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. Herstellung von TiO 2 beschichtetes Deckglas <ol><li> Anzahl die Deckgläser mit einem Diamantreißnadel. Dies trägt nicht nur zur Spur jedes Deckgläschen zu halten, sondern auch um sicherzustellen, dass die richtige Seite der Probe nach oben weist. Reinigen der Deck zunächst unter fließendem ddH 2 O, dann mit dem in Piranha-Lösung eingetaucht werden (H 2 SO 4: H 2 O 2 = 4: 1). Nach 10 Minuten, spülen Sie die Deckgläser gründlich, 8-mal in ddH 2 O. Trocknen Sie die Deckgläser unter N 2 fließen. </li><li> Eingestellt TiO 2 -Target in der Radiofrequenz (RF) Sputtersystem. Bringen Sie die Deckgläser auf einem Probenhalter der Sputteranlage mit einem Polyimid-Band. Setzen Sie den Probenhalter in der Sputterkammer. Kammer evakuieren bis der Druck 2,0 × 10 -4 Pa. </li><li> Einzuführen Ar-Gas in die Kammer und den Abscheidungsdruck auf 4,0 mTorr. Während der Verschluss geschlossen ist, schrittweise zu erhöhendie HF-Leistung bis 70 W. </li><li> Öffnen des Verschlusses und Sputter für 15 min, um eine Folie mit einer Dicke von 120-150 nm (Abbildung 1: Schritt A) zu erhalten. Wachstumsrate des Films muss für jede einzelne Maschine abgeleitet werden. </li></ol> 2. Oberflächenbeschichtung mit Handy abweisenden Film <ol><li> Hydrophilisierung der TiO 2 -Oberfläche durch Behandeln der Probe mit O 2 -Plasma, nach Anweisungen des Herstellers des Plasmareaktors bereitgestellt. Wir behandeln die Probe für 5 Minuten bei 200 W mit O 2 Fluß von 100 sccm. Tauchen Sie die Probe in ddH 2 O und bestätigen, dass die Oberfläche superhydrophil ist. Trocknen Sie die Oberfläche gründlich unter N 2 -Strom. </li><li> Vorbereitung 1 mM Octadecyltrichlorsilan (OTS) Lösung durch Zugabe von 39,6 ul OTS zu 100 ml Toluol gegeben. Eintauchen der Probe in die Lösung für 1 h bei RT. Führen Sie diesen Schritt innerhalb einer N 2 -gefüllten Handschuhbeutel (Abbildung 1: Schritt B). </li><li> Um physisorbierte Moleküle zu entfernen, sonicate die Probe in Toluol, Aceton, Ethanol und ddH 2 O für 5 Minuten jeden, in dieser Reihenfolge. Spülen Sie die Proben vier Mal in frischem ddH 2 O und trocknen Sie die Oberfläche unter einem N 2 -Strom. Sollte die Oberfläche hydrophob mit einem Kontaktwinkel von 100-110 ° betragen. </li></ol> 3. Ex-situ-Oberflächenstrukturierung <ol><li> Arbeiten in einer Sterilbank, zu ziehen mehrere Kratzspuren mit einer Diamant-Reißnadel auf der Oberfläche. Die Markierungen helfen bei der Verfolgung der bearbeiteten Regionen und bringt auch Mikroskope in den Fokus. Sterilisieren der OTS-beschichteten TiO 2 durch Eintauchen der Probe in 70% Ethanol für 5 min. Dann zweimal spülen Sie die Probe in sterilisierte ddH 2 O </li><li> Legen Sie die Probe in einem 35-mm-Schale, und 2 ml PC12 Wachstumsmedium (siehe Schritt 4.2). Inkubation für mehr als 3 Stunden in einem CO 2 -Inkubator (37 ° C). Diese Vorgehensweise soll Serumalbumine absorbiert auf der Oberfläche zu lassen. Adsorbiert Albumine hemmen anschließende Adsorption von anderen proteIns und Zellen (1: Schritt C). </li><li> Während der Wartezeit, die Einrichtung des invertierten Fluoreszenzmikroskop. <ol><li> Schalten Sie die Bogenlampe, die UV-Filterwürfel einzufügen, und stellen Sie die Objektivlinse zu 20X. </li><li> Messen der Lichtintensität I (W cm -2) unter Verwendung eines UV-Intensitätsmesser, und berechnen Bestrahlungszeit t (in Sekunden) für eine Dosis von d (in J cm -2) als: t = d / I. Um zum Beispiel bei einer Dosis von 200 J cm -2 bestrahlt unter Verwendung einer Lichtquelle von 600 mW cm -2, beträgt 333 sec Bestrahlung erforderlich. </li><li> Verwenden Sie eine Objektmikrometer und schließen Sie die Feldblende zum Einstellen der Größe der Region zu bestrahlenden, beispielsweise 200 &amp; mgr; m. </li></ol></li><li> Nach 3 Stunden Inkubation ergänzen das Medium mit 200 ul 3,0 mg ml-1-Typ-IV-Kollagen (Col-IV; Endkonzentration von 300 &amp; mgr; g ml -1). </li><li> Übertragen Sie die 35-mm-Schale auf den Mikroskoptisch. Finden Sie den Kratzer mLade Fokus des Mikroskops auf die Probenoberfläche und Bestrahlen mit UV-Licht bei einer Dosis von 200 J cm -2 (Abbildung 1: Schritt D). Der Bereich der UV-Bestrahlung kann entweder durch Einstellen der Öffnung der Leuchtfeldblende