JoVE Journal > Bioengineering
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
생체공학

Design og implementering af en automatiseret lysende, Dyrkning, og Sampling System for Mikrobiel optogenetic Applications

Published: February 19, 2017
doi:
10.3791/54894
,
1Department of Biomedical Engineering,University of Wisconsin-Madison

Summary

Vi designede en kontinuerlig dyrkning apparat til brug med optogenetic systemer til at belyse kulturer af mikrober og regelmæssigt billedceller i effluenten med et inverteret mikroskop. Dyrkning, prøveudtagning, billedbehandling og billedanalyse er fuldt automatiseret, så dynamiske reaktioner på belysning kan måles over flere dage.

Abstract

Optogenetic systemer anvender genetisk kodede proteiner, der ændrer konformation som reaktion på specifikke lysbølgelængder at ændre cellulære processer. Der er behov for dyrkning og målesystemer, der inkorporerer programmerede belysning og stimulering af optogenetic systemer. Vi præsenterer en protokol til opbygning og anvendelse af en kontinuerlig dyrkning apparat til at belyse mikrobielle celler med programmerede doser af lys, og automatisk erhverve og analysere billeder af celler i spildevandet. Driften af ​​apparatet som en chemostat tillader vækstraten og den cellulære miljø, der skal tæt kontrolleret. Effluenten af ​​den kontinuerlige cellekultur regelmæssigt stikprøven, og cellerne afbildes af flere kanaler mikroskopi. Dyrkning, prøveudtagning, billedbehandling og billedanalyse er fuldt automatiseret, så dynamiske reaktioner i fluorescensintensitet og cellulære morfologi af celler stikprøven fra kulturen spildevand måles over flere dageuden brugerinput. Vi demonstrerer anvendeligheden af denne dyrkning apparat ved dynamisk at inducere proteinproduktion i en stamme af Saccharomyces cerevisiae manipuleret med et optogenetic system, der aktiverer transskription.

Introduction

Optogenetic systemer bruger lys til at styre en voksende liste af cellulære processer, herunder genekspression, 1, 2, 3, 4, 5 proteinlokalisering, 6 proteinaktivitet, 6, 7, 8-protein binding, 8, 9, 10 og proteinnedbrydning. 11 En metode til dyrkning af celler i et kontrolleret miljø med programmeret optisk stimulation og til måling af deres respons over biologisk relevante tidsskalaer er nødvendig for at udnytte potentialet i disse værktøjer til forskning i cellebiologi og bioteknologi. Vores fremgangsmåde drager fordel af chemostasis at opretholde en konstant celle vækst i en velblandet, aevurderet, og temperaturstyret glas dyrkningsbeholder 12, 13, der er udsat for programmeret belysning. Vi billeddata individuelle celler i kulturen spildevand med et inverteret mikroskop til måling af, kulturen til programmeret belysning. Dyrkning, prøveudtagning, billedbehandling og billedanalyse er fuldt automatiseret, så fluorescensintensiteten og cellulær morfologi spildevandet cellekultur kan måles over flere dage uden brugerinput.

Denne protokol kan implementeres i de fleste laboratorier er fortrolige med voksende cellekultur og mikroskopi, og det anvendte apparat er billig og lavet af let tilgængelige komponenter. En gennemsigtig dyrkningsbeholder anbringes over en matrix af lysemitterende dioder (LED), der kan udsende 1 pW / cm2 -10 mW / cm2 af lys. Mikrober dyrkes i dyrkningsbeholderen kontinuerligt; en peristaltisk pumpe bruges til at tilføje medier påfortynding rate, en anden bruges til at trække kulturen på en mindre sats til mikroskopet, og forskellen undslipper gennem et overløb stikkontakt. En varmepude opretholder temperaturen. Luft kontinuerligt pumpet ind i dyrkningsbeholderen at opretholde et positivt tryk samt at blande og lufte kulturen. Bortset fra luftpumpen, er strøm til disse enheder reguleret af en microcontroller, der også modtager input fra et termometer og en tilsluttet computer. Effluenten cellekultur pumpes til en mikrofluidanordning på scenen af ​​et omvendt mikroskop. Ikke-fluorescerende og fluorescerende billeder automatisk erhverves. Cellerne i billederne er karakteriseret ved en algoritme, der lokaliserer hver celle som et område af interesse (ROI) og måler egenskaberne for hver ROI.

