Name: a rapid image-based bacterial virulence assay using amoeba
Uploaded: 2018-06-27T15:00:00
Description: Watch this Scientific Journal Video about A Rapid Image-based Bacterial Virulence Assay Using Amoeba at JoVE.com

KP och som skrev och reviderade manuskriptet. KP utfört experimenten och analys. Detta arbete stöds av National Institutes of Health (NIH) karriär övergång tilldelning (K22AI112816) till AS.

Alla experimentella rutiner som genomfördes på University of California, Irvine.
1. buffertar och lösningar
<ol><li>Förbered Lysogeny buljong (LB) i en 1 L glasflaska genom att lägga till 25 g av LB-Miller mix i 1 L dubbeldestillerat vatten (ddH2O). För petriskålar, lägga till en ytterligare 20 g agar. Autoklav att sterilisera. Häll 25 mL av smält agarsubstrat i 10 cm-diameter petriskålar och låt stelna i rumstemperatur. Lagra flytande media vid rumstemperatur och agar...

<ol>
	<li>Rasko, D. A., Sperandio, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-virulence+strategies+to+combat+bacteria-mediated+disease.">Anti-virulence strategies to combat bacteria-mediated disease.</a> Nature Reviews Drug Discovery. 9 (2), 117-128 (2010).</li><li>Coburn, B., Grassl, G. A., Finlay, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella,+the+host+and+disease:+a+brief+review.">Salmonella, the host and disease: a brief review.</a> Immunology and Cell Biology. 85 (2), 112-118 (2007).</li><li>Alibaud, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+virulence+genes+identified+in+a+Dictyostelium+host+model.">Pseudomonas aeruginosa virulence genes identified in a Dictyostelium host model.</a> Cellular Microbiology. 10 (3), 729-740 (2008).</li><li>Pukatzki, S., Kessin, R. H., Mekalanos, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+pathogen+Pseudomonas+aeruginosa+utilizes+conserved+virulence+pathways+to+infect+the+social+amoeba+Dictyostelium+discoideum.">The human pathogen Pseudomonas aeruginosa utilizes conserved virulence pathways to infect the social amoeba Dictyostelium discoideum.</a> Proceedings of the National Academy of Sciences U S A. 99 (5), 3159-3164 (2002).</li><li>Cosson, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+Virulence+Analyzed+in+a+Dictyostelium+discoideum+Host+System.">Pseudomonas aeruginosa Virulence Analyzed in a Dictyostelium discoideum Host System.</a> Journal of Bacteriology. 184 (11), 3027-3033 (2002).</li><li>Pukatzki, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+conserved+bacterial+protein+secretion+system+in+Vibrio+cholerae+using+the+Dictyostelium+host+model+system.">Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system.</a> Proceedings of the National Academy of Sciences U S A. 103 (5), 1528-1533 (2006).</li><li>Steinert, M., Heuner, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dictyostelium+as+host+model+for+pathogenesis.">Dictyostelium as host model for pathogenesis.</a> Cellular Microbiology. 7 (3), 307-314 (2005).</li><li>Hagele, S., Kohler, R., Merkert, H., Schleicher, M., Hacker, J., Steinert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dictyostelium+discoideum:+a+new+host+model+system+for+intracellular+pathogens+of+the+genus+Legionella.">Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella.</a> Cellular Microbiology. 2 (2), 165-171 (2000).</li><li>Matz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+uses+type+III+secretion+system+to+kill+biofilm-associated+amoebae.">Pseudomonas aeruginosa uses type III secretion system to kill biofilm-associated amoebae.</a> ISME Journal. 2 (8), 843-852 (2008).</li><li>Hauser, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+type+III+secretion+system+of+Pseudomonas+aeruginosa:+infection+by+injection.">The type III secretion system of Pseudomonas aeruginosa: infection by injection.