Name: design and use of a low cost, automated morbidostat for adaptive evolution of bacteria under antibiotic drug selection
Uploaded: 2016-09-27T15:00:00
Description: Watch this Scientific Journal Video about Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection at JoVE.com

The authors would like to thank Prof. Sze-Bi Hsu and Ms. Zhenzhen for useful discussions and help in the theoretical analysis and numerical simulation. Y. T. Y. would like to acknowledge funding support from the Ministry of Science and Technology under grant numbers MOST 103-2220-E-007-026 and MOST 104-2220-E-007-011, and from the National Tsing Hua University under grant numbers 103N2042E1, 104N2042E1, and 105N518CE1.

1. Montering och pretesting av Morbidostat Device <ol><li> Montering av Morbidostat <ol><li> Stansa 3 hål på locket av odlingsflaskan med en 18 G sprutnål. Skär tre bitar av polyeten slang ~ 7 cm i längd. Infoga dessa tre bitar av polyeten slang på locket. </li><li> Använd tejp för att linda kanten av locket för att tjäna som gjutna för polydimetylsiloxan (PDMS) blandning. Blanda 5 g av A-komponenten och 0,5 g B-komponenten av PDMS i en 150 ml plastbehållare genom omrörning för hand med en tandpetare. Fy...

<ol>
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En låg fotavtryck morbidostat enhet från lågkostnadskomponenter demonstreras. Ökningarna i läkemedelsresistens nivå som registrerats av enheten överensstämmer med de tidigare rapporter 5. Designad för evolutionära studier av läkemedelsresistens, är enheten potentiellt tillämpas på många andra experiment. För det första kan fastställas en omfattande databas av läkemedelsinducerade mutationer för ett stort antal kliniskt relevanta antibiotika. Till exempel, kan det evolutionära reaktionsvä...

Sedan introduktionen av den första antibiotikum penicillin, har mikrobiell antibiotikaresistens utvecklats till ett globalt hälsoproblem 1. Även förvärvet av antibiotikaresistens kan i efterhand studeras in vivo, är villkoren för dessa försök ofta inte kontrolleras hela evolutionen 2. Alternativt kan adaptiv laboratorium evolution avslöja molekylär evolution av en mikrobiell art enligt miljöpåfrestningar eller selektionstryck från ett antibiotikum läkemedel 3. På senare tid har många välkontrollerade evolutionära experiment av antibiotika resistens klar uppkomsten av antibiotikaläkemedelsresistens. Till exempel, Austin grupp visade snabb framväxt i en korrekt konstruerad mikroflödesfack miljö 4. Den nyutvecklade morbidostat inducerar systematiska mutationer enligt läkemedelsselektionstryck 5,6. Den morbidostat, en mikrobiell selectionsanordning som kontinuerligt justerar antibiotikakoncentrationen att upprätthålla en nästan konstant befolkning, är ett stort framsteg från fluktuationer test som används i mikrobiologi 7,8. I fluktuationen test, ett antibiotikum läkemedlet injiceras vid hög koncentration, och de överlevande mutanter screenas och räknas. Istället mikrober i en morbidostat ständigt utmanas och förvärva flera mutationer. Den morbidostat fungerar på samma sätt som kemostat, en kultur anordning uppfanns av Novick och Szliard 1950 som upprätthåller en konstant befolkning genom att kontinuerligt tillföra näringsämnen samtidigt späda den mikrobiella populationen 9. Sedan introduktionen har kemostaten förts fram och förbättras. Aktuella mikroflödes kemostater har nått nanoliter och encelliga kapacitet. Emellertid är dessa anordningar är olämpliga för adaptiv evolution experiment, vilka kräver en stor cellpopulation med många mutationshändelser 10,11. Nyligen, mini-kemostater med arbetsvolymer ~ 10 ml har också utvecklats för att fylla gapet mellan liter skala bioreaktorer och mikroflödes kemostaten 12,13. Här presenterar vi utformningen och användningen av en låg kostnad, automatiserad morbidostat för en antibiotisk läkemedelsresistens studien. Den föreslagna Modulen kan användas i en skakinkubator i ett mikrobiologiskt laboratorium med krav minimal hårdvara. Öppen källkod firmware är också lätt anpassas till specifika tillämpningar av adaptiv evolution, såsom metabolisk teknik 3. Slutligen är morbidostat integreras i en multiplexerad mikroflödes plattform för antibiotikakänslighetstestning 14.