For at demonstrere en anvendelse af denne protokol, vi målte reaktion på varierende lysintensiteter af Saccharomyces cerevisiae-celler manipuleret med en blå lys responsive optogenetic system, som kontrollerer transkriptionen af ​​fluorescerende protein. S. cerevisiae, almindeligvis kendt som bagegær, blev valgt, fordi der allerede findes flere optogenetic systemer til kontrol af genekspression i dette system 14, 15, 16. Desuden er denne model organisme almindeligt anvendt til studier i systembiologi 17 og som et chassis til bioteknologiske applikationer 18, 19, 20. Vores repræsentative resultater viser, at denne protokol kan anvendes til at styre transkription af en kultur over flere dage ved at variere input lysintensiteter og måling af produktion af en fluorescerende reporter.

Protocol

Figur 1: Den kontinuerlige dyrkning apparat. Denne forenklede diagram viser, hvordan apparatet skal samles, når det anvendes til kultur, belyse og måle optiske egenskaber af mikrober. Klik her for at se en større version af dette tal. <img al…

Representative Results

Dette apparat blev anvendt til at stimulere en kultur af S. cerevisiae, der udtrykker gult fluorescerende protein (YFP) som respons på blåt lys via en inducerbar optogenetic transskription system baseret på CRY2 / CIB1 proteinpar 30. Celler blev dyrket chemostatically i phosphat-begrænset medium med en gennemsnitlig fortynding på 0,2 ± 0,008. Fosfat begrænsning er almindeligt anvendt i S. cerevisiae Kemostater eksperimenter for at kontroll…

Discussion

Vi har designet dette apparat med fleksibilitet i tankerne. Alt den anvendte kode er gratis og open-source. Standard billedanalyse proces at segmentere celler er enkel og kører hurtigt. Tilpasset analyse kunne gennemføres ved at optage brugerinput mens analysere et repræsentativt billede med FIJI grafisk brugergrænseflade, omdanne input til en Beanshell script, og derefter indstille plugin til at kalde scriptet. Når det kaldes, vil dette script blive sendt en String-array kaldet "billeder", der indeholder…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi vil gerne anerkende Molly Lazar og Verónica Delgado om hjælp til at teste protokollen, Kieran Sweeney for nyttige diskussioner og redigering, og Taylor Scott, My An-adirekkun, og Stephanie Geller for kritisk læsning af manuskriptet. Megan Nicole McClean, Ph.D. har en karriere Award på den videnskabelige interface fra Burroughs Wellcome fonden.