</a> Nature Reviews Microbiology. 7 (9), 654-665 (2009).</li><li>Moore, P. L., MacCoubrey, I. C., Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+pH+insensitive,+two+color+fluorescence+viability+(cytotoxicity)+assay.">A rapid pH insensitive, two color fluorescence viability (cytotoxicity) assay.</a> Journal of Cell Biology. 111, 58 (1990).</li><li>Kaneshiro, E. S., Wyder, M. A., Wu, Y. -. P., Cushion, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+calcein+acetoxy+methyl+ester+and+ethidium+homodimer+or+propidium+iodide+for+viability+assessment+of+microbes.">Reliability of calcein acetoxy methyl ester and ethidium homodimer or propidium iodide for viability assessment of microbes.</a> Journal of Microbiological Methods. 17 (1), 1-16 (1993).</li><li>Siryaporn, A., Kuchma, S. L., O'Toole, G. A., Gitai, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+attachment+induces+Pseudomonas+aeruginosa+virulence.">Surface attachment induces Pseudomonas aeruginosa virulence.</a> Proceedings of the National Academy of Sciences U S A. 111 (47), 16860-16865 (2014).</li><li>Decherchi, P., Cochard, P., Gauthier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+staining+assessment+of+Schwann+cell+viability+within+whole+peripheral+nerves+using+calcein-AM+and+ethidium+homodimer.">Dual staining assessment of Schwann cell viability within whole peripheral nerves using calcein-AM and ethidium homodimer.</a> Journal of Neuroscience Methods. 71 (2), 205-213 (1997).</li><li>Braut-Boucher, F., Pichon, J., Rat, P., Adolphe, M., Aubery, M., Font, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-isotopic,+highly+sensitive,+fluorimetric,+cell-cell+adhesion+microplate+assay+using+calcein+AM-labeled+lymphocytes.">A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes.</a> Journal of Immunological Methods. 178 (1), 41-51 (1995).</li><li>Grieshaber, P., Lagr&#232;ze, W. A., Noack, C., Boehringer, D., Biermann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staining+of+fluorogold-prelabeled+retinal+ganglion+cells+with+calcein-AM:+A+new+method+for+assessing+cell+vitality.">Staining of fluorogold-prelabeled retinal ganglion cells with calcein-AM: A new method for assessing cell vitality.</a> Journal of Neuroscience Methods. 192 (2), 233-239 (2010).</li><li>Witkin, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inherited+Differences+in+Sensitivity+to+Radiation+in+Escherichia+Coli.">Inherited Differences in Sensitivity to Radiation in Escherichia Coli.</a> Proceedings of the National Academy of Sciences U S A. 32 (3), 59-68 (1946).</li><li>Loomis, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+Dictyostelium+discoideum+to+nucleic+acid+analogues.">Sensitivity of Dictyostelium discoideum to nucleic acid analogues.</a> Experimental Cell Research. 64 (2), 484-486 (1971).</li><li>Rahme, L. G., Stevens, E. J., Wolfort, S. F., Shao, J., Tompkins, R. G., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+virulence+factors+for+bacterial+pathogenicity+in+plants+and+animals.">Common virulence factors for bacterial pathogenicity in plants and animals.</a> Science. 268 (5219), 1899-1902 (1995).</li><li>O'Loughlin, C. T., Miller, L. C., Siryaporn, A., Drescher, K., Semmelhack, M. F., Bassler, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quorum-sensing+inhibitor+blocks+Pseudomonas+aeruginosa+virulence+and+biofilm+formation.">A quorum-sensing inhibitor blocks Pseudomonas aeruginosa virulence and biofilm formation.</a> Proceedings of the National Academy of Sciences U S A. 110 (44), 17981-17986 (2013).</li></ol>

