iGEM 2022 — Team szu-china

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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Notebook

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[Overview](#part-1)

[Teamlog](#part-2)

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# **Overview**

## **2021.12**

Set up the 2022-SZU-China team.

## **2021.12-2022.2**

Experimental skills training and literature reading training.

## **2022.2-2022.4**

Brainstorming.

Project discussion and literature research were carried out in groups.

The projects of each group are summarized and finally the topics to be explored by the team are determined.

## **2022.5**

The preliminary design of the complete project system and a large number of background research and literature research were completed.

The first new parts were designed and built for the project.

Some experiments were started.

## **2022.6-2022.9**

The second batch of new elements was constructed following suggestions from interviews with professionals and professors.

Feedback from experiments and human practice is used to continuously improve our project (click here to jump to
the Experiments and Human practice section).

The results of the vast majority of wet versus dry experiments covered by our project were obtained.

Write the first draft of the wiki.

## **2022.10**

Complete the final wiki version and upload it.

Complete the poster production.

All experiments were completed.

2022 iGEM Giant Jamboree!

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# **Teamlog**

## **Brainstorming and meeting**

**3.10**

**SZU-China Project Concept Conference**

Time: March 10 20:59-23:30.
Venue: Room 202, College of Life and Marine Science Building, Lihu Campus, Shenzhen University.
Minutes of the meeting: Xinying Zhang.

Highlights:

1. Discuss and determine the complete process of team brainstorming and establish the "three-step" strategy:

* Step 1. Release your thoughts and record them.
* Step 2. Search and organize a lot of data.
* Step 3. Initially select the project with the most development potential for further advancement, and finally
  select the most suitable one as our project after reviewing each pre-project from multiple perspectives.

2. Created a brainstorming summary sheet to record our interesting ideas.

**3.25**

**SZU-China Preliminary Topic Selection Conference**

Time: March 25, 21:00-23:00.
Venue: Room 202, College of Life and Marine Science Building, Lihu Campus, Shenzhen University
Minutes of the meeting: Xinying Zhang.

Highlights:

1. All members of SZU-China2022 introduced the project content and the specific design and investigation in turn.

* Bingru Feng: Early detection of plant related diseases.
* Bingru Feng: Oral diseases: prevention and adjuvant treatment of dental caries and periodontitis.
* Ruoyu Chen: Phage drug therapy for trachoma.
* Ruoyu Chen: Diagnosis of β-thalassemia.
* Yi Peng: Detection and degradation of microplastics in mangroves.
* Yi Peng: Rapid diagnosis and management of yellow Dragon disease in citrus crops.
* Xinying Zhang: Bacterial resistance and antibiotic resistance management.
* Xinying Zhang: Bioenrichment and degradation of industrial dust.
* Yingfeng Wu: Optimizing DNA-based data storage methods.
* Linzhen Bao: Detection of heartworm infection in pets.
* Xihe Wang: Treatment of cassava brown streak disease (CBSD).
* Minxi Zeng: Management of pine wood nematodes.
* Zhiying Zhang: The treatment of banana wilt disease.

2. After wonderful speeches and heated discussions, we finally confirmed the following four projects for further promotion:

Project 1: Detection of plant pathogens

Person in charge: Bingru Feng, Ruoyu Chen, Xihe Wang
Optimization direction: Technical research should be placed at the second-best level, and specific application scenarios
(pathogens of cabbage, banana and other plants) should be prioritized.

Project 2: Bacterial resistance and antibiotic resistance

Person in charge: Bao Linzhen, Zhang Xinying
Optimization direction: Application scenarios such as phage therapy

Project 3: Detection and degradation of microplastics in mangroves

Person in charge: Yi Peng, Yingfeng Wu
Optimization direction: To find a better technical route.

Project 4: Management of pine wood nematodes

Person in charge: Minxi Zeng, Zhiying Zhang, Yixi Wang
Optimization direction: Continue to deepen according to the original planning plan

**4.9**

**SZU-China Project Approval Conference**

Time: 20:00-23:50, April 9
Venue: Room 202, Life and Marine Science Building, Lihu Campus, Shenzhen University
Minutes of the meeting: Xinying Zhang

Highlights:

1. The members of the four teams submitted the project planning plan that they were responsible for promoting,
and gave project speeches in front of Advisor and PI in turn.

2. Under the joint decision of team members, Advisors and PI, we initially selected the new project "Early
detection, prevention and treatment of rice sheath blighty", which was extended from the original plant pathogen
detection project, as our preferred project. It will be further advanced in the coming week.

**4.16**

**Project Planning Meeting**

Time: 9:00-12:00, April 16th
Venue: Room 202, Life and Marine Science Building, Lihu Campus, Shenzhen University
Minutes of the meeting: Xinying Zhang

Highlights:

1. Project Discussion

* (1) Suggestions and preferences for the current project.
* (2) Rice sheath blight project of Rhizoctonia solani.

2. Discussion of the rules of the Synthetic Biology card game

3. Assignment of research tasks to various parts in the early stage of the project

* (1) Find ways to buy R.solani and order it; Find culture Protocol：Zhiying Zhang.
* (2) Vector, synthetic gene and Protocol in Trichoderma vermiliformis：Bingru Feng, Minxi Zeng, Linzhen Bao.
* (3) RNAi design and related implementation scheme：Ruoyu Chen, Yi Peng.
* (4) Problems related to electronic nose system：Yingfeng Wu, Xinying Zhang, Zhiying Zhang.
* (5) Whether the application scenario needs to be changed：Zhiying Zhang, Xinying Zhang, Yixi Wang.
* (6) Life history and appropriate action time points of Rhizoctonia solani in rice field (secondary emphasis)：Xihe Wang, Yixi Wang.
* (7) How to design the spraying time of RNAi and Trichoderma lucidum (whether there is mutual influence and how to
  maximize the effect)：Wang Xihe, Bao Linzhen.

**4.30**

**Preliminary Survey Summary and Experiment, HP Work Arrangement Meeting**

Time: 9:00-12:00, April 30th
Venue: Room 202, Science of Life and Marine Sciences Building, Lihu Campus, Shenzhen University
Minutes of the meeting: Xinying Zhang

Highlights:

1. Coordinate and arrange follow-up work with PI: start to cultivate all kinds of bacteria needed in May, synthesize
part of the designed components and biobrick, and define the complete technical route.

