Describe how and why you chose your iGEM project.
Describe how and why you chose your iGEM project.
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Our main goal is to produce CBD efficiently via Saccharomyces cerevisiae. To make CBD there are two substances required. These substances are Geranylgeranyl-PP(GPP) and olivetolic acid (OA). GPP is produced via terpenoid backbone biosynthesis and in this pathway, two rate-limiting enzymes are present. The first one is EC 2.3.3.1. This enzyme catalyzes the reaction where acetyl-CoA turns into HMG-CoA. acetoacetyl-CoA + acetyl-CoA + H2O = (3S)-hydroxy-3-methylglutaryl-CoA + CoA + H+
To overcome these rate-limiting factors, our team thought of overexpressing these enzymes by inserting them into the plasmid and transforming the yeast (H9A1V3 · H9A1V3_CANSA). By doing so the production of GPP increases.
The second substance is OA. This substance can be bought and put directly into the culture medium, but OA is very expensive (5g is approximately $60) thus we thought of producing OA in Saccharomyces cerevisiae. By doing thorough research we found that OA is made by adding Hexanoic acid to the medium and this will be taken in by the Saccharomyces cerevisiae. Hexanoic acid and CoA (present in the Saccharomyces cerevisiae) will react with the help of the enzyme, H9A1V3 · H9A1V3_CANSA, and produce hexanoyl-CoA (this substance is also expensive). (Hexanoic acid+CoA=Hexanoyl-CoA)
In total, 7 enzymes will be inserted into 3 different plasmids and these plasmids will be inserted into Saccharomyces cerevisiae. The enzymes that will be inserted are EC 2.3.3.10EC 1.1.1.34, H9A1V3 .H9A1V3_CANSA, EC 2 3.1 206, EC 4.4.1.26, EC 2.5.1.102, EC 1.21.3.8. Our main goal is to increase the amount and decrease the production of CBD by resolving the rate-limiting factors and producing the expensive substrates via other pathways.
The first step is to construct the plasmid in the E. coli. After this step is proved to be successful, the recombinant plasmid is then introduced into the Saccharomyces cerevisiae. The overexpression of the inserted genes occurs here. The reference material used for the method of extraction is from-
The sample is then treated with zymolase, followed by ethyl acetate/formic acid. Afterward, bead-beating is performed as an extraction method. The organic and inorganic layers are then separated by centrifuge and the samples will be extracted three times. The combine organic layers are then evaporated in a vacuum oven and the remainders will be resuspended in acetonitrile/H2O/formic acid. Then samples will be filtered with PVDF membrane. The extracted product is then analyzed via High-performance liquid chromatography with UV detection. Cannabinoids can be detected by diode array detection at 270nm.
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iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you thought about your project and what works inspired you.