Bioreactor
Overview
As our primary product is a bacterially produced collagen-mimetic protein, it is essential that we can provide a safe and efficient place for our bacteria to grow. This bacterial home must maintain a precise balance of several variables and react to the changing environment of the bacteria in real time. In order to produce a large amount of bacteria without needing a giant growth chamber, we have chosen to create a continuous flow bioreactor. In essence, we supply nutrients and dilute basic solution to meet the needs of the growing E. coli, which produce our Scl2 protein. When existing E. coli have expressed the desired Scl2 protein, they are pumped out of the growth tank to be replaced by new generations of bacteria to continue the process. This whole process is monitored by a fleet of sensors, which connect to multiple support systems in order to optimize our protein production.
Hardware
Design ProcessOur initial idea involved engineering yeast (Saccharomyces cerevisiae) to express two types of eukaryotic collagen that would differ in structure and function by using varying intensities of UV light. By varying low intensities of UV light, we could stimulate the yeast to produce a certain variety of collagen, thus adding a tunability aspect to our project. This led to our first bioreactor design, which included a light control system inside the fermentation chamber that would differentially stimulate the bacteria as the light intensity changed. We then altered this design so that the light control system would be outside the fermentation chamber, allowing for uniform projection and reduced cleaning requirements.
Figure 1 First bioreactor design for eukaryotic collagen production
Figure 2 Altered version of initial bioreactor design with light control system surrounding the fermentation chamber
However, after further research into the production of eukaryotic collagen in yeast, we discovered that collagen’s length and rigid triple helix structure required export pathways not native to yeast. We decided this angle of the project was not worth pursuing due to this reason, as well as additional foreseen difficulties in getting the collagen to form a dense hydrogel in this manner. We shifted our project towards using a prokaryotic collagen-mimetic protein (Scl2), inspired by results from a scientific study conducted by Cosgriff-Hernadez et. al called Bioactive hydrogels based on Designer Collagens [1]. Scl2 protein is useful in that it provides the binding sites for the ECM proteins of mammalian cells, which can be engineered to directly influence the attachment properties and growth patterns of mammalian cells. Existing research has proposed this idea [1]. While this concept is being explored in current research, Collatrix by Cornell iGEM focuses on the development of spatial Scl2 protein distributions on the hydrogel surface. This process involves our gradient maker, which “prints” an Scl2 gradient that directs the growth patterns of different cells on the basis of ECM protein interactions.
Through this process we were able to find other bioreactor designs [2],[3],[4],[5],[6] to draw inspiration from. After receiving guidance from our previous iGEM lead, Rahul Rambhatla, we decided a chemostat bioreactor, which has continuous inflow and outflow, would best fit our needs. To complement this type of bioreactor, we decided to make it a continuously stirred tank reactor (CSTR) to establish uniformity of the nutrients, bacteria, bases, and other added mediums. To ensure optimal conditions for bacteria growth we would monitor the temperature, pH, and dissolved oxygen levels using sensors all powered by an Arduino.
Figure 3 Initial bioreactor sketch for prokaryotic collagen
Figure 4 Final CAD model of bioreactor
We learned from a previous iGEM group [4] that there was the chance of our bioreactor killing our bacteria, so we took precautionary steps to prevent this by sterilizing all of components with 70% ethanol and we chose lysogeny broth (LB) as our growth medium. Additionally, we were able to improve upon their design by creating a water bath, using a sous vide machine to adjust the temperature for different time intervals as needed, as well as peristaltic pumps that could add LB medium (feed) during specific time intervals and NaOH (our base), as indicated by the pH sensor.
Figure 5 Bioreactor implementation
For our bioreactor testing we ran the bioreactor with 10 mL of our cell culture and 1000 mL of the LB growth medium. Since we based our bioreactor design on continuous inflow and outflow, we set the feed to pump into the bioreactor every 3 hours, while simultaneously the contents of the bioreactor were being pumped out at the same rate. We then took concentration readings every 4 hours to see how much our bacteria grew. Unfortunately, in between two data collection points, our base, sodium hydroxide (NaOH), leaked out of the bioreactor. NaOH is “leak-seeking” and prone to spilling through cracks, which most likely indicates that there was a crack in the container holding the jars that we were not aware of. Since NaOH is very corrosive, this caused our electrical components to partially disintegrate, thereby making our bioreactor unusable.
Although we were not able to finalize our bioreactor data, we plan on recreating our bioreactor and re-attempting to observe bacteria growth. This opens the opportunity for us to improve upon our own bioreactor design. These improvements can potentially include optimizing our circuit schematic by using a printed circuit board (PCB) and designing a liquid-proof box to store and protect our electrical components. The PCB would ensure that the bioreactor would run most efficiently with a lower likelihood of an electrical component not working. Moreover, another potential route in the future would be to test our bioreactor out using different bases or growth mediums to see which best maximizes E. coli growth.
