Overview
Our project aims to solve the problem of cannabis addiction caused by ingestion of cannabis products.
Our lab's work focused on detecting the presence of Δ9-THC in the intestine and degrade it, and inducing the engineered bacteria to kill themselves and prevent them from leaking into the environment when the body no longer needs them.
Our model consists of four part: Detection, Quorum Sensing, Degradation, and Suicide module.
We developed an easy and reliable hardware for intestinal colonization and detection, we chose hydroxypropyl methylcellulose capsules to bury the frozen fungus powder. It has been proved both on the mechanism and the applicability.
As more and more countries legalize cannabis, protection and treatment of cannabis addiction are urgent and necessary, which heralds the broad prospects of our project.
Module function verification
Our lab work consists of three parts, connected to complete the verification of the platform function.
The first part is to detect the presence of Δ9-THC in the intestine. In the first part of the experimental proof, we verified that engineered bacteria can be induced to secrete C-phycocyanin by IPTG, while the modeling results shows that our engineered bacteria could sense Δ9-THC and then express C-phycocyanin as reporter protein.
The second part is to degrade Δ9-THC in the intestine. In the second part of the experimental proof, we successfully verified that our engineered bacteria can surface display CYP2C9, CYP2C19 and UGT1A3 under the induction of IPTG, and we expect to prove their activity in metabolizing the psychoactive Δ9-THC. As for whether our pathways can degrade Δ9-THC efficiently under Δ9-THC induction, the modeling results show that it works well.
In the third part of the experiment, we proved that arabinose of appropriate concentration can induce suicide in engineered bacteria.
We also designed a cold-triggered toxin system, preventing engineered bacteria from polluting the environment. The engineered bacteria were cultured at 28°C and 37°C for the same time, though the number of colonies was different, they failed to achieve the expected effect.
So we obtained two hypotheses by analyzing the experimental results and literature. We wanted to verify one of the hypothesis, so the engineered bacteria were diluted and coated on LB-Kanr solid medium and cultured at 28°C, and then a petri dish was taken every hour and placed at 37°C for a total of 8 hours.
As the outcome shows, we verify that all two colonies treated for less than 7 hours recovered compared to the control group grown in 28℃. And those treated for more than 6 hours suffer from proliferation inhibition to different degrees. Results of our current exploration are shown below in Fig.6. Later experiments of higher concentration will be conducted for further verification.
Model prediction
Before we determined our project design, we participated in the online meeting up hosted by ZJU-China, CPU_China, HiZJU-China, ZJUintl-China, ZJUT-China. During this meetup, we heard many excellent and eye-opening project ideas from the teams as well as advanced synthetic biology techniques, which inspired us a lot. In addition, we learned the importance of project design and experimental design, and reviewed more in-depth information to prepare for the subsequent progress of the project.
We use modeling to predicted the effect of sensation, degradation and suicide system. Since each module has its own unique biometrics, we developed different models to meet the theoretical and experimental requirements of each module. By establishing the model, we are able to validate the feasibility of our project design.
Application of hardware
After our extensive research, we designed a capsule as hardware. We took the the vacuum freeze-drying method as our living bacteria preparation form, and we chose the microcapsule form to coat the bacteria with hydroxypropyl methylcellulose as a container which has been proved to have certain feasibility and application prospect.
