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Figure 1: Engineering cycle diagram of our project
Design
Figure 2: Snapgene diagrams of AgBS, αPS, βPS, LIS, MsLIMS, PaFS, PtPS, SaSS, SaSS-CYP736A167
Build
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Figure 3: Colony PCR results for all our samples
Test
0.5mM IPTG is added to BL21(DE3) to induce the terpene synthases expression. Through SDS-PAGE, we tested less than only two out of the five tested terpene synthases were be expressed in BL21(DE3).
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Figure 4: SDS-PAGE protein check gel electrophoresis result of AgBS, SaSS, PaFS, αPS
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Figure 5: Gas chromatography results of terpene production by E.coli(The results of α-Pinene is shown here as an example).
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Because less than half of the samples we tested expressed terpene synthases and no samples successfully synthesized terpenes. We needed to find a more efficient way to produce terpenes. Through discussions with our advisorss, we learned that E. coli contains very few of the key precursors for terpene synthesis and is not an ideal chassis for expressing terpenes. Cyanobacteria, although genetically complex to manipulate, are undoubtedly much better at expressing terpene synthases. Therefore, we decided to move on to use cyanobacteria, Synechocystis sp. PCC 6803, as our chassis in the next steps.
Design
Build
- Cyanobacteria Electroporation Transformation 0809
- Streaking Cultivation 0810
- Gel Electrophoresis 0803
Figure 6: Streak monocultures of transformed Synechocystis sp. PCC 6803 with terpene synthases genes AgBS, αPS, βPS, LIS, MsLIMS, PaFS, PtPS, SaSS, SaSS-CYP736A167
- Cyanobacteria Colony PCR 0811
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Figure 7: Colony PCR of AgBS, βPS, PaFS, PtPS, SaSS in Syn. PCC 6803 monocultures
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Figure 8: Colony PCR of αPS, SaS-CYP736A167, MsLIMS, LIS in Syn. PCC 6803 monocultures
- Culture of Monocultures in BG-11 Culture Medium in Erlenmeyer Flask for 2-3 Weeks at 30◦C in Light Bioreactor
Test
Through gas chromatography, we can see whether the products that our cyanobacteria produced are the same as what we engineered it to produce. Both PaFS and βPS were successful in producing the wanted terpenes. The others are still in the process of testing.
Figure 9: picture of induced Syn. PCC 6803 we engineered. The transparent layer at the top is dodecane, which can dissolve the terpene molecules, making them easier to test.
Figure 10: Gas Chromatography results confirming production of beta pinene and farnasene
Though we are able to produce the scent molecules, we are still unable to directly fulfill the purpose of our project, which is to relieve stress through aromatherapy, and to bring our project into people's daily lives. Part of it is because of the safety restrictions of the competition, but even more so is that we do not have a way to do it. From our HPs at our schools, we further confirmed that students face a lot of stress and would really like to have our prospective product in their daily lives. Therefore, we decided to build different bioreactors.