Det här protokollet beskriver en snabb och kvantitativ metod för att assay virulens i P. aeruginosa. Detta protokoll kan testas med andra bakterier. Det är dock viktigt att komma ihåg att odlingsmedium bör vara förenliga med amoeba tillväxten villkorar. Vi i synnerhet har optimerat det protokoll använder PS:DB som bakterietillväxt medium. Om andra medier används, kan det nödvändigt att utföra en tillväxt endast media-kontroll där det finns ingen bakterieceller som är närvarande för att kontroll...

Bakteriell infektion är en av de ledande dödsorsakerna i människa och djur1,2. Förmågan att mäta virulens av bakterier i kulturer eller biofilmer är viktigt i sjukvård och forskning inställningar. Här beskriver vi en mångsidig, snabb och relativt enkel metod för att kvantifiera bakteriell virulens. Den eukaryota organismen Dictyostelium discoideum (amöba) används som modell värd organismen. D. discoideum har använts som en värd för att identifiera virulensfaktorer i Pseudomonas aeruginosa (P. aeruginosa)3,4,5 och andra bakterier6,7 ,8 och är mottagliga för i stort sett de samma virulensfaktorer som döda däggdjursceller inklusive typ III sekretion9,10. Tidigare virulens analyser med hjälp av D. discoideum har involverat långvarig exponering av bakterier med D. discoideum celler under loppet av timmar3,4,5. Protokollet, här presenterar en snabb metod för att bestämma virulens använder denna amöba. Detta protokoll (figur 1) beskriver hur du: (1) växer endoparasiterna axenically (i avsaknad av bakterier), (2) växa bakterier för analysen, (3) förbereda bakterier och värdceller för mikroskopi, (4) utföra epifluorescence mikroskopi och (5) analysera amoeba fluorescens.
Endoparasiterna är initialt stryks ut från fryst lager och odlas på en gräsmatta av Escherichia coli (E. coli), där endoparasiterna producera sporer. Dessa sporer plockas och inokuleras i ett berikat medium för axenic tillväxt. Endoparasiterna underhålls genom axenic tillväxt i näringsrika förhållanden tills de är redo att blandas med bakterier för bedömning av bakteriell virulens. Överlevnad eller döden av endoparasiterna kvantifieras genom mätning av fluorescensen av calcein-acetoximetyl (calcein-AM), som klyvs av intracellulära esteraser och, därmed, aktiverad för fluorescens11,12. Levande endoparasiterna uppvisar liten eller ingen fluorescens betonade och döende celler fluorescerar intensivt. Detta resultat beror på en liten eller ingen införlivandet av calcein-AM i friska endoparasiterna och införlivande och klyvning av substratet i stressade endoparasiterna13. Detta är särskilt skild från calcein-AM fluorescens i däggdjursceller11,14,15,16.
Bakterier som kommer att bedömas för virulens odlas separat. Här, beskriver vi hur man mäter virulens opportunistisk patogen P. aeruginosa och detalj hur att kvantifiera virulens plankton (simning) och surface-anslutna delpopulationer. Detta protokoll kan anpassas att testa virulens hos andra bakterier. I avsnittet representativa resultat visar vi att virulens är aktiverad i celler yta-sitter och är låg i plankton celler, som rapporterades tidigare13. Virulens-aktiverade surface-anslutna P. aeruginosa dödar endoparasiterna medan icke-virulent plankton celler förbrukas av endoparasiterna. Om är enbart att analyseras virulens hos plankton bakterier, kan bakterier vara odlade i vanliga kultur rör i stället för att använda petriskålar som beskrivs i protokollet.
Tillväxten av endoparasiterna och P. aeruginosa kulturer måste samordnas så att P. aeruginosa kulturer nå den avsedda tillväxtfasen medan endoparasiterna växer vid steady state i näringsrika förhållanden. Detta tillstånd kräver vanligtvis endoparasiterna kulturer spädas minst 1 dag före när de blandas med bakterier. Endoparasiterna och bakterier är orörlig använder agar kuddar, är samtidig ruvade för 1 h och avbildas med hjälp av en låg upplösning (10 X, numeriska bländaröppningen 0,3) objektiva, grönt fluorescerande protein (GFP) filter och en tänkbar kamera. Analys kan utföras med hjälp av fritt tillgängliga ImageJ programvara eller anpassad bild analys programvara. Vår analys utfördes med hjälp av vår egen programvara som skrivs med hjälp av en vetenskaplig analys paketet13. Programvaran bör skapa en mask som använder fas kontrast bilden och extrahera fluorescens värden från de maskerade områdena i bilden fluorescens. Fluorescens är medelvärdet över minst 100 celler, vilket resulterar i ett index för dödandet av numeriska värden.

