Team:NYU New York/Design
New York University
Design
After hearing feedback from research scientists about the useful qualities of flavonoids and the lack of an efficient method to obtain them, our team decided to engineer E. coli to be able to produce flavonoids at high concentrations. The focus of our research was the optimization of flavonoid pathways using an optogenetic system. We also designed a 2-liter bioreactor that would grow the cells and activate the optogenetic genes. This is to demonstrate the ability of our process to be scaled up to industrial levels.
Gene Circuit Design
There are thousands of flavonoids, but for the purpose of our project, we focused on flavonoids that could be synthesized from naringenin. Naringenin is a colorless flavonoid found in a variety of plants and herbs. It is the precursor to many other flavonoids, and its conversion to another flavonoid can often be carried out with the expression of only one additional enzyme. Naringenin’s strategic location as an intermediate in the biosynthetic pathway provided us with a basis for the synthesis of other flavonoids. Figure 1 shows the range of flavonoids that can form from the reaction of naringenin with just three other enzymes; these secondary flavonoids can then be further converted using other enzymes. We saw this modular conversion model as a great starting point for our research.
Figure 1: Flavonoid Pathways Originating from Naringenin
We decided to focus on the production of apigenin because of its potential in the healthcare field. As shown in Figure 1, apigenin can be produced by the reaction of naringenin with the enzyme flavone synthase (FNS).
Figure 2: Constitutive apigenin-producing operon
Optogenetic control of genes is ideal because of the higher efficiency and yield that comes from this level of control that cannot be compared to any other genetic control system. The optogenetic system we chose to work with was a two-component system of the following plasmids: pNO286 and pSR58.6. These plasmids were designed by Dr. Jeffrey Tabor and we obtained them through AddGene.
Figure 3: pNO286-3 plasmid from Tabor lab
Figure 4: pSR58.6 plasmid from Tabor lab
Figure 5: Optogenetic system plasmids
This is a green-light-activated two component signal transduction system. CcaS is a cyanobacteriochrome sensor histidine kinase that has a photosensing cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF) region. When exposed to green light, CcaS goes into a conformation that phosphorylates the response regulator CcaR. CcaR is then responsible for binding to the output promoter which then activates downstream transcription of sfGFP.
By utilizing the CcaS/CcaR system to produce sfGFP, we were able to demonstrate the efficacy of the green-light activated optogenetic system. We constructed a fully-functional bioreactor with green lights inside. Originally, we had planned to use it to grow cells that could produce naringenin constitutively and have green-light-inducible genes to convert the naringenin to apigenin once an adequate amount of naringenin was produced. However, due to time constraints, we were not able to complete the construction of the naringenin-producing operon. Thus, we created a part that could produce flavone synthase constitutively to demonstrate the potential of incorporating this gene into an optogenetic system.
Bioreactor Design
The bioreactor is a crucial element to any biochemical process in which enzymes or cell systems are used to manufacture chemicals. A bioreactor’s performance is dependent on the supply of nutrients, aeration, pH control, temperature control, and agitation. One of the simplest and most important bioreactor designs used today is the conventional stirred tank bioreactor.
Figure 6: Stirred tank bioreactor schematic
Industrial reactor vessels are made of stainless steel. The height to diameter ranges from 2:1 to 6:1. Reactor vessels are fitted with baffles to prevent central vortexing.
Figure 7: Baffle function
The width of the baffle is usually between 1/10th and 1/12th of the tank diameter. Generally, only 75% of the reactor volume is used as working volume. The remaining 25% of the volume is used for gas space.
We decided that our reactor would have 2L of working volume. Our design features a stainless steel tank with a height of 24cm and diameter of 12cm along with baffles that are 1cm in width.
To achieve adequate agitation, the Froude number must be greater than 0.1. The Froude number is a dimensionless number used to describe the mixing in a reactor.
Figure 8: Froude number equation. N is the agitator speed in Hz, Di is impeller diameter, and g is gravitational acceleration
Our impeller has a diameter of 8cm. This means that the impeller must rotate at a rate of at least 210 rpm. It is best to stay close to this lower limit as higher speeds would result in larger shear stresses that could be detrimental to the living bacteria.
Figure 9: Impeller design
For efficient mass transfer, a gas sparger is used at the air inlet. The exact design of the sparger is of secondary importance as the impeller will capture and disperse the oxygen.
