Overview
In this iGEM project, we have developed BI-BYE, a probiotic solution for the treatment of Fn-mediated colorectal cancer. We have also developed two hardware: the CRC prognosis kit for evaluating the therapeutic effect of BI-BYE, and the colorectal tumor-on-a chip (CRT-chip) for our experiment to simulate colorectal environment.
We have confirmed the negative effect of Fn on SW480, and the anti-tumor and anti-bacterial activities of LL-37, and the self-assembly ability of mFadA. These results have proven the rationality and feasibility of our design. Furthermore, with the guidance of dry experiments, we have built FRPs and Cytotoxic Peptides, and have provided preliminary evidence for their functions and activities at the molecular level. We have shown that FRPs have the ability to interact with their target molecules. In addition, DEH has been shown to have the ability of dual-targeting and dual-killing. We also explored the tumor killing mechanism of LL-37 and discovered that it is mainly caused by apoptosis and pyroptosis.
Because the amount of data and text of our experimental results is too large, we have organized this results collection for everyone to read.
Howerver, there is still a long way to go. Here are our future plans and out look.
Future Plans
First, we should test the hypoxia-responsive system and the suicide systems, and preliminary build functional plasmid and suicide plasmid. Next, we will fully characterize the engineered BL and verify its functions. We will complete colorectal tumor-on-a chip (CRT-chip) and employ it in the simulated colorectal environment test.
2. Improve Project Design
(1)Protein optimization
We will conduct more comprehensive and rigorous dry experiments to optimize our new basic parts. First, we plan to explore modify two targeting fragments (GL-BP and B-domain).Next, In previous wet experiments, the results of conditional cleavage validation experiments for DEH were suggesting that the current linker design is flawed. Therefore, we plan to improve the linker of DEH, such as changing the conditional cleavage conditions, and further evaluating the stability of DEH. In addition, we will try to replace FK-13 with other anti-cancer peptides and compare their killing activity.
(2) Gene circuit & logic gate optimization
As the design of the gene circuit is only at a theoretical level, we cannot predict the actual performance of them, so we expect to further optimize the gene circuit once it has been fully characterized. Additionally, we plan to conjugate PH-responsive gel [1] (a kind of nanogel that can carry drugs) with hypoxia-responsive system to construct a “AND” gate logic.
(3) Hardware improvementWe will complete CRT-chip, which would be an important hardware in the simulated colorectal environment test. Moreover, we plan to optimize Asymmetric RT-RPA Model, which can better simulate nucleic acid isothermal amplification, to improve the characteristics of CRC prognosis kit.
Outlook
We hope that after BI-BYE is perfected, we can conduct some preclinical experiments on mice to initially prove the safety and therapeutic efficacy of the solution. In addition, in the future, we hope to set up a biotechnology company whose core concept is pilus self-assembly, and develop a serious of cell-cell adhesion toolboxes, contributing to the synthetic biological field. CPU-CHINA (formerly CPU_CHINA) has participated in iGEM for many years, all of them had great results in Jamboree. Today, thanks to the competition, most of the former team members are dedicated to the field of synthetic biology.iGEM 2023 also left unforgettable memories for the CPU-CHINA team members as well!
