Team:NUS Singapore-A/Hardware/Pump
Introduction
Our hardware team developed two sets of hardware to address two problems in synthetic biology, and complement the work of the wet lab team to complete our optogenetic biomanufacturing platform.
Problem #1
The first problem is that while there is a rapidly-growing interest in using optogenetics for biomanufacturing, development of custom tools to support the research of optogenetic circuits cannot match this pace, and is insufficient to meet user needs[2]. An example of the most current hardware tools available is a modified Tecan microplate reader, which provides controlled illumination on top of its usual measurement capabilities[3]. Such an approach is costly and requires specialized knowledge of the microplate reader model. Another example would be the open-source light exposure tool constructed for a 24-well plate[4]. To our team, it seemed that scaling-up in optogenetic research (Figure 1) was not well-supported by current hardware solutions, which only cater to microwell plates.
Yet, the biomanufacturing industry is expected to deliver products to the market, in high volumes, at high quality, and at competitive prices[5]. If we are ever to bring our optogenetic biomanufacturing platform to an industrial scale, it is necessary to bridge the gap between the microplate and the industrial bioreactor, and adapt our cells for actual large-scale bioreactor conditions. We thus designed a suite of three devices, called PDF-LA!, which enables the characterization of optogenetic circuits at different scales - 12-well Plate, petri Dish, and conical Flask. We also created a bench-top optogenetic bioreactor, Light Wait. It is our vision that these devices will empower optogenetic researchers to make great leaps forward in their research, although we acknowledge that there is a still-greater leap between our humble bioreactor and an industrial bioreactor (Figure 2). For now, it is enough for us to have taken the first few steps.
Problem #2
The second problem is that although a proof-of-concept already exists for optogenetic biomanufacturing, the process can be further optimized to bring the vision of an industrial-scale optogenetic bioreactor closer to reality.
For some background, Zhao et al. have increased yield of isobutanol from yeast by using a blue light repressible system in a simple bioreactor, showing the potential of optogenetics in biomanufacturing[6]. However, they did not optimize the duration or intensity of blue light, instead shining blue light periodically. We discovered that dynamic regulation is a good method for optimizing biomanufacturing, because prioritization of growth and production can be achieved simultaneously. We distilled this observation from both literature[7] and our Human Practice activities. Dynamic regulation can be achieved through computer-assisted feedback control, and we found that Argeitis et. al developed automated optogenetic feedback control for precise and robust regulation of gene expression and cell growth[8]. So far this is the most recent and sophisticated feedback system for optogenetics. However after examining his method, we found that while his feedback control system was closed-loop, his physical system was open. Measurement samples were discarded as waste. This is not advantageous to biomanufacturing, as this will lead to much product being wasted, lowering effective yield.
To solve this, we combined the insights and design features from these two systems (Zhao and Argeitis) to create an automated, closed-loop feedback control system for Light Wait.
PDF-LA!
Function
Plate-Dish-Flask Light Apparatus (PDF-LA!) supports optogenetic research by allowing researchers to investigate cells cultured in 12-well plates, petri dishes, and Erlenmeyer flasks.
Product Demonstration
The utility and functionality of PDF-LA! was validated by user feedback. We also used it when characterizing the behaviour of EL222 in repressible and inducible systems, thus producing what you see on our Results page.
How it Works
Operation
This operation guide assumes that all electronics have been assembled and programmed. Ensure that this has been completed before operation, else results may vary. Instructions may be found on our dedicated page for PDF-LA!.
- Place your container into the required holder. If using an Erlenmeyer flask, first rest the flask on D-LA!, then place the flask adapter over the flask to form F-LA!. Keeping a firm grip on F-LA!, pull the flask upwards sharply to ensure a tight fit.
- Connect the AC adapter to the Arduino and wall socket.
- Turn on the wall switch controlling the AC adapter.
The devices should light up as shown in the product demonstration video above.