oder durch Ersetzen der Feldblende mit einer Metallmaske Schiedsgeometrie verändert werden. </li><li> Ersetzen Sie das Medium mit frischem Wachstumsmedium (ohne Col-IV), und legen Sie die Probe wieder in den Inkubator. </li></ol> 4. Cell Culture <ol><li> Routinekultur von PC12-Zellen wird auf einem kollagenbeschichteten Plastikschale durchgeführt. <ol><li> Zur Beschichtung eines 60-mm-Gewebekulturschale mit Col-IV, zuerst die Oberfläche nass mit ddH 2 O und saugen alle ddH 2 O. </li><li> Bereiten 300 &amp; mgr; g ml -1 Col-IV durch Verdünnen der ursprünglichen Lösung (3 mg ml -1) 10x mit ddH 2 O. </li><li> Fügen Sie 200 ul von 300 &amp; mgr; g ml -1 Col-IV pro 60-mm-Schale. Lassen Sie die durch die Oberfläche verteilt Lösung. </li><li> Dry-Ter in einer Laminarströmungshaube Teller für ca. 1 Std. </li><li> Die Oberfläche zweimal gründlich mit 4 ml Dulbecco-phosphatgepufferter Kochsalzlösung (D-PBS). Bei Nichtgebrauch des beschichtete Schale sofort, spülen Sie die Teller einmal mit 4 ml ddH 2 O. Nach Absaugen ddH 2 O, speichern Sie die Schüssel in den Inkubator. Versuchen Sie nicht, halten Sie es für mehr als zwei Wochen gelagert. </li></ol></li><li> PC12-Zellen werden in einem Wachstumsmedium, das besteht aus gewachsen: Dulbeccos modifiziertes Eagle-Medium (low glucose) + 10% fötales Rinderserum + 5% Pferdeserum + 1% Penicillin-Streptomycin-Lösung. Inkubieren Sie die Zellen in einem CO 2 Inkubator (37 ° C, 5% CO 2), und ersetzen Sie jeden zweiten Tag die Hälfte des Mediums. Passage-Zellen, bevor sie zu erreichen Fluss. <ol><li> Saugen Sie das Wachstumsmedium, und fügen Sie 2 ml PBS + (D-PBS + 10 mg ml -1 Rinderserumalbumin (BSA) + 10 mM EDTA) vorgewärmt auf 37 ° C. Inkubation für 5 min bei 37 ° C. Klopfen Sie leicht das Gericht auf alle Produkte zu lösenlls. </li><li> Sammeln Zellen in einem 15 ml konischen Röhrchen. Spülen Sie die Schale mit 3 ml frischem D-PBS, auf 37 ° C vorgewärmt. </li><li> Zentrifugieren Sie die Röhrchen bei 150 · g für 4 min. </li><li> Den Überstand aspirieren, und fügen Sie 1 ml des Wachstumsmediums. </li><li> Für Routinekultur, teilen Sie die Zellen 1: 3 bis 1: 5. </li></ol></li><li> Um Zellen Platte auf dem UV-modifizierte OTS / TiO 2 Probe zu zählen Zelldichte und fügen Sie 3,0 x 10 5 Zellen in einer 35-mm-Schale (Abbildung 1: Schritt E). Inkubiere die Schale in den befeuchteten Inkubator (37 ° C, 5% CO 2) für 1-2 Tage. Naiven und der Nervenwachstumsfaktor (NGF) -differentiated PC12-Zellen können für die Strukturierung Experimente eingesetzt werden. NGF Differenzierung werden 100 ng ml -1 von 7S-NGF in das Wachstumsmedium mehrere Tage vor Ausplattieren der Zellen auf die Probe gegeben. NGF-Differenzierung scheint die Haftfähigkeit der PC12-Zellen zu erhöhen und erleichtert die Handhabung, insbesondere bei der in-situ Strukturierungs eXperimente. </li></ol> 5. In-Situ Oberflächenstrukturierung <ol><li> Nach 1-2 Tagen Kultur auf die ex-situ modifizierten Oberfläche, zu bestätigen, dass die Zellen für die Befestigung und wächst nur auf dem UV-Bestrahlungsbereich. Richten Sie das Mikroskop, wie in Schritt 3.4 beschrieben. </li><li> Übertragung der Probe auf eine neue 35-mm-Gewebekulturschale, die das Wachstumsmedium, 100 ng ml -1 NGF (in dem Fall der Verwendung NGF-differenzierten Zellen) und 100 &amp; mgr; g ml -1 Col-IV. </li><li> Die Schale auf dem Mikroskoptisch. Finden Sie die passende Position, und strahlen UV-Licht bei einer Dosis von 200 J cm -2 (Abbildung 1: Schritt F, G). Die neu erstellte permissive Bereich ist mit einem Sternchen in Figur 1 angegeben: Schritt G. </li><li> Versuchen, die Verarbeitung einer einzelnen Probe innerhalb von 30 Minuten zu vervollständigen. Nach der Bestrahlung, übertragen Sie die Probe wieder in das Medium ohne Col-IV. </li><li> Seien Sie besonders vorsichtig bei der Durchführung die Schale mit cultured Zellen und Überführung der Probe vom Gericht, austeilen Ablösen der strukturierten Zellen zu verhindern. </li></ol><img alt="Abbildung 1" src="/files/ftp_upload/53045/53045fig1highres.jpg" width="700" /> Abbildung 1. Schematische Darstellung des gesamten Prozesses. Siehe Text für Details. <a href="https://www.jove.com/files/ftp_upload/53045/53045fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a>

