método de deposición, ya que nos permite preparar de manera reproducible una fotocatalítica de TiO2 película con una rugosidad sub-nm. Aunque los procesos de deposición por pulverización catódica son familiares para los científicos de materiales e ingenieros electrónicos, puede que no sea bastante accesible para los biólogos. En ese caso, TiO 2 película s...

Procesos de litografía de semiconductores y sus derivados - tales como 1,2 fotolitografía, la litografía por haz de electrones 3-6, y microcontacto impresión 7.10 - se han convertido en una herramienta establecida en la biología celular a crecer las células vivas en una posición y geometría definida. El método del patrón se basa en el uso de sustratos microfabricados, que consta de micro-isla de recubrimiento permisiva celular en un fondo no permisiva. Tal sustrato sirve como una plantilla para patrón de las células. Estas tecnologías nos han proporcionado los nuevos métodos para diseñar las células y su función en un nivel de una o varias celular, para extraer las propiedades intrínsecas de las células, y para aumentar el rendimiento de cribado de fármacos basado en células 11. El grado de libertad en la morfogénesis celular aumentaría enormemente si la geometría patrón de plantilla podría ser alterado in situ, es decir, mientras que las células se cultivan sobre los sTu cara. Los métodos convencionales para la formación de patrones no pueden aplicarse directamente aquí, ya que procesan muestras en la atmósfera o en el vacío. Por lo tanto se han propuesto diversas técnicas nuevas modificación de la superficie, que se basan, por ejemplo, en compuestos fotorreactivos 12,13 5,14 o ablación con láser, sólo para nombrar unos pocos. Los métodos propuestos han sido muy bien revisado por Robertus et al. 15, y más recientemente por Choi et al. 16 y 17 por Nakanishi. Aquí en este artículo, se describe un nuevo protocolo de modificación in situ de la superficie, que se aprovecha de la descomposición fotocatalítica de moléculas orgánicas en un dióxido de titanio (TiO2) Superficie 18,19. En este método, se inserta una película de TiO 2 entre el sustrato de vidrio y la película orgánica que interconecta las células, y la película orgánica se descompone in situ por localmente irradiar luz ultravioleta (UV)luz para una región de interés (λ &lt;388 nm). Se demuestra que el nuevo protocolo se puede utilizar para crear micropatrones de proteínas de matriz extracelular y células vivas tanto ex situ como in situ. TiO2 es biocompatible, químicamente estable y ópticamente transparente, características de lo que lo hace amigable para introducir en los experimentos de cultivo celular. Este protocolo proporciona una alternativa de ciencia de los materiales de base para la modificación de los andamios de cultivo celular en medio de cultivo celular.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass coverslip</td>
			<td>Warner Instruments</td>
			<td>CS-15R15</td>
			<td>15 mm, #1.5 thickness</td>
		</tr>
		<tr>
			<td>Diamond scriber</td>
			<td>Ogura Jewel Industry</td>
			<td>D-Point Pen</td>
			<td></td>
		</tr>
		<tr>
			<td>RF sputtering system</td>
			<td>ANELVA</td>
			<td>SPC350</td>
			<td></td>
		</tr>
		<tr>
			<td>TiO2 sputtering target</td>
			<td>Kojundo Chemical Lab</td>
			<td>Titanium (IV) oxide, target</td>
			<td>Purity, 99.9%</td>
		</tr>
		<tr>
			<td>Plasma reactor</td>
			<td>Yamato</td>
			<td>PR301</td>
			<td></td>
		</tr>
		<tr>
			<td>n-octadecyltrichlorosilane 
			(OTS)</td>
			<td>Aldrich</td>
			<td>104817</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Wako</td>
			<td>204-01866</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture dish (35 mm)</td>
			<td>Greiner</td>
			<td>627160</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture dish (60 mm)</td>
			<td>BD Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>Type-IV collagen</td>
			<td>Nitta Gelatin</td>
			<td>Cellmatrix Type IV</td>
			<td></td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>12483-020</td>
			<td>Heat-inactivate and pass through a 0.22 mm filter before use</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Gibco</td>
			<td>26050-088</td>
			<td>Pass through a 0.22 mm filter before use</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (100x)</td>
			<td>Nacalai tesque</td>
			<td>26253-84</td>
			<td></td>
		</tr>
		<tr>
			<td>7S nerve growth factor (NGF)</td>
			<td>Alomone Labs</td>
			<td>N-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Dojindo</td>
			<td>N001</td>
			<td>Stock solution in 0.5 M</td>
		</tr>
		<tr>
			<td>TiO2 nanoparticle</td>
			<td>Tayca</td>
			<td>TKD-701</td>
			<td></td>
		</tr>
	</tbody>
</table>

photopatterning proteins and cells in aqueous environment using tio2 photocatalysis