2. Early investigation and summary of the project.

* (1）Plasmid Design：The initial version has been completed and will continue to evolve iteratively.
* (2）RNAi research：Plants, bacteria RNAi uptake rate, spray modeling.
* (3）How to design the spraying time of RNAi and Trichoderma atroviride：no literature.
* (4）Experimental simulation of ethyl acetate volatilization：Explosion concentration test.
* (5）Toxicity (to rice), degradation and removal of ethyl acetate.

3.HP event planning:

* （1）iHP scheme: Construct IHP related framework of RNA part.
* （2）Rice greenhouses visit: Establish the time arrangement and the specific contents to be investigated.
* （3）To solve the problems related to the publication of the synthetic biology booklet prepared in cooperation
  with the team of Southern University of Science and Technology.

**5.4**

**Conference on Plasmid Design**

Time: 14:30-16:30, May 4th
Location: Room 202, College of Life and Marine Sciences Building
Minutes of the meeting: Xinying Zhang

Highlights:

1. Discussion on plasmid design:

* Overexpression of PRB1 (protease PRB1 gene).
* A protease Prb1 derived from T. Harzianum (IMI 206040) has been shown to be an effective biocontrol agent against
  a variety of pathogens. The transformation of this gene PRB1 in other Trichoderma strains increased the biocontrol
  potential of the transformed strain against R. Solani by many times compared with the wild-type strain.
* Overexpression of EPL1 (surface hydrophobic protein gene).
* Expression of antimicrobial peptide Snakin-1 (SN1) in Trichoderma atroviride.
* RNAi kills candidate genes of Rhizoctonia ruthenicum:
  (1) PMK1 homolog (RPMK1-1 / RPMK1-2) [two and four alternative shRNA molecules, respectively].
  Host-delivered RNA interference (HD-Rnai) technology was used to target two pathogenic MAP kinase 1 (PMK 1) homologs
  from R. solani, RPMK 1-1 and RPMK 1-2, using a hybrid RNAi construct. Homologs of PMK1 play an important role in other
  fungal pathogens for episome formation, fungal infection structures required to penetrate plant cuticle, and invasive
  growth once inside plant tissues and overall pathogen viability within plants.
  (2) PG (AG1IA\_04727) [There are two alternative shRNA molecules]
  Polygalacturonic acid (endonuclease) enzyme (PG) is a cell wall structural protein that catalyzes the cleavage of
  pectin molecule poly α-(1,4) -polygalacturonic acid, participates in the degradation of pectin, and disintegrates the
  cell wall structure. The synthesis of PG enzymes by pathogenic fungi is key to initiating the host infection process.

2. Discussion on the project experimental design scheme

* (1) The culture of Rhizoctonia orbicularis was started.
* (2) R-body (electron microscope +OD value), which was verified separately from RNAi.

3. Rice plant acquisition, field investigation and electronic nose system arrangement:

* (1) Rice greenhouses can be built in the base, after successful verification on rice plants.
* (2) Difficulty: It is difficult to grow rice from scratch in the culture room with constant conditions.

**5.8**

**Weekly team meeting and South China Exchange Conference to host seminars**

Meeting time: 9:00-12:00, May 8th
Venue: Room 202, College of Life and Ocean Sciences, Shenzhen University
Minutes of the meeting: Zhang Xinying

Highlights:

1. Weekly work report of each member.

2. Human practice, meeting up and Collaboration:

* (1) Liaise with other teams: Bingru Feng, Zhiying Zhang.
* (2) Social account operation and contact: Xihe Wang.
* (3) Human Practice: divide the work according to the type of activities and participation.

3. Discuss the overall issues of holding the Meeting up:

* (1) Confirm attendance list.
* (2) Exchange meeting process arrangement.
* (3) Invite guests to participate in comments related matters.
* (4) Making and planning of the 2022 conference manual of South China Exchange Conference.
* (5) Confirm whether it will be held offline.
* (6) The specific time will be finalized.

## **Experiment**

**5.28-5.31**

—Yi Peng: Amplification and culture of Escherichia coli containing pPIC-P-T empty plasmid; Small amount
extraction and verification of pPIC-P-T plasmid; Preparation and transformation of Trichoderma protoplasm.
—Xinying Zhang: Incubation and planting of the first batch of rice seeds.

**6.1-6.4**

—Bingru Feng: PCR of Rhizoctonia solani colony (preliminary experiment), failed. After investigation and analysis,
we planned to use TPS simple method and CTAB method to extract DNA of T. paraquat, and then perform colony PCR.

**6.6-6.9**

—Bingru Feng: pCAMBIA1302 was transferred into Escherichia coli BL21(DE3) and verified. It failed due to the failure of kanamycin.
—Xinying Zhang & Yi Peng & Yingfeng Wu: The second batch of rice sowing.

**6.10-6.12**

—Yi Peng & Ruoyu Chen: Determination of the growth curve of Rhizoctonia solani.
—Ruoyu Chen: Plate inoculation of Rhizoctonia Aquarius (Rotary plate method with hole punch).
—Ruoyu Chen: A culture of Rhizoctonia solani was inoculated into isolated rice leaves.
—Yi Peng & Xinying Zhang: Protoplast transformation of Trichoderma atroviride. (empty plasmid pPIC-P-T)

**6.14-6.15**

—Xinying Zhang: Trichoderma protoplast transformation (all processes were changed to ice).
—Xinying Zhang: The transformed Trichoderma suspension was inoculated onto resistant plates for screening.

**6.16-6.18**

—Xinying Zhang: Sowing the third batch of rice.
—Xinying Zhang: Activation and cultivation of the deep green Trichoderma preservation collection.
—Xinying Zhang: Observation of Trichoderma pseudotransformants on PDA plates containing hygromycin resistance.

**7.7**

—Xinying Zhang: Purification of Trichoderma atroviride, single colonies of Trichoderma atroviride were
obtained by dilution of colonies on bevel medium.

**7.16-7.19**

—Xinying Zhang: Plasmid Pan-7 and PPIC were extracted and prepared for the second transformation.
—Xinying Zhang: Preparation of Trichoderma protoplasts.
—Xinying Zhang: PDA plate inoculation of Trichoderma dark green.
—Ruoyu Chen: RNAi-associated plasmids were transformed into Escherichia coli DH5a and selected using a medium containing kanamycin resistance.
—Ruoyu Chen: Rhizoctonia ligulatum culture infected isolated rice leaves (optimized infection device).
—Ruoyu Chen: PDA plate inoculation of Rhizoctoromyces erectus.

**7.20-7.21**

—Xinying Zhang: Three plasmids designed for engineering Trichoderma were transformed into Escherichia coli DH5a for verification.