Physical
The core of the bioreactor is two separate half-gallon glass growth chambers, each containing a separate variety of E. coli. Leading into each jar are a series of sensors monitoring pH, temperature, and dissolved oxygen concentration. All of these sensors provide real-time feedback to the Arduino control system, which sends commands to multiple components in order to maintain homeostasis. The temperature is controlled by submerging the jars into a heated water bath, which responds to a thermometer inside of the bioreactors and maintains an ideal growth temperature of 37°C. Oxygenation is controlled by an aquarium air pump, pushing air into gas diffusion stones at the bottom of each tank. This setup attempts to keep the dissolved oxygen concentration as high as the solution can maintain, around 6.5 mg/L in the 37°C tank with our solutes. Three pairs of pumps controlled by an Arduino Mega serve various purposes; maintaining a pH of 7 inside each bioreactor by supplying a dilute basic solution of NaOH, supplying fresh nutrients with a solution of LB growth medium and proteins, and maintaining the total volume by pumping out fully-reacted cells filled with Scl2 proteins. Thin 3mm tubes are used to transport the feed solution and dilute NaOH, while thicker 6mm tubes are used to transport the finished cellular media out of the tank. Each tank is also equipped with a stirring rod that ensures proper distribution of nutrients and growth factors.
Electrical
Each of the bioreactor chambers consists of a pH sensor and a temperature sensor, and the right chamber additionally has an oxygen sensor.This oxygen sensor is assumed to accurately depict the dissolved oxygen level in both chambers, since the oxygen for both chambers is being provided by one aquarium air pump. All of these components are connected to an Arduino Mega, which is programmed to display the sensor readings and collect data on the different conditions of the bioreactor. Each chamber is connected to four 12V DC motors controlled by an L298N motor driver, one for the stirring rod, two for pumping feed and basic solution into the bioreactor, and the last one for pumping effluent out of the bioreactor. There are on/off buttons for each of the chambers, which have been programmed by the Arduino Mega to stop the motors as needed. The entire bioreactor is supplied by an external 12V 10A power supply.
Figure 6 Bioreactor circuit schematic (the 9V battery should be 12V)
In addition to our circuit schematic, we coded an Arduino Mega to control our bioreactor system, which can be found here. Our exponential feeding schedule is implemented by our control system, as is our individualized temperature and pH controls for our two bioreactor chambers. For ease of use, we included an LED display in order to reveal the temperature, pH, and dissolved oxygen data, in addition to on and off buttons used to shut down the entire bioreactor.
Miscellaneous
In the beginning stages of our design process, when we were planning on using eukaryotic collagen we had come up with a design for a 3D bioprinter that could be used to produce 3D collagen structures for biomedical applications, such as organ scaffolds.
Figure 7 Eukaryotic collagen 3D printer using UV projector to crosslink collagen layers
By using 3D printing and consecutively layering sheets of collagen on top of one another we could control the dimensions of the collagen in order to yield a much more consistent and effective product. In this design the yeast would be lysed by high intensity UV light, which would subsequently crosslink the collagen in place.
Resources
- 1. E. Cosgriff-Hernandez, M.S. Hahn, B. Russell, T. Wilems, D. Munoz-Pinto, M.B. Browning, J. Rivera, M. Höök, Bioactive hydrogels based on Designer Collagens,Acta Biomaterialia, Volume 6, Issue 10, 2010, Pages 3969-3977, ISSN 1742-7061, https://doi.org/10.1016/j.actbio.2010.05.002.
- 2. “Open Source Bioreactor.” Open Bioeconomy Lab, Open Bioeconomy Lab, https://openbioeconomy.org/projects/open-source-bioreactor/
- 3. Molloy, Jenny, et al. “Microbial Bioreactor.” Project Hub, Arduino, 19 Nov. 2018, https://create.arduino.cc/projecthub/open-bioeconomy-lab/microbial-bioreactor-d7f61b
- 4. “Bioreactor.” Oviita, 2020 iGEM Team Calgary, 2020, https://2020.igem.org/Team:Calgary/Bioreactor
- 5. “Bioreactor.” 2015 iGEM team: Aachen, 2015 iGEM Team Aachen, 2015, https://2015.igem.org/Team:Aachen/Lab/Bioreactor
- 6. “Bioreactor Design.” 2018 iGEM Team:Exeter, 2018 iGEM Team Exeter, 2018, https://2018.igem.org/Team:Exeter
Gradient Machine
Overview
The goal of our second piece of hardware is to create the bone-cartilage scaffold. After the formation of the hydrogels, we aimed to make a 3mm x 1cm x 1cm scaffold with the two hydrogels containing different binding motifs. To do so, we require a device with a controllable level of precision and ability to deal with the intricate structure of hydrogels. Our first thought was to build a dynamic mixing machine; however commercial versions were prohibitively expensive. Additionally, these machines seemed too complex for our purpose and not specific enough for the small magnitude of our scaffold. Therefore, we decided to create our own kind of mixer: the gradient machine. Its relatively simple 3D-printed design allows us to achieve two goals. First, the device is significantly less cost-prohibitive. Second, the device can effectively generate a gradient transition between collagen-mimetic proteins (customized Scl2 proteins) with different binding motifs.