Los contaminantes orgánicos adsorbidos en la superficie de dióxido de titanio (TiO 2) se pueden descomponer mediante fotocatálisis bajo radiación ultravioleta (UV). Aquí se describe un nuevo protocolo que emplea la fotocatálisis TiO2 para alterar localmente afinidad de células de la superficie del sustrato. Para este experimento, una delgada película de TiO 2 fue revestido por bombardeo iónico sobre un cubreobjetos de vidrio, y la superficie de TiO 2 fue posteriormente modificado con una monocapa de organosilano derivado de octadecyltrichlorosilane (OTS), que inhibe la adhesión celular. La muestra se sumerge en un medio de cultivo celular, y se centró la luz UV se irradió a una región octogonal. Cuando un neuronales células PC12 línea celular se sembraron en la muestra, células adheridas sólo en la zona UV-irradiado. Además, muestran que esta modificación de la superficie también se puede realizar in situ, es decir, incluso cuando las células están creciendo en el sustrato. Modificación adecuada de la superficie requiere una matriz extracelular protein colágeno para estar presente en el medio en el momento de la irradiación UV. La técnica que aquí se presenta potencialmente se puede emplear en múltiples tipos de células de modelado para la construcción de sistemas de cocultivo o manipular arbitrariamente células en cultivo.

La Figura 2A muestra una imagen de microscopía electrónica de barrido de la sección transversal (SEM) de la película de TiO 2 bombardeo iónico depositado. De la observación, el espesor de la película se estimó en aproximadamente 150 nm. Notable aquí es la planitud del depositada TiO2 película. Un análisis posterior mediante microscopía de fuerza atómica (AFM) reveló que la rugosidad de la raíz cuadrada media (RMS) de la superficie era de 0,2 nm (Figura 2B).</...

Watch this Scientific Journal Video about Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis at JoVE.com

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

Photopatterning proteínas y células en el medio ambiente acuoso Usando TiO<sub&gt; 2</sub&gt; Fotocatálisis

Organic contaminants adsorbed on the surface of titanium dioxide (TiO2) can be decomposed by photocatalysis under ultraviolet (UV) light. Here we describe ...

photopatterning-proteins-cells-aqueous-environment-using-tio2

Research

JoVE Journal

Bioengineering

Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

7.8K vistas.  Tohoku University. We describe a protocol for modifying cell affinity of a scaffold surface in aqueous environment. The method takes advantage of titanium dioxide photocatalysis to decompose organic film in the photo-irradiated region. We show that it can be used to create microdomains of scaffolding proteins, both ex situ and in situ.

Video: Photopatterning Proteins and Cells in Aqueous Environment Using TiO2 Photocatalysis

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

Fabricación de ADN electroquímico biosensores para la detección Reagentless de ácidos nucleicos, proteínas y moléculas pequeñas

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Investigación

Bioingeniería

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

Visualización de las proteínas y complejos macromoleculares de Negative EM Mancha: desde la preparación de la cuadrícula para adquisición de imágenes

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Células vivas imágenes de la migración de las células que expresan proteínas de fluorescencia etiquetado en una matriz tridimensional

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

Un juego de herramientas para la conversión de hidrocarburos en ambientes acuosos

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days.
We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging1 (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers2. This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution.
Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors3.

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

Time-lapse de imágenes de fluorescencia del crecimiento de raíces de Arabidopsis con la manipulación del ambiente rápido y raíz mediante la RootChip

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Co-cultivo celular Patterning Uso acuoso de dos fases Sistemas

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Trasplante De InducidaS Pluripotentes Derivadas De Células Madre Mesoangioblastos Como Progenitores Miógenos En Modelos De Ratón De La Regeneración Muscular

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

La luminiscencia de energía de resonancia de transferencia para estudiar cambios conformacionales en proteínas de membrana expresado en células de mamífero

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

Gotas acuosas utilizan como enzimática microreactores y su accionamiento electromagnético

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Un método versátil de Patterning proteínas y células

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...