**7.22**

—Xinying Zhang: Validation of Escherichia coli DH5a transformants by colony PCR.
—Xinying Zhang: Washing and germination of spores of Trichoderma deep green.
—Linzhen Bao: Colony PCR for snakin-1 plasmid and plasmid PCR after plasmid extraction.

**7.23**

—Xinying Zhang: Three plasmids designed for engineering Trichotilbe were transformed into Escherichia coli DH5a
for verification (the second batch of plate).
—Xinying Zhang: Trichoderma spore count.

**7.24**

—Xinying Zhang: The spores germinated overnight were observed under microscope, and the germinated Trichoderma atroviride
were inoculated on PDA plates.
—Xinying Zhang: Determination of the relationship curve between spore OD550 and spore concentration of Trichoderma atroviride.
—Ruoyu Chen: Rhizoctonia litmus culture infected isolated rice leaves (infection observation device was optimized again).

**7.25**

—Xinying Zhang: Colony PCR validation of Escherichia coli DH5a recombinant (epl1, prB1, snakin-1).

**7.26-7.27**

—Xinying Zhang: Expanded culture of Escherichia coli DH5a recombinant (epl1, prB1, snakin-1).

**7.28**

—Xinying Zhang: Small volume extraction of engineered Trichoderma Plasmids.
—Xinying Zhang & Yi Peng: Agarose gel electrophoresis verification of small amount of plasmid extract.
—Xinying Zhang & Yi Peng: Preparation of reagents for mass plasmid extraction.
—Xinying Zhang & Yi Peng: Mass extraction of Plasmids from engineered Trichoderma atroviride.
—Xinying Zhang & Yi Peng: Validation of large amount of plasmid extract by agarose gel electrophoresis.
—Xinying Zhang & Yi Peng: Verification of double digestion of a large amount of plasmid extract.
—Linzhen Bao: Verification of double digestion of small amounts of plasmid extracts.
—Ruoyu Chen: Transformation of E. coli HT115(DE3) (RNAi partial plasmids: PG2, RPMK1-1-2, RPMK1-2-2).

**7.29**

—Ruoyu Chen: Retransformation of HT115(DE3) : RPMK1-1-1, RPMK1-2-1.
—Ruoyu Chen: Mix 50% glycerol with equal volume of bacterial solution and store at -80°C refrigerator.
—Xinying Zhang & Yi Peng: Mass extraction of plasmids from engineered Trichoderma fungi (Repeat 7.28 to prepare subsequent
plasmids for transformation) .
—Xinying Zhang & Yi Peng: PCR verification and double digestion verification of a large number of plasmid extracts.
—Xinying Zhang: Inoculate Trichoderma atroviride.

**7.30-7.31**

—Ruoyu Chen: Colony PCR of HT115 (DE3) transformants.
—Ruoyu Chen: IPTG induces microRNA production in Escherichia coli.
—Ruoyu Chen: Trizol method was used to extract Escherichia coli RNA.
—Xinying Zhang: Transformation of Trichoderma verticillum by carbon nanotube delivery method; Transformation of
Trichoderma atroviride by transmembrane peptide method.

**8.1**

—Ruoyu Chen: Colony PCR validation of HT115 (DE3) transformants transferred into RNAi-associated plasmids.
—Ruoyu Chen: IPTG induces a large number of micrornas in HT115 (DE3).
—Ruoyu Chen & Yi Peng: Large volume extraction of Escherichia coli RNA.
—Linzhen Bao & Wang Xihe: Small amount extraction of snakin-1 plasmid.
—Xinying Zhang & Linzhen Bao & Xihe Wang: Plasmid transformation into Trichoderma dark green by nanomaterial transformation method.

**8.2**

—RuoYu Chen & Yi Peng: IPTG induces HT115 (DE3) to produce a large number of micrornas.
—RuoYu Chen & Yi Peng: Large scale extraction of E.coli RNA.
—Xinying Zhang: Verification of Snakin-1 plasmid digestion.

**8.3**

—Xinying Zhang: Transformation of Trichoderma atroviride by carbon nanotube delivery method; Transformation of
Trichoderma atroviride by transmembrane peptide method.

**8.5**

—Ruoyu Chen: Chemical transformation of HT115 (DE3).
—Ruoyu Chen: Experiment on the infection of rice leaves in vitro by Rhizoctonia solani culture.
—Xinying Zhang: Transformation of snakin-1 plasmid into BL21 (DE3).

**8.6**

—Ruoyu Chen: Colony PCR validation of RNAi partial transformants.
—Yi Peng: Bulk extraction of PG-1 microRNA.
—Yi Peng: Stability experiments of complexes obtained from CNT coated micrornas.

**8.7**

—Ruoyu Chen: Bulk extraction of RNA from Escherichia coli.
—Xinying Zhang: Resuscitation and expanded culture of transformants stored in a refrigerator at -80°C (prb 1 and epl 1).
—Xinying Zhang: Germination of spores of Trichoderma atroviride.

**8.8-8.10**

—Xinying Zhang: Bulk extraction of epl 1, prb 1 plasmids.
—Xinying Zhang: Transformation of protoplasts from Trichoderma atroviride.
—Xinying Zhang: Washing dark green Trichoderma spores.
—Yi Peng: Time and stability of CNT encapsulated RNA experiment.

**8.11**

—Yi Peng: Investigation on the duration and encapsulation rate of RNA wrapped by CNT.
—Xinying Zhang: Confrontation experiments between Trichoderma atroviride and Rhizoctonia solani at different concentrations on plates.
—Ruoyu Chen: Repeated experiments on the infection of rice leaves by Rhizoctonia solani.
—Ruoyu Chen: Transformation of GFP plasmid in HT115(DE3).
—Xinying Zhang: Preparation of Trichoderma atroviride protoplasts.

**8.13**

—Ruoyu Chen: IPTG induces a large number of micrornas in HT115 (DE3).
—Ruoyu Chen: Mass extraction of Escherichia coli RNA.
—Ruoyu Chen: Repeated experiments on the infection of rice leaves by Rhizoctonia solani.
—Xinying Zhang: Preparation of magnesium silicate nanoclay.

**8.14**

—Xinying Zhang: Transformation of Trichoderma deep green by magnesium silicate nanoclay method.
—Ruoyu Chen & Yi Peng: Large-scale extraction of Escherichia coli RNA and verification by agarose gel electrophoresis.
—Xinying Zhang: Germination and washing of spores of Trichoderma atroviride.

