Overview
After we sent a cooperation invitation from iGEM communication platform in China, we received a response from ZJUintl-China. Their project focuses on using CAR-T to target the killing circuit of senescent cells to delay the aging of the body, and to modify the CAR-T initiation pathway and negative feedback regulation circuit. Since our projects are all related to chimeric antigen receptors, we hope to carry out continuous and in-depth cooperation and become good partners. After preliminarily understanding the projects of both parties, we cooperated in the four parts——circuit optimization, dry experiment，education and cost analysis.
Circuit optimization
During the online communication with the team of International Union Department of Zhejiang University, we fully understood each other's project content and promotion process, and put forward valuable suggestions. In the initial construction of the plasmid, they found that we were using three plasmids, which would make transduction inefficient, and suggested that we find a way to combine the two plasmids to improve the efficiency. We took their suggestion and constructed a new plasmid by reviewing the literature.
At the same time, we also made a great contribution to their project. They encountered a problem with poor transfection results, namely, IL6R and CAR were simultaneously transfected on the same circuit, resulting in a single positive IL6R. After discussion and analysis, we found that the promoter of IL6R was MND and the promoter of CAR was SV40. The effect of MND was much greater than that of SV40, leading to abnormal transfection. We pointed out the problems with these two promoters, and they worked on them.
They redesigned two schemes: 1. The promoter of the CAR gene was changed to the more active p2A. 2. The promoter of IL-6R gene was changed to CMV to enable the normal expression of CAR.
The modeling part
UniProt webscraping
After learning about our project, ZJUintl-China found that the number of cell library after negative selection was still very large, and it could not guarantee that CAR-NK library injection would not cause any damage to normal human somatic cells. They suggest that we have only used bioinformatic methods to screen out genes that are highly expressed in cancer cells for specific target proteins. The red part on the right of the volcano map shows the genes highly expressed in tumor cells, which can be extracted for further screening. But it is not certain whether the gene expression products can be used as targets. And then they recommended UniProt webscraping to us. UniProt Scraping is a web scraping tool that specialized for UniProt Database.
It requires only the uniprot entry number of relevant protein. Using UniProt webscraping, we can automatically enter the gene names into Uniprot to obtain Uniprot ID, and then parse the subcellular localization of the gene products based on that ID. Finally, the genes expressed in the cell membrane were derived into tables. The binding affinity of specific CARs in CAR-NK library and target sites can also be predicted according to their expression levels, as well as the injury rate of normal cells due to a large number of CAR types. And based on that, we can get the target that localizes to the cell membrane and is highly expressed in cancer cells.
Modeling optimization
The ZJUintl-China team discussed with us the oscillations of the initial simulation results of the prey and predator model, and suggested that we add a logistic term to take into account the relationship between the environment and the cell population to make the model more comprehensive. The simulation results of the improved model are consistent with the real situation.
Education
After we take the invitation from ZJUintl-China. we worked with six teams, CPU-China, TJUSLS-China, HiZJU-China, ZJU-China, ZJUT-China and ZJUintl-China to compile tha altas of synthetic biology . This atlas systematically introduces the content of synthetic biology from the aspects of overview, technology, idea, crossover, application and outlook. In this way, we hope to promote the popularization of synthetic biology and let more people know about synthetic biology. Also, it is a good way to perform our educational method.
Cost analysis
In our initial conversation, we explained to ZJUintl-China that off the shelf is an outstanding feature of whack a mole, and it would be reasonably priced compared to the high cost of CAR-T customization. ZJUintl-China asked whether the cost of CAR-NK could be quantified to prove our point. They also said the final price of a patient's purchase is also affected by the market, patent and other factors.Finally，we decided that CAR-NK antibody libraries should be present in the form of information repository instead of cell therapy drug, which will contain sequences information library obtained from healthy individuals or NK cell library so we can save the patient’s cost due to market profit and market factors. And we also talked about what steps in building the antibody library can be completed before treatment for a single patient, such as construction of a sequence library, so that we can save the cost of the step for patient. After a series of investigations and inquiries, we completed the cost analysis.
After we completed the cost analysis, we fully shared with ZJUintl-China and helped them complete their cost analysis.
