Wet lab
Enhancement of the existing part
In order to achieve the effect of breaking the extracellular barrier of tumors and activating T cell-mediated cellular immunity, we constructed a plasmid named pET28A. The plasmid structure consists of Ptac (promoter) (BBa_K864400)-RBS (Ribosome Binding Site) (BBa_B0034)-HYAL2 ()-His tag ()-Terminator (BBa_K1897030)-Ptac (promoter) (BBa_K864400)-RBS(BBa_B0034)-Flab(BBa_K4833100)-Flag tag ()-Terminator (BBa_K1897030), which will minimize potential risks while improving the efficacy of immunotherapy.
Introduction of a new component
In order to specifically break down the extracellular “barrier” formed by excess TECM, we introduced a new component, HYAL2 (BBa_5512104), an enzyme encoded by a human gene that has the ability to break down the ECM to remove the extracellular barrier of tumors.
To ensure that these proteins are specifically released within the tumor without affecting normal tissues or organs, we introduced pTAC (BBa_K864400), His tag (BBa_), and RBS ribosome-binding site (BBa_B0034) as basic parts of the plasmid.
We inserted the tumor microenvironment targeting PpepT promoter and lactate-responsive PsodA promoter into the plasmid for integration into E. coli ECN1917, and by doing so ensured that the target proteins, Hy and FlaB, would be expressed only in the high-lactate environment of the TME. In addition, we introduced another cleavage system controlled by an arabinose manipulator (araC-PBAD) into the plasmid. Patients can stop this biological reaction by ingesting a certain amount of arabinose after taking the drug for some time.
We have obtained composite elements after connecting HYAL to other parts, which are designed to enable engineered bacteria produce Hy, which can broke the ECM of TME, and ultimately enhance anti-tumor immune system.
Dry lab
To abundance the result of our research as well as exploring deeply into the various aspects of the OSCC, we applied dry lab in our project. We downloaded the OSCC genome data from the GSE41613 in the GEO dataset. 5266 differential genes was identified using the R package Depseq2 (Fig 1).
The GSEA analysis showed the gene set was enriched in some biological pathway (Fig 2). For example, the gene set was significantly enriched in mononuclear cell differentiation. Monocytes are an important part of the immune system and play a complex role in the tumor microenvironment. In the tumor microenvironment, monocytes can differentiate into different types of cells such as tumor-associated macrophages ( TAMs ) and tumor-associated dendritic cells ( TADCs ). These cells affect the tumor microenvironment through a variety of mechanisms, including inducing immune tolerance, promoting angiogenesis, and helping tumor cell metastasis.
Subsequently, we focus on the p53 pathway in this gene set. The p53 signaling pathway is a very important regulatory network in cells. It plays a key role in cell response to DNA damage, cell cycle control, apoptosis, senescence, and cell metabolism. Patients with high level of p53 pathway show better survival comparing to those with low level of p53 pathway(Fig 3-4).
In the dry lab, we discovered information such as the differential expressed genes and enriching biological pathways regarding OSCC. Furthermore, we combined our research with clinical data, which may guide the clinical treatment of OSCC. In the future, we will expand our dry lab, making efforts to find out more pioneering results.
Educational Outcomes
We invite students into the biology laboratory, organize visits to the Biological Science Museum, conduct knowledge popularization activities, and engage in community outreach to promote cancer prevention and control knowledge. Through these efforts, we aim to introduce the allure of biosynthesis to those who have never been exposed to it.
Promoting biosynthesis in schools and raising awareness of cancer prevention in the community not only enhances the public's scientific literacy and health awareness but also fosters social support and collaboration. This, in turn, drives research and technological development, creating a society rich in scientific atmosphere.
The popularization of cancer prevention and control knowledge, along with research in biosynthesis, plays a significant role in enhancing human health, promoting scientific progress, and advancing new drug development. We hope that through continuous scientific research and social education, we can better address the global health challenge posed by cancer in the future.