**8.15**

—Ruoyu Chen: Re-transformation of GFP plasmid in Escherichia coli HT115 (DE3).
—Ruoyu Chen: Repeated experiments on the infection of rice leaves by Rhizoctonia solani.
—Xinying Zhang: Antifungal experiment of Trichoderma atroviride.

**8.16-8.18**

—Ruoyu Chen & Yi Peng: Large-scale RNA extraction from Escherichia coli (Plasmid：GFP, PG-1, PG-2, RPMK1-1-1,
RPMK1-1-2, RPMK1-2-1，RPMK1-2-2).
—Xinying Zhang: Transformation of Trichoderma deep green by magnesium silicate nanoclay method.
—Xinying Zhang: Preparation and transformation of protoplasts from Trichoderma atroviride.
—Xinying Zhang: Antifungal experiment of Trichoderma atroviride.
—Yi Peng: Encapsulation assay of LDH-RNA complex.

**8.19**

—Xinying Zhang: Screening of resistance genes in protoplast transformants.
—Ruoyu Chen: Repeated experiments on the infection of rice leaves by Rhizoctonia solani.

**8.21**

—Ruoyu Chen: Transformation of the second batch of RNAi partial plasmids.
—Xinying Zhang: Resuscitation and expansion culture of glycerol bacteria containing Snakin-1 plasmid.

**8.22**

—Xinying Zhang: A small amount of plasmid was extracted from the resuscitation solution of glycerin preserving
bacteria containing snakin-1 plasmid.
—Xinying Zhang: Snakin-1 transformants were verified by PCR and double restriction enzyme digestion.

**8.23-8.24**

—Ruoyu Chen: IPTG induced Escherichia coli HT115 (DE3) to produce a large amount of target RNA (plasmids: C6, E3, Psm1, β1).
—Ruoyu Chen & Yi Peng: Large scale extraction of RNA from Escherichia coli.
—Xinying Zhang: Protoplast transformation of Trichoderma atroviride.
—Xinying Zhang: Subculture of Trichoderma atroviride.

**8.25**

—Ruoyu Chen: Effect of RNA on the morphology of dead cells under the microscope.
—Yi Peng: Preparation and transformation of Trichoderma atroviride protoplasm.

**8.26**

—Ruoyu Chen: The effect of shRNA against dryness at the micro level.
—Ruoyu Chen: The effect of shRNA on the growth of vegetative cells on plates.
—Xinying Zhang: Transformation and screening of protoplasts from Trichoderma atroviride.
—Xinying Zhang: Antifungal experiment of Trichoderma atroviride.

**8.28**

—Yi Peng: Transformation of Agrobacterium GV3101 (plasmid: epl 1 and prb 1).
—Ruoyu Chen: Large scale RNA extraction from Escherichia coli (plasmid: PG-1, PG-2, Psm1, β1, GFP).

**8.30**

—Yi Peng: Colony PCR validation of Agrobacterium GV3101 transformants.
—Yi Peng: Amplification and culture of Agrobacterium GV3101 transformants.

**8.31-9.2**

—Yi Peng: Trichoderma deep green spores were transformed using Agrobacterium GV3101 transformants.
—Ruoyu Chen: Bulk extraction of RNA from Escherichia coli.
—Ruoyu Chen: Stability verification of shRNA.
—Ruoyu Chen: Verification of shRNA stability after binding LDH.
—Xinying Zhang: Protoplast transformation of Trichoderma atroviride.
—Ruoyu Chen & Xinying Zhang: An experimental study on the inhibitory effect of shRNA on Rhizoctonia solani on plates.
—Xinying Zhang: Resistance plate screening for protoplast transformation of Trichoderma atroviride.
—Xinying Zhang: Transformation of Agrobacterium tumefaciens GV3101 (plasmid: snakin-1).
—Ruoyu Chen: Microscopic examination showed that shRNA could enter the cells of Trichoderma atroviride.

**9.3**

—Xinying Zhang: Agrobacterium containing PRB 1 and epl 1 plasmids on culture plates was resuscitated and expanded.
—Xinying Zhang & Yi Peng: Transformation of Trichoderma atroviride spores using Agrobacterium Tumefaciens GV3101 transformants.

**9.5-9.12**

At this stage, the previous agrobacterium transformation experiment and in vitro rice leaf infection experiment were
repeated continuously, so no duplicate records were made.

—Xinying Zhang & Yi Peng: Transformation of Trichoderma atroviride spores or protoplasts using Agrobacterium
Tumefaciens GV3101 transformants.
—Ruoyu Chen: Observation of rice leaves under stereomicroscope after mycelium inoculation.
—Ruoyu Chen: RNA extraction from Rhizoctonia solani.

**9.13-9.14**

To our great excitement, we successfully obtained the first Trichoderma atroviride pseudotransformants transferred
into the prb 1 plasmid on September 13th! Moreover, the transformants transferred into epl 1 and snakin-1 plasmids
also showed signs of growth.

—Xinying Zhang & Yi Peng: Screening of the secondary rotation plate of transformants by Trichoderma atroviride.
—Ruoyu Chen: RNA was extracted from Rhizoctonia solani and cDNA was obtained by reverse transcription.
—Ruoyu Chen & Bingru Feng: Screening assay for standard primers (18S, GADPH).

**9.15**

—Ruoyu Chen & Bingru Feng: Re-extraction of cDNA from Rhizoctonia solani.
—Ruoyu Chen & Bingru Feng: Revalidation of standard primers.

**9.18**

—Xinying Zhang & Yi Peng: DNA extraction and PCR of the pseudotransformants of Trichoderma atroviride.
—Xinying Zhang & Yi Peng: The proposed transformants were transferred to PDA plates without resistance for expanded culture.

**9.22**

—Yi Peng: The proposed transformants were transferred into PDB liquid medium containing hygromycin B resistance for
expanded culture.

**9.24**

—Yi Peng & Xinying Zhang: Extraction of whole protein from the transformants of Trichoderma deep green. The
target protein containing His-tag was purified using an affinity purification kit.
—Yi Peng：The purified target protein was verified by SDS-PAGE.

**9.26-10.1**

—Yi Peng: LC-MS technology was used to further verify the target protein and relative quantitative analysis
of its overexpression.

## **Products**

**7.25-8.3**

—Minxi Zeng: Preliminary experimental research on inhaling powder.
—Minxi Zeng: Spore production induced by Trichoderma atroviride.
—Minxi Zeng: Drawing of standard curve of absorbance and spore number.
—Bingru Feng: Simple method was used to extract the DNA of Rhizoctonia solani and verify it by PCR.