Materials

Extensive lab manual GitHub NA An extensive, regularly updated lab manual is available in the “Optogenetic Chemostat Files” GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files). This also includes a description of the microfluidic mold used to generate the representative results.
Fritzing Design Viewer Fritzing NA The free, open-sourced software to view and edit the .fzz type circuit board designs is available at "http://fritzing.org/download/"
Arduino Uno R3 (Atmega328 – assembled) Adafruit 50 Microcontroller. 1 required.
Arduino Stackable Header Kit SparkFun Electronics 10007 Female pin headers for connecting PCB to microcontroller. 1 required.
Adjustable 30W 110V soldering iron – XY-258 110V Adafruit 180 For making electrical connections to the PCB. 1 required.
Soldering iron stand Adafruit 150 For making electrical connections to the PCB. 1 required.
Mini Solder spool – 60/40 lead rosin-core solder 0.031" diameter – 100g Adafruit 145 For making electrical connections to the PCB. 1 required.
0.1 μF capacitor SparkFun Electronics COM-08375 Stabilizes voltage in PCB. 1 required.
10 μF capacitor SparkFun Electronics COM-00523 Stabilizes voltage in PCB. 1 required.
MAX7219CNG LED Matrix/Digit Display Driver – MAX7219 Maxim MAX7219CNG LED driver. 1 required.
8 pin IC Socket Mouser Electronics 575-144308 16 required. These will be stacked on top of each other to support the culture vessel above the LED matrix.
24 Pin IC socket Mouser Electronics 535-24-3518-10 Optional. Use this to reversibly attach the MAXIM 7219CNG driver to the PCB.
Digital multimeter Adafruit 2034 For troubleshooting electronics. 1 required.
Break Away Headers – 40-pin Male (Long Centered, PTH, 0.1") SparkFun Electronics PRT-12693 Male pin headers for connected LED matrix to printed circuit board. Ends can be trimmed with wire cutters. 1 set required. 
Flush diagonal wire cutters Adafruit 152 For trimming long pin headers and cutting power cables. 1 required.
Premium Female/Female Jumper Wires – 40 x 12" (300mm) Adafruit 793 Wire ribbon for connecting breadboard to LED matrix. Can be connected end-to-end with male pin-headers to be longer. 1 required.
Half-size breadboard Adafruit 64 The LED matrix will connect to this and the culturing vessel will rest above it.
Miniature 8×8 Blue LED Matrix Adafruit 956 Light source. Dominant wavelength is 470nm (blue). 1 required. Alternative miniature LED matrices from the same vendor are available with dominant wavelengths: 624 nm (red), 588 nm (yellow), 525 nm (green), 572 nm (yellow-green), and white.
Stackable header-3 pin SparkFun Electronics 13875 8 required.
Resistor Kit – 1/4W (500 total) SparkFun Electronics 10969 For electronics. 1 required.
 IRL520N MOSFET International Rectifier IRL520N Voltage regulating switch for controlling DC current. 4 required.
Hook-Up Wire – Assortment (Solid Core, 22 AWG) SparkFun Electronics PRT 11367 Wire for electronics. 1 required.
5V 2A (2000mA) switching power supply – UL Listed Adafruit 276 Power supply for the heating pad and Arduino. 2 required.
12 VDC 1000mA regulated switching power adapter – UL listed Adafruit 798 For peristaltic pumps. 2 required.
Electric Heating Pad – 10cm x 5cm Adafruit 1481 For heating the bioreactor. 1 required.
Low flow variable flow peristaltic pump Fisher Scientific 13-876-1 For pumping media. 1 required
Medium flow variable flow peristaltic pump Fisher Scientific 13-876-2 For pumping culture. 1 required.
9 VDC 1000mA regulated switching power adapter – UL listed Adafruit 63 For microcontroller power supply. Order 1.
High Temp Waterproof DS18B20 Digital temperature sensor + extras Adafruit 642 Thermometer for the bioreactor. 1 required.
Micromanager Micromanager NA The free, open-sourced microscope control software is available at "https://micro-manager.org/wiki/Download_Micro-Manager_Latest_Release"
FIJI ImageJ NA The free, open-sourced image analysis software is available at "http://fiji.sc/"
Arduino Integrated Development Environment Arduino NA The free, open-sourced IDE is available at "https://www.arduino.cc/en/Main/Software"
Custom code GitHub NA The custom microcontroller code and "Bioreactor Controller" plugin are available in the “Optogenetic Chemostat Files” GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files).
USB Cable A to B – 6 Foot SparkFun Electronics CAB-00512 Used to download data to microcontroller. 1 required.
bioreactorTimecourse_example.csv GitHub NA The advantage of loading LED matrix values from a CSV file is that a program can be called by the plugin to update those values based on image analysis results, and those values can be reloaded to the microcontroller, enabling feed-back control. It is available from the “Optogenetic Chemostat Files” GitHub repository (https://github.com/McCleanResearch/Optogenetic-Chemostat-Files).