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	<tbody>
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			<td height="21" style="height:21px;width:220px;">Reagents</td>
			<td style="width:108px;"></td>
			<td style="width:91px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Bacto agar, dehydrated</td>
			<td style="width:108px;">BD Difco</td>
			<td style="width:91px;">214010</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Antibiotic-Antimycotic (100X)</td>
			<td style="width:108px;">Life Technologies</td>
			<td style="width:91px;">15240062</td>
			<td style="width:297px;">Aliquot &#60; 1 mL and store at -20 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Calcein-acetoxymethyl ester (calcein-AM)</td>
			<td style="width:108px;">Life Technologies</td>
			<td style="width:91px;">C34852</td>
			<td style="width:297px;">Calcein Acetoxymethyl (AM)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Calcium chloride, anhydrous</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">C1016</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">D-Glucose</td>
			<td style="width:108px;">Fisher Chemical</td>
			<td style="width:91px;">D16500</td>
			<td style="width:297px;">Dextrose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Dimethyl sulfoxide</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">D5879</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Folic acid</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">F8758</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">LB-Miller</td>
			<td style="width:108px;">BD Difco</td>
			<td style="width:91px;">244620</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Magnesium chloride</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">M8266</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Yeast extract</td>
			<td style="width:108px;">Oxoid</td>
			<td style="width:91px;">LP0021</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Special peptone</td>
			<td style="width:108px;">Oxoid</td>
			<td style="width:91px;">LP0072</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Potassium hyroxide</td>
			<td style="width:108px;">Fisher Chemical</td>
			<td style="width:91px;">P250</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Potassium phosphate monobasic&#160;</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">P0662</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Sodium phosphate dibasic heptahydrate</td>
			<td style="width:108px;">Fisher Chemical</td>
			<td style="width:91px;">S373</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Vitamin B12</td>
			<td style="width:108px;">Sigma-Aldrich</td>
			<td style="width:91px;">V2876</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Strains</td>
			<td style="width:108px;"></td>
			<td style="width:91px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:220px;">Dictyostelium discoideum</td>
			<td style="width:108px;">Siryaporn lab</td>
			<td style="width:91px;">Strain AX318</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:220px;">Escherichia coli</td>
			<td style="width:108px;">Siryaporn lab</td>
			<td style="width:91px;">Strain B/r17</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:220px;">Pseudomonas aeruginosa</td>
			<td style="width:108px;">Siryaporn lab</td>
			<td style="width:91px;">PA14</td>
			<td style="width:297px;">PA14 strain19</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Pseudomonas aeruginosa &#916;lasR</td>
			<td style="width:108px;">Siryaporn lab</td>
			<td style="width:91px;">AFS20.1</td>
			<td style="width:297px;">PA14-derived strain20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Supplies</td>
			<td style="width:108px;"></td>
			<td style="width:91px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">0.22 &#181;m filter&#160;</td>
			<td style="width:108px;">Millipore</td>
			<td style="width:91px;">SCGPT01RE</td>
			<td style="width:297px;">For filter sterilization</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:220px;">Conical tube, 15 mL</td>
			<td style="width:108px;">Corning</td>
			<td style="width:91px;">352097</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Glass storage bottles</td>
			<td style="width:108px;">Pyrex</td>
			<td style="width:91px;">13951L</td>
			<td style="width:297px;">250 mL, 500 mL, 1000 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Petri dish, 6 cm diameter</td>
			<td style="width:108px;">Corning</td>
			<td style="width:91px;">351007</td>
			<td style="width:297px;">60 x 15 mm polystyrene plates</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:220px;">Petri dish, 10 cm diameter</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">FB0875712</td>
			<td style="width:297px;">100 x 15 mm polystyrene plates</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:220px;">Plastic containers with lid</td>
			<td style="width:108px;">Ziploc</td>
			<td style="width:91px;">2.57E+09</td>
			<td style="width:297px;">Square 3-cup containers</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:220px;">Glass plates</td>
			<td style="width:108px;">Bio-Rad</td>
			<td style="width:91px;">1653308</td>
			<td style="width:297px;">For preparing agar pads. Other glass plates may be used with similar dimensions.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:220px;">Wooden sticks</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">23-400-102&#160;</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Equipment</td>
			<td style="width:108px;"></td>
			<td style="width:91px;"></td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Eclipse Ti-E microscope&#160;</td>
			<td style="width:108px;">Nikon</td>
			<td style="width:91px;">MEA53100</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">10X Plan Fluor Ph1 objective&#160; 0.3 NA</td>
			<td style="width:108px;">Nikon</td>
			<td style="width:91px;">MRH20101</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Fluorescence excitation source</td>
			<td style="width:108px;">Lumencor</td>
			<td style="width:91px;">Sola light engine</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:220px;">Fluorescence filter set</td>
			<td style="width:108px;">Semrock</td>
			<td style="width:91px;">LED-DA/FI/TX-3X3M-A-000&#160;</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Orca Flash 4.0 V2 Camera</td>
			<td style="width:108px;">Hamamatsu</td>
			<td style="width:91px;">77054098</td>
			<td style="width:297px;">Microscope setup</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">ImageJ</td>
			<td style="width:108px;">NIH</td>
			<td style="width:91px;">v. 1.49</td>
			<td style="width:297px;">Software for image analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">MATLAB</td>
			<td style="width:108px;">Mathworks</td>
			<td style="width:91px;">R2013</td>
			<td style="width:297px;">Software for image analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Orbital shaker incubator</td>
			<td style="width:108px;">VWR</td>
			<td style="width:91px;">89032-092</td>
			<td style="width:297px;">For growth of bacteria at 37 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Platform shaker</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">13-687-700</td>
			<td style="width:297px;">For growth of amoebae at 22 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Spectrophotometer</td>
			<td style="width:108px;">Biochrom</td>
			<td style="width:91px;">Ultrospec 10</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Undercounter refrigerated incubator</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">97990E</td>
			<td style="width:297px;">For growth of amoebae at 22 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:220px;">Isotemp waterbath&#160;</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:91px;">15-462-21Q</td>
			<td style="width:297px;">For cooling media to 55 &#176;C</td>
		</tr>
	</tbody>
</table>