**8.4-8.10**

—Minxi Zeng: To determine the effect of suction powder on different kinds of common solutions.
—Minxi Zeng: Determination of encapsulation rate and loading rate of suction powder microspheres.
—Minxi Zeng: Explore the balance process of suction and swelling powder content detachment.
—Minxi Zeng: To explore the degradation effect of suction powder.
—Minxi Zeng: Selection of gas producing materials.
—Bingru Feng: Various methods were used to extract DNA from Rhizoctonia solani and verified by PCR and electrophoresis.
—Bingru Feng：Preform LAMP reactions and verified by electrophoresis.

**8.11-8.21**

—Minxi Zeng: Determination of adsorption radius of adsorptive powder.
—Minxi Zeng: Short-term release of Trichoderma spore.
—Minxi Zeng: Determination of sedimentation rate of suction powder.
—Bgru Feng: Perform LAMP reaction and verified by electrophoresis.
—Bgru Feng: Use varieties of methods to extract DNA from R.solani, and the DNA was verified by PCR and electrophoresis.

**8.23-8.31**

—Minxi Zeng: T.ACE quality control.
—Minxi Zeng: Calculate the effective concentration of each recommended method.
—Bingru Feng: pre-experiment of LFD.
—Bgru Feng: Perform LAMP reaction and verified by electrophoresis.
—Bgru Feng: Use varieties of methods to extract DNA from R.solani, and the DNA was verified by PCR and electrophoresis.

**9.1-9.10**

—Minxi Zeng: Measurement and calculation of morphology, particle size and other parameters of T.ACE prescription II.
—Minxi Zeng: Determination of the release rate of T.ACE with different particle sizes.
—Minxi Zeng: T.ACE product effect monitoring.
—Minxi Zeng: Temporary photographic monitoring of the release process of T.ACE.
—Bingru Feng: Perform LAMP reaction and verified by electrophoresis.
—Bingru Feng: Use varieties of methods to extract DNA from R.solani, and the DNA was verified by PCR and electrophoresis.

**9.10-9.20**

—Bingru Feng: CTAB method and TPS method were used to extract DNA of R.solanum AG-1, respectively.
—Bingru Feng: PCR verification of DNA of Rhizoctonia solani.
—Bingru Feng: PCR gradient experiment of DNA extraction from R.solanum AG-1 by TPS.

## **Model，software and hardware**

**5.1-7.10**

—Yingfeng Wu：Algorithm design and system architecture design of electronic nose.

**7.11-7.23**

—Yingfeng Wu：The appearance design of the electronic nose.
—Yingfeng Wu：The circuit principle design of electronic nose.
—Yingfeng Wu：Design of electronic nose supporting APP (version 1.0).
—Yingfeng Wu：Prediction model of co-culture concentration of R.solani and T.atroviride.

**7.24-8.21**

—Yingfeng Wu & Yi Peng：Write the user guide of electronic nose supporting APP (version 1.0).
—Yingfeng Wu：Modeling of disease spot detection and culture medium colony detection of rice infected by R.solani.
—Yingfeng Wu：Endurance curve modeling and pollen spore release modeling of fish suction.

**8.24-9.17**

—Yingfeng Wu:Determine the overall framework of the wiki.
—Yingfeng Wu & Yixi Wang：Design of the second generation electronic nose supporting APP.
—Yingfeng Wu & Yi Peng：Write the user guide of the second generation electronic nose supporting APP.
—Yingfeng Wu：Build the wiki template.
—Yingfeng Wu：Electronic nose hardware assembly and debugging.
—Yingfeng Wu：Electronic nose data acquisition experiment.
—Yingfeng Wu：Modification of the spore release model of T.atroviride.
—Yingfeng Wu：The algorithm development of the hardware part of the electronic nose.

**9.18-9.30**

—Yingfeng Wu:Refinement of all models.
—Yingfeng Wu：Electronic nose data acquisition experiment perfection.
—Yingfeng Wu：Analyze the differences in data collection between R.solani and Pirospora oryzae.

## **Wiki**

After the initial confirmation of our wiki framework on August 24th, all of us started writing the manuscript for
our respective pages. We started making demos of each page on September 10th, and the first phase of Wiki Freeze was
launched on September 20th. Between September 20th and October 12th, we made further changes and content additions to
each page, and finally completed the entire wiki.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

Engineering

#

[Overview](#part-1)

[RNAi Silence System
  RNA V1: siRNA](#part-2)

[RNA V2: shRNA](#part-3)

[RNA V3: CNT-shRNA](#part-4)

[TACE (*Trichoderma*'s acest carrier ever)
  TACE1.0:Starch Graft Acrylate Polymer](#part-5)

[TACE2.0:Hypromellose (HPMC)](#part-6)

#

#

# **Overview**

In our project, we built two biological systems to comprehensively control *Rhizoctonia solani* in rice sheath blight.
Among them, the engineered *Trichoderma* system is used for the prevention of *Rhizoctonia solani*, and the RNAi system
is used for the biological killing of *Rhizoctonia solani*. In order to make them better perform the expected functions,
we followed the DBTL principle, tested the functions of RNAi and *Trichoderma*'s ace carrier ever (TACE) during the project
cycle, and improved and iterated according to the test results, so that they finally achieved our expected results.

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-1.png)

**Figure 1 Engineering cycle**

#

# **RNAi Silence System**

## **RNA V1: siRNA**

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-design.png)

## DESIGN

Because of the high specificity, safety and sustainability of RNAi, we chose this technology to achieve the
treatment of rice sheath blight. After screening, we selected the key gene, PG, which plays a role in the process
of rice infection by *R.solani* as our target gene. For the selection of sRNA molecules that can trigger RNAi process,
because siRNA is more widely used and the application system is more mature, we initially decided to use siRNA to
achieve our silencing of target genes of *R.solani*. On the basis of previous studies proving that *R.solani* can
independently absorb sRNA molecules, we hope that through spraying, siRNA molecules from the outside world can
enter *R.solani* and start the RNAi process in its body, so as to achieve the silencing effect of target gene PG,
and then inhibit the infection of *R.solani* on rice.