<ol>
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The authors have nothing to disclose.

In unserem aktuellen Protokoll wurde TiO 2 Film von RF-Magnetron-Sputtern gebildet. Wir bevorzugen diese Methode der Abscheidung, da es uns erlaubt, reproduzierbare Herstellung einer photokatalytischen TiO 2 -Schicht mit einer sub-nm Rauheit. Obwohl Sputter-Abscheidungsverfahren vertraut Materialwissenschaftler und Elektronik-Ingenieure sind, kann es nicht ganz zugänglich Biologen sein. In diesem Fall würde spinnbeschichteten TiO 2 -Films 23 eine Alternative sein. Bei diesem...

Halbleiter-Lithographieverfahren und seine Derivate - wie Photolithographie 1,2, Elektronenstrahllithographie 3-6 und 7-10 Mikrokontaktdruck - sind mittlerweile ein etabliertes Werkzeug in der Zellbiologie, lebende Zellen in einer definierten Position und Geometrie wachsen. Die Musterungsverfahren beruht auf der Verwendung von mikro Substrate, bestehend aus Mikro-Insel Zelle permissive Beschichtung in einem nicht-permissiven Hintergrund. Ein solches Substrat dient als Vorlage, um Muster der Zellen. Diese Technologien haben uns zur Verfügung gestellt, die die neuen Methoden, um Zellen und ihre Funktion Ingenieur bei einem Ein- und Mehrzellebene, um die inhärenten Eigenschaften von Zellen zu extrahieren, und um den Durchsatz von zellbasierten Wirkstoff-Screening-11 zu erhöhen. Der Grad Freiheits in Zellmusterungs würde erheblich zunehmen, wenn die Schablonenmustergeometrie in situ verändert werden, das heißt, während die Zellen auf den n kultivierturface. Die herkömmlichen Verfahren zur Musterbildung kann hier nicht direkt angewendet werden, da sie Verfahrensproben in der Atmosphäre oder im Vakuum. Deshalb werden verschiedene neue Oberflächenmodifikationstechniken sind vorgeschlagen worden, die basieren, zB auf photoreaktiven Verbindungen 12,13 oder Laserablation 5,14, um nur einige zu nennen. Die vorgeschlagenen Verfahren sind gut durch Robertus et al von Choi et al. 16 und Nakanishi 17 überprüft. 15, und in jüngerer Zeit. Hier in diesem Artikel beschreiben wir eine neue Methode der in-situ-Oberflächenmodifizierung, die Vorteile der photokatalytischen Zersetzung von organischen Molekülen auf einem Titandioxid nimmt (TiO 2) Fläche 18,19. In diesem Verfahren wird ein TiO 2 -Film zwischen dem Glassubstrat und dem organischen Film, der die Zellen eine Schnittstelle eingeführt und der organische Film in situ durch lokales Bestrahlen mit ultravioletten (UV) zerlegtLicht in einem interessierenden Bereich (λ &lt;388 nm). Wir zeigen, dass das neue Protokoll kann verwendet werden, um Mikrostrukturen der extrazellulären Matrixproteinen und lebenden Zellen sowohl ex situ und in situ zu erzeugen. TiO 2 ist biokompatibel, chemisch stabil und optisch transparente, Features, von denen macht es freundlich in Zellkulturexperimenten einzuführen. Dieses Protokoll stellt eine Materialwissenschaften basierte Alternative zur Modifizierung von Zellkultur-Gerüsten in Zellkulturumgebung.

