Results

Overview

The aim of this project was to produce apigenin from naringenin via enzymatic catalysis using Flavone Synthase (FNS) through an optogenetic two-part system of plasmids pNO286-3 and pSR58.6. We wanted to test the hypothesis that the use of the CcaS/CcaR system would allow us to control the production of apigenin using green light as an activator. Once we knew that the plasmids worked, we tested the production of apigenin using cells engineered to contain the gene encoding flavone synthase.

Testing the Optogenetic System

The pNO286-3 and pSR58.6 plasmids were created by Dr. Jeffrey Tabor at the Department of Bioengineering at Rice University. We ordered the plasmids from Addgene as a bacterial stab and streaked them onto plates with the appropriate antibiotics. We also co-transformed them into MegaX E. Coli cells and grew the cells overnight exposed to green light at 520 nm.

Figure 1: Plates containing the pSR58.6 (left) and pNO286-3 (right) plasmids under UV light

Figure 2: Plate containing cells co-transformed with both pSR58.6 and pNO286-3 plasmids

An AMS ambient light sensor was used to measure the light emitted from the plates. Five plates of cells were grown in a 37°C incubator with 530 nm lights inside.

Plates grown:

pNO286-3
pSR58.6
Co-transformed pNO286-3 and pSR58.6 grown in green light
Co-transformed pNO286-3 and pSR58.6 grown in covered dish (dark)
Plate containing no cells, used as calibration for light sensor

Figure 3: Light sensor readings from plates containing optogenetic plasmids

Testing Naringenin Conversion to Apigenin

We wanted to design a test to determine the rate of conversion from naringenin to apigenin in the cells. To accomplish this, we transformed BL21 E. coli cells with our composite part, BBa_K2956000. This part consists of the J23100 promoter, ribosome binding site, and gene for flavone synthase.

To confirm that this part had successfully been inserted into the vector, the plasmid was digested with a single enzyme to linearize it, and then run on a gel. The length of the plasmid was determined using a GeneRuler 1 kb Plus DNA Ladder from Thermo Fisher Scientific.

Figure 4: Gel containing BBa_K2956000 in pSB1C3 destination plasmid

Three tubes of cells were grown in liquid media at 37°C:

Control: Untransformed cells
FNS A: Cells containing BBa_K2956000, without the addition of naringenin
FNS B: Cells containing BBa_K2956000, with the addition of naringenin

Over the course of the 10 hours, we took periodic measurements using ultraviolet-visible spectrometry to determine the OD600 values of the samples. As the cells entered the exponential phase of their growth, 1 mg of naringenin was added to one of the tubes, labeled “FNS B.” We decided to add naringenin only after the cells started to enter this phase because we did not want to waste the naringenin if the cells did not grow.

After the addition of naringenin, the samples were measured every 30 minutes for OD600 as well as naringenin levels. Gas chromatography was used to measure the amounts of naringenin in the samples.

Figure 5: Fitted diagram of FNS B cell growth over 10 hours

Figure 6: Conversion of naringenin to apigenin in FNS B, measured by gas chromatography

Figure 7: GC results of collected samples. Naringenin peak is circled

Inserting FNS into the Optogenetic System

After collecting results that confirmed the efficacy of the CcaS/CcaR system as well as the functionality of our FNS part, we wanted to design a construct that would allow us to have optogenetic control of the FNS gene. We attempted to use 3A Assembly to ligate the different parts together; unfortunately, DNA gel electrophoresis revealed that the ligation had failed, as different colonies produced different bands on the gel.

Figure 8: Results of gel electrophoresis of different colonies of CcaR ligated with FNS. All bands are produced from samples of the CcaR-FNS except for the third from the left, which was the GeneRuler 1 kb Plus DNA Ladder

Although our attempt to ligate the parts together using 3A Assembly did not succeed, we thought that it was important to document the results of the attempt on our wiki to ensure that future teams do not make the same mistakes that we did in ligating the parts together.

Final Bioreactor Prototype

The final prototype of our bioreactor has all of the components necessary to keep bacteria alive. The reactor has a temperature control system that keeps the bacteria at 37°C, a constant input of filtered air to supply the bacteria with oxygen, and an impeller to agitate the bacteria and distribute oxygen and nutrients. The reactor also features waterproof LEDs to take advantage of our optogenetic system.

Figure 9: Final bioreactor prototype

The reactor is controlled by a Raspberry Pi 4 computer attached to an Arduino microcontroller. The Arduino Serial Monitor allows the user to input commands and control the reactor operations.

Figure 10: Reactor control system