##

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-build.png)

## BUILD

|

After determining the target gene, we searched the cDNA database of *Rhizoctonia solani* AG1-IA according to the
sequences provided by the literature, and found the homologous cDNA sequences of AG1-IA strain. Next, the sequences
found by this method were analyzed, queried or predicted on the National Center for Biotechnology Information (NCBI)
website whether there were multiple spliced versions of mRNA, and if there were, the homologous region was taken as
the target region of RNAi interference target. Through BLAST, we checked the biosafety of the target sequence. After
confirming that its good specificity, and then by professional siRNA Design websites to analyze these gene fragments.
Based on the principle of shRNA design, we selected the fragments with high potential siRNA activity from a series of
sequences(Table1). Then we sent the sequence to the biological company and obtained our siRNA molecule by chemical
synthesis.

|  |
|

**Table 1. siRNA information targeting the PG gene of *R.solani***

|![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-table-1.png)

##

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-test.png)

## TEST

|

The rice leaves infected with *R.solani* were sprayed with siRNA, and the expression level of target genes of
*R.solani* on the leaves was detected for 5 consecutive days after spraying. From the results of continuous
qRT-PCR, it can be seen that the target gene was silenced after siRNA treatment for 3 days, with a silencing
rate of 85.6%, and began to rise rapidly after the third day(Figure 2). (More details can be viewed
in [Proof of Concept](%7B%7Burl_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%7D%7D).)

|  |
|![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-2.jpg)
|

**Figure 2 Silencing level of siRNA to target gene**

##

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-learn.png)

## LEARN

Seen from the silencing effect, siRNA can not effectively silence target genes for a long time, which means that
we need to spray siRNA several times in practical applications to achieve our desired effect. However, chemical
synthesis of siRNA in vitro is expensive, so it is not practical to achieve large scales of continuous spraying. In
addition, the exposed ends of siRNA make it easy to be degraded by RNase in the air. Therefore, we have to consider
selecting other sRNA molecules and developing new sRNA production methods.

#

## **RNA V2: shRNA**

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-design.png)

## DESIGN

We further screened out more target genes. Silencing these target genes can achieve the purpose of inhibiting
infectivitity and killing pathogens. Considering that shRNA is more stable than siRNA, we decided to use shRNA
to realize the RNAi process in *Rhizoctonia solani*, and designed different target sequences according to the
selected target genes. Because a large amount of shRNAs are required during spraying, they need to be produced by
a low-cost and high-yield method. So we thought that we could use fermentation engineering technology to obtain
a large amount of shRNA molecules. We selected the most common strain, *E.coli*, for fermentation production, and
used nuclease deficient *E.coli* HT115. The designed target sequence is constructed through the order of sense fragment loop-antisense fragment,
and connected to the plasmid pET-28a (+) to obtain our shRNA expression vector.
During the wet experiment, we also decided to build an ODE model of RNAi to predict the effect of shRNA molecules.

##

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-build.png)

## BUILD

|

In order to verify our successful transformation, we used specific primers to amplify the plasmid extracted
from *E. coli* by PCR, and proved that our shRNA expression vector successfully entered *E. coli* HT115 by
agarose gel electrophoresis. Then, after IPTG induction, we extracted the RNA of *Escherichia coli*, and
confirmed the successful production of shRNA in *Escherichia coli* according to the band size(Figure 3).

|  |
|![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-3.png)
|

**Figure 3 Electrophoresis of RNA extracted from HT115 (DE3)**

|

In order to ensure that our shRNA can enter the cell of *Rhizoctonia solani* smoothly, we also observed its
spraying under the laser confocal microscope (Figure 4).

|![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-4.png)
|

**Figure 4 Adding GFP-shRNA of Cy5 labeled bundled CNT to *R.solani* on leaves**

##

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-test.png)

## TEST

|

At the same time, the shRNA produced was sprayed on the leaves infected by *R.solani*, and the inhibition
effect of shRNA on target genes of *Rhizoctonia solani* was detected by qRT-PCR. The result shows that the
target gene was silenced after spraying shRNA for 3 days, with a silencing rate of 89.7%, and began to rise
rapidly after the third day(Figure 5).The data showed that shRNA had a better inhibition effect than siRNA,
and had a longer effect.（More details can be viewed in [Proof of Concept](%7B%7Burl_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%7D%7D)).

|  |
|![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-5.jpg)
|

**Figure 5 Silencing level of shRNA to target gene**

|

We substituted the data in *E.coli* into the ODA model of RNAi, and obtained the theoretically silent effect
and action duration of shRNA(Figure 6).The effect of model simulation is highly similar to the experimental results.

|![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-6.png)
|

**Figure 6 RNAi ODE model results**

|

More details about the RNAi ODE model, please click [model](%7B%7Burl_for%28%27pages%27%2C%20page%3D%27model%27%29%7D%7D).

##

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-learn.png)

## LEARN

Our actual application scenario is in the field, and it is difficult to ensure the stability and sustainability
of shRNA molecules under complex environmental conditions. In the face of this problem, we have to find a more
effective way to deliver our shRNA.

#

## **RNA V3: CNT-shRNA**

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-design.png)

## DESIGN

Nanomaterials can contribute to the stability of sRNA molecules, and their applications have been widely verified
in delivering RNAi drugs in medicine. However, few people have completed this work in the field of crop protection.
We hope that by binding with nanomaterials, we can help our shRNA molecules to be more stable in the air and
complete steady release for a period of time, so that they can be applied to spraying in the field.

##

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-build.png)

## BUILD

|

We obtained our nanomaterial shRNA particles by co incubating the nanomaterial and shRNA in a fixed proportion
at room temperature for 30 minutes. For the binding of LDH, we verified its successful packaging by agarose
gel electrophoresis (Figure 7). In addition, we also sprayed carbon nanotube(CNT) separately to ensure that they are
not toxic to our rice leaves and *R.solani* (Figure 8).

|  |
|![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-7.png)
|

**Figure 7 Research on binding conditions with LDH**

|![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-8.png)
|

**Figure 8 Incidence of leaves after CNT treatment**

|

Similarly, in order to ensure that our shRNA can still enter the cells of *Rhizoctonia solani* smoothly after
binding CNT, we also carried out verification (Figure 9).

|![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-9.png)
|

**Figure 9 Adding GFP shRNA of Cy5 labeled bundled CNT to *R.solani***

##

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-test.png)

## TEST

|

Spray shRNA bound with nanomaterials on the leaves infected by *R.solani*, and detect its inhibition effect on
target genes by qRT-PCR (Figure 10). From the results, CNT shRNA has a strong inhibitory effect on target genes.
On the fourth day, the inhibition rate reached the maximum of 98.13%, and the inhibition rate was slow, ensuring
that CNT shRNA can continue to play its role for a period of time (More details can be viewed in [Proof of Concept](%7B%7Burl_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%7D%7D)).

|  |
|![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-10.jpg)

**Figure 10 Silencing level of CNT-shRNA to target gene**

##

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-learn.png)

## LEARN

From the above results, our nanomaterial-shRNA has a excellent inhibitory effect on target genes, and the silencing
effect lasts longer. Therefore, we can ensure that the nanomaterial-shRNA can be sprayed in the field as our RNAi
product to achieve a precise and continuous attack on pathogenic fungi. Our engineering cycle of RNAi Silence
System also ends after the verification of this step.

