Team:ZJU-China/Demonstrate
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Demonstrate
Overview
The general functions of our project:
Detecting the phytopathogens or the unhealthy situation of the plant by Trichoderma atroviride or our device.
Signal transduction and amplification
Expression of downstream genes
When our engineered Trichoderma atroviride meets phytopathogens, (take Phytophthora nicotianae as an example), some of report genes will be activated and give warning to our device.
How we prove it?
☑︎Cloned the ech42 promoter (the promoter can be elicited when Trichoderma atroviride meets phytopathogens) from Trichoderma atroviride and performed confrontational coculture to test its phytopathogen sensitivity.
Fig.1 The relative fluorescent intensity of the hyphae contain the plasmids within Pech42
Fig.2 The fluorescence variation before and after activating the ech42 promoter
Our device detects the change of VOC(Volatile Organic Compounds) released by plants and estimate whether our plants are infected.
How to prove it?
☑︎ We have constructed a classification model which can tell the health situation from the VOC they released.
Once the device can transmit the order to our engineered Trichoderma atroviride with chemical or electromagnetic signals.
Chemical signals：
☑︎ Cloned the phlABCD cluster and carried out the bio-synthesis of DAPG in E.coli.
☑︎ Constructed the plasmids for DAPG bio-synthesis in Saccharomyces cerevisiae and Trichoderma atroviride.
☑︎ Constructed pho promoter and expressed phlF repressor.
Fig.3 HPLC result of DAPG bio-synthesis in E.coli | Fig.4 Western blot of phlF protein
Future work
☐︎ Test pho-phlF system with DAPG
☐︎ Tested the function of phlABCD in Saccharomyces cerevisiae and Trichoderma atroviride and detect the bio-synthesis of DAPG.
Electromagnetic signals
☑︎ Expressed TRPV-Ferritin in Saccharomyces cerevisiae and tested the its function with heat shock and capsaicin.
☑︎ Constructed Pcdre-mRFP in Saccharomyces cerevisiae and measured the relative intracellular calcium content needed to activate CDRE promoter.
☑︎ Proved that the calcium influx induced by TRPV1 is strong enough to activate CDRE promoter.
Fig.5 Relative calcium content of differet groups | Fig.6 Relative fluorescent intensity of two groups
Future work
☐︎ Test TRPV1-Ferritin system with medium radio frequencies in Saccharomyces cerevisiae.
☐︎ Construct TRPV1-Ferritin-CDRE system in Saccharomyces cerevisiae and Trichoderma atroviride.
Once our Trichoderma atroviride has received the signal, the downstream gene will be expressed.
How to prove it?
☑︎ We managed to express a special Serine protases in Saccharomyces cerevisiae and tested its activity.
Fig.7 The values of OD253 increased with time
Fig.8 The values of OD253 increased with time and this solution did not add the PMSF.
These were the OD253 of the solutions after a period of reaction.The red one was the solution that did not add the PMSF; the blue one was the solution that added. Obviously, the OD253 of the former one is higher than the later one, so that, we could say that the yeast produced the serine protase successfully and effectively.
Future work
☐︎ Express this serine protase in T.atroviride.
☐︎ Search and express more downstream genes to equip our T.atroviride with more functions.
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