Diseño e implementación de un sistema automatizado de Iluminador, cultivo y sistema de muestreo para Aplicaciones microbiana Optogenética
Summary
Se diseñó un aparato de cultivo continuo para uso con sistemas de optogenética para iluminar cultivos de microbios y periódicamente celdas de imagen en el efluente con un microscopio invertido. El cultivo, el muestreo, tratamiento de imágenes y análisis de imágenes son totalmente automatizadas para que las respuestas dinámicas a la iluminación pueden ser medidos durante varios días.
Abstract
optogenética sistemas utilizan proteínas codificadas genéticamente que cambian la conformación en respuesta a longitudes de onda específicas de luz para alterar los procesos celulares. Hay una necesidad para el cultivo y la medición de los sistemas que incorporan programados iluminación y la estimulación de los sistemas de optogenética. Se presenta un protocolo para la construcción y el uso de un aparato de cultivo continuo para iluminar las células microbianas con las dosis programadas de la luz, y automáticamente adquirir y analizar imágenes de las células en el efluente. El funcionamiento de este aparato como un quimiostato permite que la tasa de crecimiento y el medio ambiente celular para ser estrechamente controlados. El efluente del cultivo celular continua se muestrea regularmente y las células se imágenes de microscopía de múltiples canales. El cultivo, el muestreo, tratamiento de imágenes y análisis de imágenes son totalmente automatizadas para que las respuestas dinámicas de la intensidad de la fluorescencia y la morfología celular de células de la muestra del efluente de la cultura se miden a través de múltiples díassin intervención del usuario. Se demuestra la utilidad de este aparato de cultivo mediante la inducción de la producción de proteínas de forma dinámica en una cepa de Saccharomyces cerevisiae con un sistema de ingeniería optogenético que activa la transcripción.
Introduction
Sistemas Optogenética utilizan la luz para controlar una lista creciente de procesos celulares que incluyen la expresión génica, 1, 2, 3, 4, 5 la localización de proteínas, actividad de la proteína 6, 6, 7, 8 la proteína de unión, 8, 9, 10 y degradación de proteínas. 11 Un método para el cultivo de células en un entorno controlado con la estimulación óptica programada y para medir su respuesta en escalas de tiempo biológicamente relevantes es necesario para explotar el potencial de estas herramientas para la investigación en biología celular y biotecnología. Nuestro método se aprovecha de chemostasis para mantener una tasa de crecimiento constante de celda en un bien mezclada, aeclasificar y recipiente de cultivo de vidrio de temperatura controlada 12, 13 que está expuesto a la iluminación programado. Nosotros imagen células individuales en el efluente de cultivo con un microscopio invertido para medir la respuesta de la cultura a la iluminación programado. El cultivo, toma de muestras, imágenes y análisis de imágenes son totalmente automatizadas de manera que la intensidad de fluorescencia y la morfología celular del cultivo celular efluente se pueden medir a través de múltiples días sin intervención del usuario.
Este protocolo se puede implementar en la mayoría de laboratorios familiarizados con creciente cultivo celular y microscopía, y el aparato utilizado es barato y está hecho de componentes fácilmente disponibles. Un recipiente de cultivo transparente se coloca por encima de una matriz de diodos emisores de luz (LED) capaces de emitir 1 mW / cm 2 -10 mW / cm 2 de la luz. Los microbios se cultivan en el recipiente de cultivo de forma continua; una bomba peristáltica se utiliza para agregar los medios de comunicación en eltasa de dilución, otro se usa para retirar de cultivo a una tasa menor para el microscopio, y la diferencia se escapa a través de una salida de rebosamiento. Una almohadilla de calefacción mantiene la temperatura. Se bombea aire continuamente en el recipiente de cultivo para mantener una presión positiva, así como para mezclar y airear el cultivo. A excepción de la bomba de aire, la energía a estos dispositivos está regulado por un microcontrolador que también recibe la entrada desde un termómetro y un ordenador de sobremesa conectado. El cultivo celular efluente se bombea a un dispositivo de microfluidos de la etapa de un microscopio invertido. No fluorescente y las imágenes fluorescentes se adquieren automáticamente. Las células en las imágenes se caracterizan por un algoritmo que localiza cada célula como una región de interés (ROI) y mide las propiedades de cada ROI.
Para demostrar una aplicación de este protocolo, se midió la respuesta a diferentes intensidades de luz de células de Saccharomyces cerevisiae ingeniería con una responsa de luz azulve sistema optogenético que controla la transcripción de la proteína fluorescente. S. cerevisiae, comúnmente conocida como la levadura de panadero, se seleccionó debido a múltiples sistemas optogenética para controlar la expresión génica en este sistema ya existen 14, 15, 16. Además, este organismo modelo se utiliza comúnmente para estudios en la biología de sistemas 17 y como un chasis para aplicaciones biotecnológicas 18, 19, 20. Nuestros resultados representativos demuestran que este protocolo se puede utilizar para controlar la transcripción de un cultivo durante varios días mediante la variación de las intensidades de luz de entrada y la medición de la producción de un indicador fluorescente.
Protocol
Representative Results
Discussion
Hemos diseñado este aparato pensando en la flexibilidad. Todo el código utilizado es libre y de código abierto. El proceso de análisis de imagen por defecto en las células del segmento es simple y funciona rápidamente. Análisis personalizado podría ser implementado mediante el registro de entrada del usuario, mientras que el análisis de una imagen representativa con la interfaz gráfica de usuario FIJI, la conversión de la entrada a un guión beanshell y después configurar el plugin para llamar al script. Cua…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Nos gustaría reconocer Molly Lazar y Verónica Delgado para la asistencia en la prueba del protocolo, Kieran Sweeney útil para los debates y la edición, y Taylor Scott, My An-adirekkun, y Stephanie Geller para la lectura crítica del manuscrito. Megan Nicole McClean, Ph.D. posee una concesión de carrera en la interfaz de la Ciencia del Fondo Burroughs Wellcome.
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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