<table border="0" cellpadding="0" cellspacing="0" width="797">
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			<td height="21" style="height:21px;width:275px;">Environmental Shaker Incubator</td>
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			<td>tapered shape dim: 10 mm x 4mm</td>
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			<td height="42" style="height:42px;width:275px;">Glass scintillation 20ml vial</td>
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			<td>Pyrex glass 28mm(dia.)x 61 mm(h)</td>
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			<td>ValuePlastics.com</td>
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			<td>used for microfluidic control</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Tantalum capacitor, 10uF, 25V, 10%</td>
			<td>DigiKey.com</td>
			<td>478-1841-ND</td>
			<td>used for microfluidic control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Andor CCD camera</td>
			<td style="width:135px;">Andor</td>
			<td style="width:163px;">Zyla 4.2 Plus SCMOS</td>
			<td>used for microfluidic on chip imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">ELISA plate reader</td>
			<td style="width:135px;"></td>
			<td style="width:163px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">two component Silicone&#160;</td>
			<td style="width:135px;">Momentive</td>
			<td style="width:163px;">RTV 615</td>
			<td>used for microfluidic chip fabrication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">SU-8 photoresist</td>
			<td style="width:135px;">Micrchem</td>
			<td style="width:163px;">SU8 2015</td>
			<td>used for microfluidic chip fabrication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">AZ4620 photoresist</td>
			<td style="width:135px;">Clariant</td>
			<td style="width:163px;">AZ 4620</td>
			<td>used for microfluidic chip fabrication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Plasma cleaner</td>
			<td style="width:135px;">Harrick Plasma</td>
			<td style="width:163px;">PDC 32G</td>
			<td>used for microfluidic chip fabrication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">20 Gauge Syringe Needle</td>
			<td style="width:135px;">BD</td>
			<td style="width:163px;"></td>
			<td>used for microfluidic chip fabrication</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Labcycler</td>
			<td style="width:135px;">Sensoquest</td>
			<td style="width:163px;">Labcycler</td>
			<td>PCR&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">DNA polymerase</td>
			<td style="width:135px;">Toyobo</td>
			<td style="width:163px;">KDO Plus</td>
			<td>PCR amplification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Trimethoprim</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:163px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Plate reader</td>
			<td style="width:135px;">Biotek</td>
			<td style="width:163px;">Synergy H1 hybrid&#160;</td>
			<td>antibiotic resistane measurement</td>
		</tr>
	</tbody>
</table>

design and use of a low cost, automated morbidostat for adaptive evolution of bacteria under antibiotic drug selection

Vi beskriver en låg kostnad, konfigurerbar morbidostat för att karakterisera den evolutionära vägen för antibiotikaresistens. Den morbidostat är en bakteriekultur enhet som kontinuerligt övervakar bakterietillväxt och dynamiskt justerar läkemedelskoncentrationen att ständigt utmana bakterier eftersom de utvecklas att förvärva läkemedelsresistens. Enheten har en arbetsvolym av ~ 10 ml och är helt automatiserad och utrustad med optisk densitet mätning och mikropumpar till mellanstora och drug delivery. För att validera plattformen, mätte vi stegvis förvärv av trimetoprim motstånd i Escherichia coli MG 1655, och integreras enheten med en multiplex mikroflödes plattform för att undersöka cellmorfologin och antibiotikakänslighet. Tillvägagångssättet kan skalas upp till laboratoriestudier av antibiotika resistens, och är utdragbara för adaptiv evolution för stam förbättringar i metabolisk ingenjörskonst och andra bakteriekultur experiment.

Den ovan beskrivna morbidostat är schematiserad i Figur 1. De gemensamma morbidostat verksamhet, inklusive experimentell utveckling, antibiotikakänslighet test och cellmorfologin kontroll, validerades i en E. coli MG1655 kultur utsätts för trimetoprim (TMP), ett vanligt använt antibiotikum 5,6. TMP inducerar mycket distinkta stegökningar läkemedelsresistens, och mutationerna är grupperade runt dihydrofolatreduktas (DHFR) genen. Därför är TM...

Watch this Scientific Journal Video about Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection at JoVE.com

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

Utformning och användning av en låg kostnad, Automated Morbidostat för Adaptive Evolution av bakterier under antibiotikum Drug Val

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

Research

JoVE Journal

Genetics

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Förfarande för adaptiv Laboratory Evolution av mikroorganismer med hjälp av en kemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Hög genomströmning identifiering av synergistisk läkemedelskombinationer med överlappning2 Metod

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Testning Multicopy plasmider roll i utvecklingen av antibiotikaresistens

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Genome-wide övervakning av transkription fel i eukaryota organismer

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes

Producerar genen borttagningar i Escherichia coli av P1 transduktion med punktskattepliktiga antibiotikaresistens kassetter

A first approach to study the function of an unknown gene in bacteria is to create a knock-out of this gene. Here, we describe a robust and fast protocol ...

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

qKAT: kvantitativa Semi-automated typning av Killer-cell immunglobulin-like Receptor gener

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Genteknik Dictyostelium discoideum celler baserat på urval och tillväxt på bakterier

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER

Designa automatiserade, hög genom strömning, kontinuerlig cell tillväxt experiment med eVOLVER

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring ...

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

Användning av fryst vävnad i kometanalysen för utvärdering av DNA-skador

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Användning av Drosophila S2-celler för levande avbildning av cell delning

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a ...