Figure 1 Final version of the gradient machine
Hardware
The gradient machine resulted from many weeks of prototyping and reworking the initial design. This concluded with a device with the following parts: a base, two syringes, two threaded rods, an Arduino Uno, two Stepper motors, two Stepper drivers, a 12V 5A power supply, and a perf board. The base and two pusher blocks of the machine were 3D printed using ABS plastic. The base was designed to hold two threaded rods that would drive two blocks in order to push the syringes. These syringes contain the Scl2 protein in the uncrosslinked form. We idealized that, from a source, there would be some force that would drive the rods to press against the blocks connected to the syringes, thus creating an extruded product with 100% hydrogel A with nanoparticles at one end, 50% split in the middle, and 100% hydrogel B with no nanoparticles at the other end.The source would be the combination of the Arduino, Stepper components, and the 12V power supply. Through specific wiring, the two motors were connected to the drivers. In turn, these drivers along with the battery were wired to a breadboard and the Arduino. The other ends of the motor were attached to the screws, which as aforementioned is a part of the base of the device. The final step was to write computer code for the Arduino. The code would allow for the motors to run at specific intervals, which would result in the differential pumping of the syringes. After putting all the parts together, the machine went through rigorous testing in order to ensure the parts were solid enough to extrude various density hydrogels, and sufficiently delicate so as to not create bubbles or uneven gradients in the final product.
Design Process
We initially started our design process with an outline of the features we wanted our gradient machine to include. Our main goal was to create a gradient between two collagen-like hydrogels of differing viscosities [1]. Once our goals were stated, we began researching the current market options. Most commercial gradient machines and dynamic mixers cost hundreds to thousands of dollars [2],[3], prompting us to design our own cost-effective option. After sketching basic ideas, we quickly created an initial CAD model that included the primary elements we needed, without regard for scale or exact specifications (Figure. 2A). This allowed us to consult with the Wet Lab subteam and graduate student mentors to determine how to move forward with this element of the project.
Figure 2 Iterative CAD design of gradient machine body.
We determined that the m chambers holding the hydrogels should be a few milliliters in volume, prompting us to use commercially-available syringes as the holding chambers for our gels. Returning to the research phase, we found several commercial syringe pushers, as well as a selection of open-source 3D-printable syringe pushers. Once again, commercial options were prohibitively expensive [4], and all open source options were designed to push one syringe or multiple syringes all at the same rate [5]. This led us to develop our own machine with multiple syringes on a single base (Figure. 2B). After printing and testing this model, we came across several problems. The motors initially mounted were not powerful enough to drive the syringes at a high speed, and the syringe holders were not modular, limiting us to syringes of a single diameter. A second design was created (Figure. 2C), which was fitted for new motors, and included a modular syringe loading system to allow for the use of multiple different syringe models.
The gradient machine also needed to combine the two gels into a homogenous mixture after extrusion from the individual syringes, leading to the development of a mixer head (Figure. 3). This piece of equipment acted to connect the two tubes coming out of each syringe, and using a complex internal geometry mix the two gels into a homogeneous fluid that is then extruded as the final gel.
Figure 3 CAD model of gradient machine’s mixer head.
Prior to testing our physical machine, we modeled our machine’s mixer head to ensure even mixing of our two gel types. We then proceeded to run physical tests. Although we were unable to test our gradient machine with collagen hydrogels and Scl2 hydrogels due to time constraints, we successfully implemented our product using gelatin hydrogels and Jell-o made with Mountain Dew Kickstart (for visual contrast purposes).
For our first test run, we manually moved the slide as the gradient machine was extruding the gelatin and Jell-o hydrogel to see how well the motor-syringe combination would work.
Figure 4 Gradient machine syringe-mixer head setup
Figure 5 First trial run gradient hydrogel
Figure 6 Gradient hydrogel in mold
As a final test, we ran the gradient maker extruding the gelatin hydrogel in one syringe and the gelatin hydrogel infused with nHAP particles in the other syringe on both a slide (for visual understanding) and the mold (for replicating a realistic production process).
Figure 7 nHap and gelatin gradient hydrogel
Resources
- 1. “Gradient Mixer Size 50 ML: Sigma-Aldrich.” Sigma-Aldrich, https://www.sigmaaldrich.com/US/en/product/sigma/z340413?gclid=CjwKCAjw2bmLBhBREiwAZ6ugo5ZPMDSj0UyBQ7pNAkZP1PHI9raajU659LUbVgMzOdw28Tqxf3YuZxoCA8AQAvD_BwE.
- 2. “Gradient Mixers.” Cleaver Scientific, 19 Oct. 2021, https://www.cleaverscientific.com/electrophoresis-products/gradient-mixers/.
- 3. Research Syringe Pumps, SouthPointe Surgical Supply, https://www.southpointesurgical.com/infusion.aspx.
- 4. Sant, Shilpa, et al. “Biomimetic Gradient Hydrogels for Tissue Engineering.” Wiley Online Library, John Wiley & Sons, Ltd, 9 Nov. 2010, https://onlinelibrary.wiley.com/doi/full/10.1002/cjce.20411.
- 5. Wijnen, Bas et al. “Open-source syringe pump library.” PloS one vol. 9,9 e107216. 17 Sep. 2014, doi:10.1371/journal.pone.0107216