Possible Configurations
DF-LA! was designed with modularity and flexibility as fundamental guiding principles. Many configurations are possible, enabling researchers to customize their experimental setups to a greater degree. While P-LA! was designed separately and thus does not have this functionality, a final solution, PDF-LA! 2.0, to provide a truly integrated solution was designed and can be found on our dedicated page for PDF-LA!. Unfortunately, while we could not actualize this solution due to time constraints and limits on our 3D printing equipment, it is our hope that future iGEM teams may be able to experience, test, and improve PDF-LA! 2.0’s utility and functionality.
Examples of the possible configurations can be found below.
- 1 x D-LA!, bottom illu, bottom and top illu
- 1 x F-LA!, bottom illu, bottom and top illu
- N x DF-LA!, bottom-bottom and bottom and top illu
Components
P-LA! comprises a tech holder and a lighting plate
Collectively, a single unit of DF-LA! comprises a tech holder, a petri dish illumination column, and a flask adapter.
Presenting, PDF-LA! Click here for its dedicated page.
Light Wait
Function
Light Wait supports optogenetic research, especially in optogenetic biomanufacturing, by allowing researchers to scale up to a 500 ml working volume bioreactor.
Product Demonstration
Validation
Light Wait was validated through a series of experiments which first proved each component’s functionality, and then the functionality of the whole system when all the components were assembled.
Experimental Plans
Experimental Results
How it Works
This operation guide assumes that all components have been assembled and programmed. Ensure that this has been completed before operation, else results may vary. For instructions on how to set up and operate each component of Light Wait, please refer to our dedicated component pages.
Operation
- Place Light Wait in a shaking incubator unit as shown in our Product Demonstration. Take care to ensure that all wires and tubing are slack and of sufficient length, else they may become disconnected during operation.
- Fill and cover the fermentation chamber.
- Connect the pump, 2-in-1 sensor, and the fermentation chamber with the silicone tubings in a loop as shown below (Figure _). The remaining 2 small tubes are for introducing more media, and an air pump.
- Turn on the AC adapters for the pump and the LEDs in the fermentation chamber.
- The pump should begin to rotate and the LEDs should light up.
- Connect the Arduino controlling the 2-in-1 sensor to your PC.
- Load the code for the 2-in-1 sensor and open the Serial Monitor to check that the sensor is collecting data.
- After verifying that all the components are working to your satisfaction, close the shaking incubator door.
- When the OD reaches your target levels, the LEDs in the fermentation chamber will turn off. The green LED in the 2-in-1 sensor will also turn off, and the red LED will turn on. The default OD in the code is 0.6.
- When the fluorescence from the stress reporter reaches your predetermined value indicating that the cells are stressed, the LEDs in the fermentation chamber will turn on again.
- When the fluorescence from the stress reporter reaches your predetermined value indicating that the cells are NOT stressed, the LEDs in the fermentation chamber will turn off again.
- Steps 7-8 will repeat indefinitely, unless you power the system off.
Components
Light Wait comprises a peristaltic pump, a 2-in-1 OD and fluorescence sensor, and a fermentation chamber. Click on the picture of the component to be taken to its dedicated page!
Abs600 |
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Fluorescence |
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Abs600 |
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Fluorescence |
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CONFIGURATIONS
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Bio-production
It’s important to have automation in bioproduction especially in industrial level. We designed a small bioreactor system which incorporated optical density (OD) and fluorescence sensors to control the metabolic behaviours in E. coli.
Automated Control through feedbacks
IN progress.
OD/F sensor
IN progress.
Pump
IN progress.
HEAR OUR STORY
We’re using synthetic biology to change the way the world thinks and functions.
We’re a little disruptive, dangerous and maybe a tad too bold. But the world needs some shaking up every now and then, and we think we’re just the right people for that.
Together we’re going to engineer biology and create something awesome.
CONTACT US
nusigem@gmail.com21 Lower Kent Ridge Rd, Singapore 119077