In order to carry our eukaryotic protein RTAC, we decided to use yeast as biosafe carrier. After doing research and interview, we decided to use the commensal fungi, Saccharomyces Cerevisiae, because of its broadly used in food production, which represents its satisfactory biosafety to human.
Since RSPO is required for human tissue homeostasis, we implanted the self-tunable switch in R-yeast to allow the specific expression of RTAC only in response to tumor microenvironment. In the sense, RTAC expression is switched on and the level of RSPO can be mitigated to stop the cancer progression at the tumor tissue locally. Low theoretical toxicity exists and minimal threat is posed to normal tissues.
Considering both iGEM’s Release Beyond Containment and the future use of our project (probiotic product), it is necessary to kill our engineered yeast in the environment.
Since the concentration of copper ions is enriched in the intestine and is much higher than that in the environment, we designed a kill switch in the yeast by replacing the original promoter of HSP gene (an essential gene in yeast) with a CUP1 promoter (a copper specific promoter). See more in “Design”
We have demonstrated the function of our kill switch.
See more in “Results”
We need secreted proteins (4 Rspos, RTAC, etc.) during our project. Thus, we need to use mammalian cell such as HEK293 to produce and collect these proteins by plasmid transfection.
There are two transfection methods: transient transfection and stable transfection. Although stable transfection can save us time and energy, it requires to use Lentivirus which is hazardous when operating. Therefore, we chose to use transient transfection instead.
At the very start of the competition, we have accepted basic training of doing experiments in the laboratory and biosafety in synthetic biology. However, to learn more knowledge about the specific experiments we were going to apply and learn the risk is actually more important. Therefore, we have accepted proper trainings by Mr. Sheng Yang and Dr. Tao Wang who are both the laboratory safety experts of our college.
Transformation
Gene cloning
Bioinformatics
Competence cells
Different types of bacteria, yeast and mammalian cell lines
PCR and qRT-PCR
Biosafety cabinet using
Data handling
PCR
Western Blotting
All team members who will do experiments have taken both theoretical and experimental courses of Microbiology and Cell Engineering in the last year.
Theoretical course:
The principle of microbiology experiments such as:
Gram stain
Primer design
Transformation of bacteria and yeast
The choice of culture medium for bacteria and yeast
The Growth Law of microbiology
Factors influencing the growth of microbiology
Methods to kill microbiology
Resistance selection
Experimental course:
Streak plate method
Gene Cloning
Sequence query using bioinformatics
Observation by microscope
Gram stain
Neubauer Hemocytometry
Physiological and Biochemical Identification of bacteria
Extraction of protein from yeast
immunofluorescence
Theoretical course:
The principle of Cell Engineering experiments such as:
Cell culturing
Cell passage
Transformation of mammalian cells
The choice of culture medium for mammalian cells
Cell freezing and recovery
Factors influencing the growth of mammalian cells
Monoclonal antibody
Aseptic technique
Experimental course:
Cell culturing
Cell passage
Gene Cloning
Sequence query using bioinformatics
Observation by microscope
Physiological and Biochemical Identification of mammalian cells
Extraction of protein from mammalian cells
TOPFlash
MTT
After the meetup with LZU-CHINA and the interview of Professor Ding, we decided to use Saccharomyces Cerevisiae as our medicine carrier.
After communicating with SJTU-BioX-Shanghai, they advised us to consider whether it is necessary to kill the yeast in the environment. After that we decided to design a kill switch in yeast.
See more in “Collaborations” and “Human Practices”
In order to let more people know the importance of biosafety, we held debate competition and stressed biosafety when spreading synthetic biology.
See more in “Communication”
Biosafety level: BSL-2
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