<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

Organische Verunreinigungen auf der Oberfläche eines Titandioxid (TiO 2), adsorbiert durch Photokatalyse unter ultraviolettem Licht (UV) zerlegt werden. Hier beschreiben wir eine neuartige Protokoll unter Verwendung des TiO 2 Photokatalyse zur Zellaffinität der Substratoberfläche lokal zu verändern. Für dieses Experiment wurde eine dünne TiO 2 -Schicht wurde durch Sputtern beschichtet auf einem Deckglas, und die TiO 2 -Oberfläche wurde anschließend mit einem Organosilan Monoschicht aus Octadecyltrichlorsilan (OTS) ableitet, die die Zelladhäsion hemmt modifiziert. Die Probe wurde in einem Zellkulturmedium eingetaucht ist, und konzentrierte UV-Licht wurde auf einem achteckigen Bereich bestrahlt. Wenn eine neuronale Zelllinie PC12-Zellen wurden auf der Probe überzogen, Zellen, die nur auf die UV-bestrahlte Fläche haftet. Wir zeigen weiter, daß diese Oberflächenmodifikation kann auch in situ erfolgen, das heißt, auch wenn die Zellen auf dem Substrat wächst. Korrekte Modifikation der Oberfläche benötigt eine extrazelluläre Matrix protein Kollagen in dem Medium zum Zeitpunkt der UV-Bestrahlung vorliegen. Die hier vorgestellte Methode kann potenziell in Strukturierung mehrere Zelltypen für den Bau von Co-Kultur-Systeme oder willkürlich zu manipulieren Zellen unter Kultur eingesetzt werden.

2A zeigt eine Querschnittsrasterelektronenmikroskopie (SEM) -Bild der durch Sputtern abgelagerten TiO 2 -Films. Aus der Beobachtung wurde Dicke des Films auf ungefähr 150 nm zu sein. Hier fällt auf, die Flachheit des abgeschiedenen TiO 2 -Films. Weiteren Analyse durch Rasterkraftmikroskopie (AFM) ergab, dass die Wurzel aus dem mittleren quadratischen (RMS) Rauheit der Oberfläche betrug 0,2 nm (2B). Wenn der TiO 2 -Oberflä...

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.

Photostrukturierungs Proteinen und Zellen in wässriger Umgebung Mit TiO<sub&gt; 2</sub&gt; Photokatalyse

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 Aufrufe.  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.

Serie: 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.

Herstellung von elektrochemischen DNA Biosensoren für die Reagenzienfreier Nachweis von Nukleinsäuren, Proteinen und kleinen Molekülen

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. ...

Forschung

Biotechnik

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.

Visualizing Proteine ​​und Makromolekulare Komplexe von Negative Stain EM: von Grid Vorbereitung auf Image Acquisition

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

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.

Ein Toolkit zur Umwandlung von Kohlenwasserstoffen in wässrigen Umgebungen aktivieren

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.

Zeitraffer-Fluoreszenz-Imaging von Arabidopsis Wurzelwachstum mit Rapid Manipulation der Root-Umgebung mit dem 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.

Zelle Co-Kultur Strukturierung mit wässrigen Zwei-Phasen-Systeme

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.

Transplantation von induzierten pluripotenten Stammzellen-abgeleiteten mesoangioblast-ähnlichen myogenen Vorläufern in Mausmodellen der Muskelregeneration

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.

Lumineszenz-Resonanzenergietransfer zu Konformationsänderungen in Membranproteinen in Säugerzellen exprimiert Studieren

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.

Wässrige Tröpfchen als enzymatische Mikroreaktoren und deren elektromagnetischen Betätigung

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.

Eine vielseitige Methode von Patterning Proteinen und Zellen

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

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

Materialbildung von Rekombinanten Spider-Seiden durch wässrige Lösung mit Wärme und Druck

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...