#

# **TACE (*Trichoderma*'s acest carrier ever)**

## **TACE1.0: Starch Graft Acrylate Polymer**

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-design.png)

## DESIGN

In order to block the transmission of *R.solani* from aquatic interface and soil, we hope to design a carrier that can
accurately deliver engineered *T.atroviride* spores to the susceptible parts of rice, such as the rice stem at the
aquatic interface. The first material we noticed was starch Graft Acrylate Polymer, which has strong water
absorption and retention.

##

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-build.png)

## BUILD

|

We prepare the TACE1.0 according to the following steps. Firstly, prepare the spore suspension and TACE1.0,
which are 40 mesh or 80 mesh starch Graft Acrylate Polymer particles. Then add spore suspension according
to the liquid-said ratio of 45:1, and spray after its full imbibition.

|  |

##

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-test.png)

## TEST

|

To confirm whether it is the best choice for us to load *T.atroviride* spores, we tested the performance of TACE1.0
delivering spores at the aquatic interface. The experiments include the sustained release capability, adsorption
radius, floatability, encapsulation and loading rate.

For sustained release capability, the result shows that spores in TACE1.0 are almost completely
released in 100 minutes. We measured the distance from the point where TACE1.0 particles can be adsorbed to
the plant, and measured that the adsorption radius of TACE1.0 is 0.824±0.209cm.

|  |
|![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-11.png)
|

**Figure 11 TACE1.0 is adsorbed to the rice stem**

|

Then we found that the floating capacity of TACE is unstable, and it is easy to settle in advance or disengage
from rice stem. We also calculated the encapsulation and loading rate of the spores after TACE's imbibition.
However, we found that even under the optimal liquid-said ratio, the encapsulation efficiency of TACE1.0 was
only 17%, and the loading rate was only 13%.

|![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-12.png)
|

**Figure 12 Carry capacity of TACE 1.0 to *Trichoderma* spores at different liquid-said ratio**

|

In addition, we pay great attention to whether our products are environmental-friendly, so we test whether
TACE1.0 will be residues in the environment and come to the conclusion that TACE1.0 will not be completely
degraded after its dissemination in paddy fields . There remains 0.2% - 0.3% residues of refractory substances
in the environment.

|![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-13.png)
|

**Figure 13 Degradation of TACE 1.0**

##

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-learn.png)

## LEARN

The data obtained from the experimrnts shows that the duration of spore sustained release failed to meet our expectation
of long-term release at aquatic interface. In addition, the firmness of adsorption of TACE1.0 is not ideal and is
greatly affected by external forces. This means that once TACE1.0 is blown away, it is difficult to gather at the
rice stem again.As for its floatability, TACE1.0 is hard to stay afloat. Moreover, the encapsulation and loading
rate are still below our expectations.
Having learned from the shortcomings of TACE1.0 above, we successfully
iterated the TACE.

#

# **TACE (*Trichoderma*'s acest carrier ever)**

## **TACE2.0: Hypromellose(HPMC)**

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-design.png)

## DESIGN

Inspired from pharmaceutics, we designed the second generation of TACE. TACE2.0 is a hydrophilic sustained-release
matrix preparation with Hypromellose (HPMC) as the main component and *T.atroviride* spore as the effective component.
We expect TACE2.0 to have more efficient carry capacity, better sustained release capability, more stable floating
and adhesion performance.

##

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-build.png)

## BUILD

|

After investigation and experiment, we came up with a recommended prescription of TACE2.0. Through a modern
pharmaceutical process, we mixed 15% *Trichoderma* spore powder, 15% glucose, 60% HPMC400, 10% HPMC15000 and
trace magnesium stearate and tablet , obtaining TACE2.0 of different particle sizes.

|  |
|![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-14.png)
|

**Figure 14 TACE2.0 of different particle size**

##

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-test.png)

## TEST

|

After testing, we obtained the following performance data of TACE2.0:
(1)Constancy:
At the same time, we verify that the 3mm TACE2.0 could release for at least 8h and 6mm for 1~2 days, forming
a continuous local high spore concentration environment at the aquatic interface.
In order to simulate the
release process of spores in TACE2.0, we used first-order kinetic equation to fit the release profile of TACE
(More details can be viewed in [model](%7B%7Burl_for%28%27pages%27%2C%20page%3D%27model%27%29%7D%7D)). The release curve should have
three typical release phases that TACE2.0 doesn't have. From the anomaly modeling results, we have learned that
TACE2.0 is not smooth enough, leading to rapid gelation of surface and entering the intermediate rapid release phase.

|  |
|![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-15.png)
|

**Figure 15 Fitted by the first order kinetic equation**

|

(2)Floatage: The dry TACE2.0 has a measured density of about 0.87 g/ml and can suspend on the water surface itself.

|![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-16.png)
|

**Figure 16 Adhesion of TACE2.0 to Rice Stem**

|

(3)Adhesion: Due to the gelation of surface, TACE2.0 has good adhesion performance so that it is not easy to be peeled off.
(4)Encapsulation efficiency and loading rate: TACE2.0's theoretical encapsulation rate reaches 100%, and the loading rate
reaches 15%. That is, a single 3mm TACE2.0 tablet can carry approximately 3.27 million *T.atroviride* spores.
(5)Dissolution: The HPMC matrix is dissoluble and degradable, which means TACE2.0 does not leave any residue in paddy fields.

##

![](https://static.igem.wiki/teams/4286/wiki/szu-engineering-success-figure-learn.png)

## LEARN

Our TACE2.0 has marked improvements over TACE1.0 in terms of carry capacity, sustained release capability, stable
floating and adhesion performance and so on. What's more, we achieved delayed activation of the conditional triggered
switch by adding glucose. Through a series of verification, TACE2.0 has been proved to be a successful *Trichoderma*'s
carrier.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

Results

#

[Overview](#part-1)

[Prevention
Trichoderma atroviride transformation](#part-2)

[Detection
LAMP-LFD detection system](#part-3)

[Treatment
RNAi therapy](#part-4)

#

#

# **Overview**

This year, SZU-China has established a comprehensive and systematic prevention and control strategy of "
prevention-detection-treatment" for rice sheath blight. In order to support our design, we conducted many experiments
and finally got experimental results, which were sufficient to support our entire design. In the following, we will
introduce our results in three modules: prevention, detection and treatment.