a rapid image-based bacterial virulence assay using amoeba

Traditionella bakteriell virulens analyser innebär långvarig exponering av bakterier under loppet av flera timmar till värdceller. Under denna tid, kan bakterier genomgå förändringar i fysiologi på grund av exponering för tillväxt värdmiljön och förekomsten av vara värdcellerna. Vi utvecklat ett test för att snabbt mäta tillståndet virulens av bakterier som minimerar den utsträckning som bakterier växa i närvaro av värdceller. Bakterier och endoparasiterna blandas ihop och orörlig på en enda bildplanen använder en agar-pad. Proceduren använder encelliga fluorescens imaging med calcein-acetoximetyl ester (calcein-AM) som en indikator på värden cell hälsa. Fluorescensen av värdceller analyseras efter 1 h exponering av värdceller att bakterierna med epifluorescence mikroskopi. Bild analys programvara används för att beräkna en värd dödande index. Denna metod har använts för att mäta virulens inom plankton och surface-anslutna Pseudomonas aeruginosa delpopulationer under det inledande skedet av biofilm bildning och kan anpassas till andra bakterier och andra stadier av biofilm tillväxt. Detta protokoll ger en snabb och robust metod att mäta virulens och undviker många av svårigheterna som förknippas med tillväxt och underhåll av däggdjursceller linjer. Virulens fenotyper mätt här med endoparasiterna har också validerats med hjälp av musen makrofager. I synnerhet för denna analys att fastställa att ytan fastsättning uppreglerar virulens i P. aeruginosa.

Vi växte vildtyp P. aeruginosa stam PA1419 eller en ΔlasR stam20 i samma PA14 bakgrund i 6 cm-diameter petriskålar och analyseras virulens hos plankton och surface-anslutna celler. Kulturer var inokuleras från singel-kolonier i PS:DB kulturer, vuxit över natten i kultur rör i en rulle trumma vid 37 ° C till mättnad, spädd 1: 100 i PS:DB, odlas för 8 h i 6 cm-diameter petriskålar skakar vid 100 rpm, och plankton o...

Watch this Scientific Journal Video about A Rapid Image-based Bacterial Virulence Assay Using Amoeba at JoVE.com

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

En snabb Image-baserad bakteriell virulens analys med hjälp av amöba

Här presenterar vi ett protokoll för att mäta virulens hos plankton eller surface-anslutna bakterier använda D. discoideum (amöba) som värd. Virulens mäts över en period på 1 h och värd döda kvantifieras med hjälp av fluorescence mikroskopi och bild analys. Vi visar detta protokoll som använder bakterien P. aeruginosa.

