Design og implementering av en automatisert Illuminating, dyrking, og Sampling System for Microbial optogenetic Applications
Summary
Vi har konstruert en kontinuerlig dyrkingsapparat for bruk med optogenetic systemer for å belyse kulturer av mikrober og regelmessig bildet celler i avløpet med et invertert mikroskop. Dyrknings, prøvetaking, avbildning, og bildeanalyse er fullt automatisert, slik at dynamiske responser til belysning kan måles over flere dager.
Abstract
Optogenetic systemer benytter genetisk kodede proteiner som endrer konformasjon som svar på bestemte bølgelengder av lys for å endre cellulære prosesser. Det er et behov for dyrking og målesystemer som innlemme programmerte belysning og stimulering av optogenetic systemer. Vi presenterer en protokoll for å bygge og ved hjelp av en kontinuerlig dyrkingsapparat for å belyse mikrobielle celler med programmerte doser av lys, og automatisk hente og analysere bilder av celler i avløpsstrømmen. Driften av dette apparatet som et kjemostat gjør at vekstraten og den cellulære miljøet for å bli kontrollert. Avløpet fra den kontinuerlige cellekultur er regelmessig samplet og cellene blir avbildet ved flerkanals mikroskopi. Dyrknings, prøvetaking, avbildning, og bildeanalyse er fullt automatisert, slik at dynamiske responser i fluorescensintensiteten og cellulær morfologi av celler samplet fra kulturen avløpet måles over flere dageruten brukerens input. Vi demonstrerer nytten av denne dyrking apparat ved dynamisk å indusere proteinproduksjon i en stamme av Saccharomyces cerevisiae konstruert med et optogenetic system som aktiverer transkripsjon.
Introduction
Optogenetic systemer bruker lys til å styre en voksende liste av cellulære prosesser, inkludert genekspresjon, 1, 2, 3, 4, 5 protein lokalisering, 6 proteinaktivitet, 6, 7, 8-protein binding, 8, 9, 10 og proteinnedbrytning. 11 En fremgangsmåte for dyrking av celler i et kontrollert miljø med programmert optisk stimulering og for måling av deres respons over biologisk relevante tidsrammer er nødvendig for å utnytte potensialet til disse verktøy for forskning i cellebiologi og bioteknologi. Vår metode utnytter chemostasis å opprettholde en konstant cellevekst i en godt blandet, aevurdert, og temperaturregulert glassdyrkningsbeholder 12, 13 som er eksponert for programmert belysning. Vi bilde individuelle celler i kultur avløpet med et invertert mikroskop for å måle responsen av kulturen til programmerte belysning. Dyrknings, prøvetaking, avbildning, og bildeanalyse er helautomatisk, slik at det fluorescens-intensitet og cellulær morfologi av avløpet cellekulturen kan måles over flere dager uten at brukeren må.
Denne protokollen kan gjennomføres i de fleste laboratorier er kjent med økende cellekultur og mikroskopi, og apparaturen som anvendes er billig og fremstilt av lett tilgjengelige komponenter. Et gjennomsiktig dyrkningsbeholder plassert over en matrise av lysemitterende dioder (LED) som er i stand til å utsende en μW / cm2 -10 mW / cm 2 av lys. Mikrober dyrkes i dyrkningsbeholderen kontinuerlig; en peristaltiske pumpen brukes til å legge til media påfortynningshastighet, en annen brukes til å trekke tilbake kultur med en mindre hastighet til mikroskopet, og forskjellen slipper ut gjennom en overstrømsutløp. En varmepute opprettholder temperaturen. Luft blir kontinuerlig pumpet inn i dyrkningsbeholderen for å opprettholde et positivt trykk, så vel som å blande og lufte kulturen. Med unntak av luftpumpen, blir strømmen til disse enhetene regulert av en mikrokontroller som også mottar input fra et termometer og en tilkoblet datamaskin. Avløpet cellekultur pumpes til en mikrofluid enhet på scenen av et invertert mikroskop. Non-fluorescerende og fluorescerende bildene er ervervet automatisk. Cellene i bildene er kjennetegnet ved en algoritme som lokaliserer hver celle som en region av interesse (ROI) og måler egenskapene til hver ROI.
For å demonstrere en anvendelse av denne protokollen, vi målte respons til varierende lysintensitet av Saccharomyces cerevisiae celler konstruert med et blått lys responsive optogenetic system som styrer transkripsjonen av fluorescerende protein. S. cerevisiae, vanligvis kjent som bakegjær, ble valgt på grunn av flere optogenetic systemer for styring av genekspresjon i dette system som allerede eksisterer 14, 15, 16. Videre er dette modellorganisme som vanligvis benyttes for studier i systembiologi 17 og som et understell for bioteknologiske anvendelser 18, 19, 20. Våre representative resultater viser at denne protokollen kan brukes til å styre transkripsjonen av en kultur i løpet av flere dager ved å variere inngangs lysintensiteter og måle produksjonen av et fluorescerende reporter.
Protocol
Representative Results
Discussion
Vi laget denne apparat med tanke på fleksibilitet. All kode som brukes er gratis og åpen kildekode. Standard bildeanalyseprosessen å segmentere celler er enkel og går raskt. Custom analyse kan gjennomføres ved å registrere brukerundersøkelser mens analysere et representativt bilde med FIJI grafisk brukergrensesnitt, konvertere innspill til en beanshell script, og deretter sette plugin for å ringe manuset. Når det er kalt, vil dette skriptet bli sendt en String utvalg kalt "bilder" som inneholder filba…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi ønsker å takke Molly Lazar og Veronica Delgado for å få hjelp i å teste protokollen, Kieran Sweeney for nyttige diskusjoner og redigering, og Taylor Scott, My An-adirekkun, og Stephanie Geller for kritisk lesing av manuskriptet. Megan Nicole McClean, Ph.D. har en karriere Award på Scientific Interface fra Burroughs Wellcome fondet.
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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