Tota-frost gels (diffusion paper) B&H B&H # LOFSFTL
MFR # T1-72		 For LED matrix. 1 required.
Kitting Sheet Crosslink 1/4x12x24in Grainger, inc 20JL37 Black foam for culturing vessel enclosure. 4 required.
Standard Photodiode Power Sensor, Si, 200 – 1100 nm, 50 mW  Thorlabs S120VC For measuring light intensity. 1 required.
Labelling Tape Fisher Scientific 159015N For labelling and securing loose components. 1 required.
Compact Power and Energy Meter Console, Digital 4" LCD Thorlabs PM100D For measuring light intensity. 1 required.
100mL GL45 hybridization glass bottle Bellco Glass, Inc. (7910-40150) Bioreactor vessel. 1 required.
Six port assembly Bellco Glass, Inc. Custom  For the bioreactor vessel. Tubing Specs: .125" OD x .055"ID. Port A: 1.0" long above cap slug and to bottom of tube. Ports B,C,E,F: 1.0" long above cap slug, 33 mm long below. Port D: 1.0" long above cap slug, 65 mm  long below. 1 required. Includes 45 mm diameter polypropylene open top screw cap and a white silicone gasket to ensure a tight seal between the cap and the vessel. 
Scotch Magic Tape 3105, 3/4 x 300 Inches, Pack of 3 Amazon B0009F3P3U Clear scotch tape. This is available from many other vendors. It is used to cover markings on the culturing vessel and to secure the coverglass with the PDMS channel to the aluminum support frame.
1/16" ID x 3/16" OD x 1/16" Wall Tygon Sanitary Silicone Tubing United States Plastic Corp. 57288 Tubing. ~25' required.
Cole-Parmer Twistit white rubber stopper, size 10 Cole-Parmer EW-62992-32 Media flask stopper and effluent flask stopper. 2 required.
2L Laboratory Flask Pyrex 4980 Media flask and effluent flask. 2 required.
Day pinchcock Fisher Scientific 5867 For pinching tubes shut. 3 required.
Replacement tubing assembly 1/16" ID Traceable Products 3372 The peristaltic pumps come with a set of tubes, but they wear out after weeks of use.
Replacement tubing assembly 1/50" ID Traceable Products 3371 The peristaltic pumps come with a set of tubes, but they wear out after weeks of use.
Male luer with lock ring x 1/16" hose barb, Nylon, 25/pk Cole-Parmer EW-45505-00 Connectors. ~10 luers are required.
Male luer with lock ring x 1/8" hose barb, Nylon, 25/pk Cole-Parmer EW-45505-04 Connectors. 5 required, one for each rubber stopper hole to fill with tubing.
Female luer x 1/16" hose barb adapter, Nylon, 25/pk Cole-Parmer EW-45502-00 Connectors. ~10 luers required.
Female luer x 3/16" hose barb adapter Cole-Parmer EW-45502-08 Connectors. ~10 luers required.
Cole-Parmer Luer Accessory, Female Luer Cap, Nylon, 25/Pk Cole-Parmer SC-45502-28
Cole-Parmer Luer Accessory, Male Luer Lock Plug, Nylon, 25/Pk Cole-Parmer EW-45505-56
Microbore PTFE Tubing, 0.022"ID x 0.042"OD, 100 ft/roll Cole-Parmer EW-06417-21 Tubing. 1 roll required.
Masterflex platinum-cured silicone tubing, L/S 13, 25 ft Cole-Parmer EW-96410-13 Tubing. ~25' required.
3/16" ID x 1/4" OD x 1/32" Wall Tygon Sanitary Silicone Tubing United States Plastic Corp. 57293 Tubing. ~1' required.
Vacuum filter Fisher Scientific 974107 Nalgene vacuum filter for sterile filtering media.
Aquel Oxy-Boost 200 Rena Aquatic Supply AP200 Dual diaphram adjustable flow air pump for aerating and mixing media. 1 required. 
0.2 μm pore syringe filter Corning International 431229 This ensures that air from the aquarium pump does not contaminate the apparatus. 1 required.
Slygard 184 Silicone Elastomer Kit Dow Corning Slygard 184 For microfluidic device. 1 required.
American Safety Razor GEM Scientific Single-Edge Razor Blades Fisher Scientific 17989000 For cutting tubes and PDMS. 1 blade required.
Harris Uni-Core hole puncher 1.2mm ID Sigma-Aldrich WHAWB100028 ALDRICH For punching inlet/outlet in microfluidic device. 1 required.
Microscope cover glass 22×60-1.5 Fisher Scientific 12-544-G For microfluidic device. 1 required.
Rectangular aluminum frame with a square window Custom Custom To support the microfluidic channel. Outer dimensions: 3 inches x 1.25 inches.
Inner dimmensions (cut out portion): 7/8 inches x 7/8 inches
Thickness: ~1/32 inches
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Tags

Keywords: Automated SystemMicrobial OptogeneticsContinuous CulturingReal-time MeasurementGene ExpressionMetabolic EngineeringDigital ThermometerPrinted Circuit BoardLight-proof EnclosureInoculationPeristaltic TubingMedia FlaskAerationPressure Regulation
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Stewart, C. J., McClean, M. N. Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications. J. Vis. Exp. (120), e54894, doi:10.3791/54894 (2017).
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