The bacteria inside of the reactor must be kept at a constant temperature of 37°C. Industrial reactors use thermal jackets that flow warm water around the reactor vessel to heat it up. This is not practical for our reactor as we would have to hook up our reactor to a water line and engineer a system through which the water temperature is automatically adjusted. This would be very excessive and costly for our scale. Instead, we will use electric heating pads to keep our reactor at the desired temperature.
Sterility is a very important factor when designing a bioreactor. Severe contamination will result in low yields. In order to maintain sterility, air is filtered at the inlet and peristaltic pumps are used to transfer fluids into and out of the reactor. A check valve is used at the gas exit port to control the air flow such that gas may only exit the reactor. This would prevent any external air from entering the reactor.
The base and lid of our reactor are made of 3D printed ABS plastic. The design schematic is shown below:
Figure 10: Bioreactor design
Figure 11: Animated reactor design
Our bioreactor also uses LEDs to take advantage of our optogenetic system. Waterproof red and green LEDs are placed on the inside wall of the reactor vessel.
The reactor is controlled and operated with a Raspberry Pi 4 connected to an Arduino microcontroller. This allows us to automate the temperature regulation system and activate any electrical components by inputting commands on the Raspberry Pi computer.
Bioreactor Scale Up
When scaling up any reactor, certain dimensionless quantities must be kept constant. If two reactors of different volumes and designed such that all of the relevant dimensionless quantities are the same, the two reactors will operate identically. This is a fundamental principle of reactor engineering.
The following dimensionless numbers would have to be kept constant to replicate the conditions and functionality of our reactor.
Figure 12: Froude and Reynolds numbers. ⍴ and μ are the time averaged density and viscosity of the liquid contents respectively
The Froude number characterizes the mixing and distribution of nutrients in the reactor. The Reynolds number characterizes the fluid dynamics of the liquid contents of the reactor. The time averaged density and viscosity are used in this equation because the two values change with the cell concentration. The dimensionless numbers that deal with heat transfer are not included here due to the fact that industrial scale reactors would have a different temperature regulation system as we previously established.
Unlike other bioreactors, our reactor has lights that are used to take advantage of the optogenetic system engineered into our bacteria. This requires us to create a new dimensionless number to characterize the effects of the light and keep them constant when scaling up. We will define this number as the Hasan number (Ha), named after our team member who invented it. We will take a cylindrical shell volume with a height equal to the height of the reactor tank and wall thickness equal to the time averaged penetration depth (δ) of the light.
Figure 13: Defined illuminated volume of reactor
The Hasan number is defined as the ratio of this illuminated volume to the total tank volume. This ratio reduces to the following equation:
Figure 14: Hasan number. R is the tank radius
The penetration depth is the depth at which the intensity of the light falls to 1/e of its original value. It is a function of the intensity of the light and the physical properties of the liquid. Thus, this number would be can be kept constant when scaling up the reactor by changing the light intensity. The penetration depth is taken as a time averaged value due the fact that the liquid’s turbidity will increase with the cell concentration.
If all of these dimensionless numbers are kept constant during scale up, the performance of the large scale reactor can be predicted by the performance of our small scale reactor.
References
Lee, H., Kim, B. G., Kim, M., & Ahn, J.-H. (2015). Biosynthesis of Two Flavones, Apigenin and Genkwanin, in Escherichia coli. Journal of Microbiology and Biotechnology, 25(9), 1442–1448. doi: 10.4014/jmb.1503.03011
Najafpour, G. D. (2015). Bioreactor Design. In Biochemical Engineering and Biotechnology(2nd ed.). Retrieved from https://www.sciencedirect.com/science/article/pii/B9780444633576000067
Ong, Nicholas T & Tabor, Jeffrey J. (2018). A Miniaturized Escherichia coli Green Light Sensor with High Dynamic Range. A European Journal of Chemical Biology: Chem Biochem, Synthetic Biology and Bio-Nanotechnology. Vol 19, Issue 12. 10.1002/cbic.201800028
Park, Sangkyu & Ji Choi, Min & Yeol Lee, Jong & Kwang Kim, Jae & Ha, Sun-Hwa & Lim, sun-hyung. (2016). Molecular and Biochemical Analysis of Two Rice Flavonoid 3’-Hydroxylase to Evaluate Their Roles in Flavonoid Biosynthesis in Rice Grain. International Journal of Molecular Sciences. 17. 1549. 10.3390/ijms17091549.