Abstract
During this year, our team designed and characterized multiple new parts, including RNAi and plant immunity. In order to reduce the production cost of shRNA, we have designed a large-scale in vitro transcription device for shRNA as our hardware. Due to our desire to make bold innovations in the village of Agriculture and promote the development of this field, we have developed the China Industrial Standard for RNA Pesticide and established the Enterprise Alliance T.O.M.A.T.O. to provide ideas for the iGEM teams in the village of Agriculture. Finally, by documenting the difficulties we encountered during the wet experiment, we hope to assist other teams who want to do projects related to plant diseases.
Parts
1.Improve in RNAi
Based on the 6 target genes of Botrytis cinerea selected in the Design, we designed 12 shRNAs for RNAi experiments and characterized their effects after IPTG induced expression in E. coli HT115 (DE3). We initially screened out shRNA(CHSIIIa)-2 (BBa_K4653002), shRNA(cyp51)-2 (BBa_K4653004), shRNA(OAH)-1 (BBa_K4653005), shRNA(Pme1)-2 (BBa_K4653008) with better effect. In order to improve the molecular delivery efficiency of RNAi, we bundled BP100-(KH)9 (BBa_K3224003) with screened shRNAs and adding new data to this existing part. Then, we also wanted to strengthen the effect of a single RNAi molecule to reduce the production costs, so the shRNA sequence was connected in series to obtain a new bi-shRNA, shRNA(Box-survival) (BBa_K4653015), and bundled with KH9-BP100 for spraying, so as to verify the ideal effect.
| Number | Part name | Type | Description |
|---|---|---|---|
| BBa_K3324003 | BP100-(KH)9 |
Protein coding sequences
(Adding new data) |
The short peptides that can penetrate the cell membrane and deliver a wide range of cargoes into the cell. |
| BBa_K4653002 | shRNA(CHSIIIa)-2 |
shRNA (New basic part) |
The shRNA silencing chitin (CHS) of B. cinerea. |
| BBa_K4653004 | shRNA(cyp51)-2 |
shRNA (New basic part) |
The shRNA silencing ergosterol (ERG) of B. cinerea. |
| BBa_K4653005 | shRNA(OAH)-1 |
shRNA (New basic part) |
The shRNA silencing oxaloacetate hydrolase (OAH) of B. cinerea. |
| BBa_K4653008 | shRNA(Pme1)-2 |
shRNA (New basic part) |
The shRNA silencing pectin methylesterase (PME) of B. cinerea. |
| BBa_K4653015 | shRNA(Box-survival) |
shRNA (New basic part) |
The bi-shRNA silencing the genes that maintain the survival of B. cinerea. |
2.Plant immunity
At the beginning of our experiments on plant immunity, we found that there was very little literature and data available. In this project, we have characterized two immune inducing factors, and we hope that the recording of these factors will provide a reference for future teams planning to do plant immune-related content.
| Number | Part name | Type | Description |
|---|---|---|---|
| BBa_K4653102 | Flg22: From P. aeruginosa |
Protein coding sequences (New basic part) |
A 22-amino acid sequence from the N-terminal of flagellin from P. aeruginosa, a pathogen associated molecular pattern (PAMP). |
| BBa_K4653105 | BvEP |
Protein coding sequences (New basic part) |
Protein phosphomutase from Bacillus velezensis LJ02, an plant immune inducing factor. |
Hardware
We have designed and constructed an automated equipment for large-scale production of shRNA molecules. This effort seeks to make shRNA production more convenient while simultaneously lowering the related expenses. The equipment primarily comprises three peristaltic pumps, a reaction container, and a temperature control module. Our equipment has demonstrated the successful synthesis of shRNA molecules on a significantly larger scale when compared to traditional PCR instruments. This automated equipment presents a cost-effective, user-friendly, and scalable solution for shRNA molecule synthesis, thereby enhancing the accessibility and efficiency of RNA interference technology across diverse applications.
Figure 1.The sketch map of the hardware.
Figure 2.The physical picture of the hardware.
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In Agriculture Village
1.The Proposal of Chinese RNA Pesticide Industry Standard
In the process of our HP circles, we realize that there is a lack of regulations and laws for RNAi pesticides and a great imbalance between the policies of chemical pesticides and that of new biopesticides. So, we SZU-China write a review of the analysis of current regulations and laws for RNAi pesticides in China and draft a proposal for the better development of RNAi pesticides in China. Moreover, we do a overall and specific research about laws proposed by UN, EU, the United States and China and produce this review.
Our proposal: Through the research of laws, we find that there is no clear definition and classification standard for biopesticides. So, before the eruption of new products, we in haste need to launch related laws and regulations to ensure the safety and sustainability of this new field. Hence, our team propose China industrial standard for RNA pesticide (draft) better promote the development of RNA pesticides in China.
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2.From Unit to Union -- T.O.M.A.T.O
Moreover, to better lead the development, we actively cooperate with government, Chinese traditional pesticide companies, biotech companies and manage to unit them to found China biopesticide union——"T.O.M.A.T.O".
Regarding T.O.M.A.T.O-Technical Alliance (Technology-Oriented Modernization of Agriculture-Targeted Organization), we plan to push the cooperation between traditional pesticide companies and biotech companies. In this way, they can benefit each other with the former ones gaining motivation to transform and the latter ones gaining capital and markets to develop more tech.
Regarding T.O.M.A.T.O-Thought Alliance (Thought-Oriented Modernization of Agriculture-Targeted Organization), we intend to involve government and consumers in to better guide the production of companies and stimulate government's intention to assist the development of field. Also, by forming this union, consumers' demand can be better met.
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Troubleshooting
1.Plant immunity experiment
In the protein expression validation part of plant immunity, we induced the expression of BvEP protein under general induction conditions: 37 ℃, 200 rpm, induction for 4-8 hours. However, the BCA results showed very low protein expression. Therefore, we optimized the expression conditions and finally found that the optimal conditions for inducing BvEP expression were 25 ℃, 100 rpm, for 16 hours.
In the functional validation part of BvEP protein in plant immunity, we used the DAB staining to visualize the immune response triggered by the protein. We employed two methods for applying the protein: immersion and droplet application. The results showed that the immune response induced by immersion method was much higher than that induced by droplet application, even when the final protein quality was consistent.
2.B. cinerea infection experiment
(1) At the beginning, we scraped the mycelium from the B. cinerea plate with sterile forceps and dissolved it in a certain volume of sterile water. After vortexing, the mycelium solution was obtained for the tomato fruit infection experiment. However, during the experiment, it was found that the mycelium solution was difficult to mix, which made it difficult for us to quantify the B. cinerea and act on each tomato, so the tomato would have uneven incidence. Later, we used fungus cake to infect tomato fruits. Compared with mycelium liquid, fungus cake can quantify B. cinerea, and the residual medium on the fungus cake can keep the growth activity of B. cinerea in the early stage of fruit infection.
(2) The volume of RNAi solution dropped on tomato fruit should not be too large, as too much liquid is easy to slip on the surface of the fruit. At the same time, we added silwet-77 to the system (the volume ratio of total liquid to silwet-77 is 100), which can facilitate us to spread RNAi liquid on the surface of fruits and help fruits to better absorb RNAi.
(3) The tomato fruits after the experimental treatment must be moisturized, and the ambient temperature should not be too high (21-25℃ is more suitable), otherwise it is not conducive to the growth of gray mold and lead to the failure of tomato infection.