Conclusion

Our Verification

The work we have done in T.atroviride enable us to draw the conclusion that we have managed to do genetic manipulation in it and the Homologous Recombination Kit makes it much easier. It is worth mention that we have verified the function of Pech42 during mycoparasitism, which means it may be possible to utilize Pech42 as a sensor for phytopathogens.

Our Device

Besides, we have built up a device with multiple functions, such as VOC sensing, environmental index detection and message-passing. For message-passing, there are two alternative pathways, chemical transduction and medium wave transduction. After finding out the present of phytopathogens, the former pathway releases DAPG and the latter gives out medium wave.

Our Signal Transduction

Generally speaking, we have finished the preliminary verification of both chemical signal transduction and medium wave transduction.

For chemical signal, we succeeded in producing DAPG and building up the phlo-phlf system. Though our tests is done in lab, promisingly field trial will turn out well since we can use our engineering bacteria to produce enough DAPG to operate the engineered T.atroviride and lower the cost in addition.

For medium wave transduction, we have done even more. We managed to construct the TRPV-Ferritin system and verified whether everything worked and tested the whole system. The only thing uncertain is its function hasn't been tested in T.atroviride due to lime limit. Even though, for fear that it should fail to work in T.atroviride, we have made detailed experimental plans, which go like this:)

Future Development

Though we have demonstrated that TRPV1 could induce enough calcium influx to activate our CDRE promoter in yeast, we cannot conclude whether TRPV would fail to induce enough Ca²⁺ for T.atroviride promoter. AtTPC1 will help. As a calcium-permeable channel activated by depolarisation, AtTPC1 will respond to the inflow of Ca²⁺ and open, inducing more Ca²⁺ and exaggerating the signal until the downstream gene responds. To realize that, we plan to build up a Ca²⁺ signal system consists of XYR1, CRZ1, ACE1 and corresponding Xyr1 promoter. All of them are from Trichoderma reesei Rut-C30. XYR1 is a transcriptional activator upregulated by Ca²⁺. CRZ1 is calcineurin-responsive zinc finger transcription factor 1, which could bind directly to the upstream regions of Xyr1 in response to Ca²⁺ with another transcription factor, ACE1 (The putative binding consensus site is 5'-[T/G] GGCG-3' or 50-GGGC[G/T]-3'). We will change the coding sequence with target gene, transduct the system into T.atroviride and do the verification work mentioned before.

If sadly TRPV cannot express in T.atroviride or the protein is of low activation, we will change it for some promoter of heat shock protein(Hsp family, such as Hsp70, Hsp23, etc). Then the heat produced by Ferritin would induce expression of the downstream gene, operating T.atroviride to respond.

What if Ferritin in T.atroviride produce too little heat for either TRPV or promoters of heat shock proteins? (Too bad!) There is still another way. For the problem caused by Phytophthora parasitica var. nicotianae, we have designed a response system consists of elicitin and NtTPC1. Elicitin is a kind of protein secreted by P.parasitica spp. to obtain sterol from plants, which elicits immunoreaction of plants by Ca²⁺ inflow through NtTPC1. We will introduce NtTPC1 into T.atroviride, enabling it to detect P.parasitica and respond. Since the concentration of Ca²⁺ changes, helped with Ca²⁺_-depend promoter mentioned before, T.atroviride will sense P.parasitica and respond. As a result, we can operate T.atroviride without external chemicals, avoiding weather influences or inconvenience.

Downstream

We have tried several downstream genes to produce the corresponding effects and taken Serine protease as an example to do some experimental verification, and it seems to turn out well.

See You Soon! We've tried our best this year. Looking forward to the Giant Jamboree!

Reference

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