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

This method can help answer key questions in the bacterial pathogenesis field, such as when are host-killing virulence effectors expressed in bacterial cells, and what are the virulence states of surface-attached implantonic cells? The main advantage of this technique is that it is fast, quantitative and the host cells are relatively easy to handle. We first had the idea for this method when we noticed that surface-attached implantonic subpopulation of Pseudomonas have different host-killing phenotypes.To begin grow E.coli strain BR overnight as outlined in the text protocol. Then bring the E.coli culture to room temperature. Using a wooden stick, innoculate amoeba into 750 l of the culture.Immediately add 700 l of the amoeba E.coli mixture to a GYP plate. Tilt the plate to spread the culture evenly on the surface of the plate. Then place the plate in a box that maintains high humidity.Incubate the plate inside of the box at 22 C for four to five days. After the incubation period is over, use a sterile pipette tip to collect amoeba spores from half of the plate. Then pipette up and down to resuspend the amoeba on the pipette tip in 1 mL of PS medium.Transfer the inoculate mixture to a 250 mL flask with 25 mL of PS medium supplemented with antibiotic antimicotic solution. Grow the amoeba at 22 C in a rotating shaker at 100 rpm. After culturing Pseudomonas aeruginosa overnight, dilute the culture with PSB media in a 6 cm petri dish.Then shake the culture on a benchtop rotator set to 100 rpm at 37 C.First microwave 50 mL of 1%agar in DB buffer in a 250 mL flask. Add 15 L of calcium AM to the 15 mL of molten agar and mix gently. Pour the molten agar mixture into a 12 by 10 cm glass plate, and allow the agar to solidify for ten minutes at room temperature.Next use a metal ruler and a printed grid to cut the solidified agar pad into smaller 1.5 by 1.5 cm sections. Ensure the gel remains hydrated until it is ready for use. Next, to isolate the cells attached to the surface, remove the liquid medium from the petri dish.Then wash the dish with 1 mL of development buffer to remove the planktonic cells. To assay virulence of surface attached bacteria cells, add 10 L of amoeba cells from the previously obtained surface attached bacterial cells. To isolate the planktonic cells, transfer 10 L of the culture to a fresh petri dish.Next, assay the virulence of planktonic cells by mixing 10 L of amoeba cells with the previously obtained planktonic bacteria. Immediately place the small agar pad on top of the appropriate bacteria amoeba mixture on the petri dish surface. Then, remove excess liquid by gently pipetting any excess liquid, and allow the dish to dry for 20 minutes.Cover the dish with the lid and incubate it at room temperature for an additional 40 minutes. Finally, use a fluorescence microscope to acquire images for at least 100 amoeba cells. In this protocol, the virulence of planktonic and surface-attached Pseudomonas aeruginosa cells was assayed.Surface-attached wild type Pseudomonas aeruginosa killed amoeba indicated by a round amoeba cell shape and the presence of calcein fluorescence. Compared to planktonic cells surface-attached wild type Pseudomonas aeruginosa had a high host killing index. Planktonic cells were consumed by the amoeba indicated by the amorphous amoeba cell shapes and relatively little calcein fluorescence.While attempting this procedure, it is important to remember to use a fresh stock of calcein AM, and amoeba cells and keep a close track of the optical density of amoeba. Following this procedure, other methods like transcription profiling can be performed in order to answer additional questions like Which genes are activated in planktonic and surface-attached subpopulation? After watching this video you should have a good understanding of how to conduct the rapid image-based virulence assay.

a-rapid-image-based-bacterial-virulence-assay-using-amoeba

Research

JoVE Journal

Bioengineering

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.

Kännetecknar Bakteriell Flyktiga använda Sekundär elektrospray Spectrometry jonisation (Sesi-MS)

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

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Bakteriell Detection &amp; identifiering med elektrokemiska sensorer

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Snabb och billig Prototyping för medicintekniska produkter Använda 3D Tryckt Formar för Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

Mätt TCR-pMHC Bindning<em&gt; In Situ</em&gt; Med en FRET-baserad Mikroskopi analys

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis of the intrinsic photothermal response of hemoglobin molecules, the sensor enables high-sensitivity, chemical-free measurement of [Hb]. [Hb] detection capability with a limit of 0.12 g/dl over the range of 0.35 - 17.9 g/dl has been demonstrated previously. The method can be readily implemented using inexpensive consumer electronic devices such as a laser pointer and a webcam. The use of a micro-capillary tube as a blood container also enables the hemoglobin assay with a nanoliter-scale blood volume and a low operating cost. Here, detailed instructions for the PT-AS optical setup and signal processing procedures are presented. Experimental protocols and representative results for blood samples in anemic conditions ([Hb] = 5.3, 7.5, and 9.9 g/dl) are also provided, and the measurements are compared with those from a hematology analyzer. Its simplicity in implementation and operation should enable its wide adoption in clinical laboratories and resource-limited settings.

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

En snabb och kemisk-fritt hemoglobin-analys med fototermisk Vinkel Light Scattering

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...

Rapid Mix Preparation of Bioinspired Nanoscale Hydroxyapatite for Biomedical Applications

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest regarding the use of bioinspired nanoscale hydroxyapatite (nHA). However, biological apatite is known to be calcium-deficient and carbonate-substituted with a nanoscale platelet-like morphology. Bioinspired nHA has the potential to stimulate optimal bone tissue regeneration due to its similarity to bone and tooth enamel mineral. Many of the methods currently used to fabricate nHA both in the laboratory and commercially, involve lengthy processes and complex equipment. Therefore, the aim of this study was to develop a rapid and reliable method to prepare high quality bioinspired nHA. The rapid mixing method developed was based upon an acid-base reaction involving calcium hydroxide and phosphoric acid. Briefly, a phosphoric acid solution was poured into a calcium hydroxide solution followed by stirring, washing and drying stages. Part of the batch was sintered at 1,000 &#176;C for 2 h in order to investigate the products&#39; high temperature stability. X-ray diffraction analysis showed the successful formation of HA, which showed thermal decomposition to &#946;-tricalcium phosphate after high temperature processing, which is typical for calcium-deficient HA. Fourier transform infrared spectroscopy showed the presence of carbonate groups in the precipitated product. The nHA particles had a low aspect ratio with approximate dimensions of 50 x 30 nm, close to the dimensions of biological apatite. The material was also calcium deficient with a Ca:P molar ratio of 1.63, which like biological apatite is lower than the stoichiometric HA ratio of 1.67. This new method is therefore a reliable and far more convenient process for the manufacture of bioinspired nHA, overcoming the need for lengthy titrations and complex equipment. The resulting bioinspired HA product is suitable for use in a wide variety of medical and consumer health applications.

This paper describes a novel method for the rapid manufacture of high quality bioinspired nanoscale hydroxyapatite. This biomaterial is of great significance in the manufacture of a wide range of innovative medical devices for clinical applications in orthopedics, craniofacial surgery and dentistry.

Rapid Mix Framställning av Bioinspired Nanoscale hydroxyapatit för biomedicinska tillämpningar

Hydroxyapatite (HA) has been widely used as a medical ceramic due to its good biocompatibility and osteoconductivity. Recently there has been interest ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

En automatiserad metod för att utföra Mikrokärnanalys in vitro med hjälp av Multispektral avbildning flödescytometri

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

Hög genomströmning Identifiering av resistens mot Pseudomonas syringae pv. Tomat i tomat med plantning Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

Snabb montering av multigenkonstruktioner med modulär Golden Gate-kloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell cultures. Microcarrier culture technology has become one of the main techniques in cytological research and is commonly used in the field of large-scale cell expansion. Microcarriers have also been shown to play an increasingly important role in in vitro tissue engineering construction and clinical drug screening. Current methods for preparing microcarriers include microfluidic chips and inkjet printing, which often rely on complex flow channel design, an incompatible two-phase interface, and a fixed nozzle shape. These methods face the challenges of complex nozzle processing, inconvenient nozzle changes, and excessive extrusion forces when applied to multiple bioink. In this study, a 3D printing technique, called alternating viscous-inertial force jetting, was applied to enable the construction of hydrogel microcarriers with a diameter of 100-300 &#181;m. Cells were subsequently seeded on microcarriers to form tissue engineering modules. Compared to existing methods, this method offers a free nozzle tip diameter, flexible nozzle switching, free control of printing parameters, and mild printing conditions for a wide range of bioactive materials.

3D Printing of In Vitro Hydrogel Microcarriers by Alternating Viscous-Inertial Force Jetting

Presented here is a mild 3D printing technique driven by alternating viscous-inertial forces to enable the construction of hydrogel microcarriers. Homemade nozzles offer flexibility, allowing easy replacement for different materials and diameters. Cell binding microcarriers with a diameter of 50-500 &#181;m can be obtained and collected for further culturing.

3D-utskrift av In Vitro Hydrogel Microcarriers genom alternerande viskös-tröghetskraft jetting

Microcarriers are beads with a diameter of 60-250 &#181;m and a large specific surface area, which are commonly used as carriers for large-scale cell ...