![](https://static.igem.wiki/teams/4286/wiki/szu-results-icon-1.png)

# Prevention

#

# **Trichoderma atroviride transformation**

To prevent Rice Sheath Blight, we choose Trichoderma atroviride as our chassis to overexpress Epl1, a T.atroviride
derived eliciting plant response-like protein 1, which is also a hydrophobic protein and Prb 1, a type of serine
protease playing great importance in mycoparasitism. Otherwise, we express Snakin 1, a potato derived cysteine-rich
antimicrobial peptide inhibiting the growth of a variety of plant pathogens, especially R.solani. To achieve our goal,
we first need to overproduce the three plasmids and then transfer them into T.atroviride for further expression
and function verification.

## **1.Overproduction of three plasmids**

Since we needed to transfer the plasmids into Trichoderma, which would require a large number of plasmids,
we transferred pCAMBIA1302 recombinant plasmids with Epl 1, Prb 1, and Snakin 1 into E.coli DH5a, to amplify
them in large quantities, thus obtaining a constant and large number of plasmids.

After transforming the recombinant pCAMBIA1302 plasmid into DH5a competent cells, the recombinants were screened
by the kana resistance gene on the plasmid. Subsequently, we first performed colony PCR on the isolated colonies
and selected the successfully transformed isolated colonies for simple amplification with the extracted plasmids.
Then we verified them by PCR and double digestion. We designed three pairs of primers with theoretical PCR fragment
sizes of Epl 1-565bp, Prb 1-1388bp, and Snakin 1-425bp, respectively. The PCR results of three plasmids are shown
in Figure 1-1, and all the selected plasmids were in expected positions, consistent with the positions of the positive control.

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-1-1.png)

**Figure 1-1. 1% agarose gel stained with Epl 1, Prb 1, Snakin 1 intergration of pCAMCIA1302 in
E.coli was checked by PCR . Agarose gel was used to validate the pcr results. This figure shows PCR results of
Epl 1, Prb 1, and Snakin 1, respectively. E+, P+, S+ are positive controls for Epl 1, Prb 1, and Snakin 1(positive
clone is the synthetic plasmid). The capitalized word with a number represents the sample we choose.**

In the double digestion verification, we used EcoRI and Bgl II enzymes to cut the plasmid into two segments, the
longer segment was 9729bp, and the shorter segments were (Epl 1) 1252bp, (Prb 1) 2065bp, and (Snakin 1) 1102bp,
but after the PCR test when digested, we found that the plasmid band of Snakin 1 was always at a lower position.
As shown in the electrophoresis diagram of Prb 1 plasmid in Figure 1-2, the lower plasmid is in the superhelical
state, followed by a band in the target position, which is probably the linear band of the plasmid. Therefore,
we decided to single-cleave our Snakin 1 plasmid at the same time. However, this time the obtained agorase gel
results showed that the plasmid of Snakin 1 was in the position of the theoretical linear plasmid. The results
are shown in Figure 1-2.

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-1-2.png)

**Figure 1-2. 1% agarose gel stained with epl 1, prb 1, snakin 1 intergration of pCAMCIA1302
in E.coli was checked by enzyme digestion. (A) The results of double digestion of Epl 1 plasmid. E5-0 and E6-0
are controls that were not treated with enzymes. E5-2 and E6-2 represent the results of double digestion of E5
and E6. (B) The results of double digestion of Prb 1 plasmid. P5-0 and P6-0 are controls that were not treated
with enzymes. P5-2 and P6-2 represent the results of double digestion of P5 and P6. (C) The results of double
digestion of Snakin 1 plasmid. S0 is control ,S1 is the result of single enzyme digestion and S2 is double
digestion of Snakin 1.**

These results show that the selected separated colonies are positive and we then amplified and cultured these
bacteria, and then extracted the plasmids in bulk for subsequent transformation of Trichoderma.

## **2. OD550nm-spore count curve**

When attempting transfermation of Trichoderma, we found that among other things, the quantification of Trichiderma
material took a large amount of time. Because the number of spores required for transformation of Trichoderma is
`10^7` spores/ml and we need to try a lot of times to succeed transforming Trichoderma, it is necessary to
count the spores. Traditionally, spore counting is done by hematocrit, which adds a little more work to the
already tedious task of transforming Trichodrma. At this point we thought of the instrument used in the laboratory
to measure concentrations: a spectrophotometer. We then wondered if we could use a spectrophotometer to make
measurements of the spore count of Trichoderma. After reviewing papers, we found that the idea was feasible
and that the spores of Trichoderma atroviride were suitable at OD550 as the absorption nanometer value for
counting, so we decided to make this spore counting curve. We measured the absorbance values at OD550 nm of
the spores at each gradient concentration after counting them with a hemocytometer plate, and each concentration
was repeated three times. Then, after processing, we obtained the following relevant equations:

![](https://static.igem.wiki/teams/4286/wiki/szu-results-eq-1-1.png)

In this equation, `y` represents the value of OD550, `X`represents the concentration of T.a spore(`10^7` per ml)

## **3.Genetic transformation of Trichoderma**

To transfer recombinant plasmids into Trichoderma, we first tried nanomaterials-mediated transformation as
well as using cell penetrating peptides to transfer the plasmids, but neither of them succeeded. After that,
we tried a more traditional way protoplasted-mediated transformation. However, this CaCl2-PEG induction method
didn't work. All of these methods and tries can be viewed in [Protocol](%7B%7Burl_for%28%27pages%27%2C%20page%3D%27protocol%27%29%7D%7D)
and [Notebook](%7B%7Burl_for%28%27pages%27%2C%20page%3D%27notebook%27%29%7D%7D). Finally, we decided to use
Agrobacterium-mediated transformation (AMT).

We first transferred the three recombinant plasmids into agrobacterium GV3101 and these were screened by Kanamycin and colony PCR.

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-1-3.png)

**Figure 1-3. Agarose gel stained with epl 1, prb 1, snakin 1 integration of pCAMCIA1302 in
GV3101 was checked by colony PCR. (A) E1 and E2 represent the isolated Epl 1 transformed GV3101 colony. Their
were in expected positions. (B) P1 and P2 represent the isolated Prb 1 transformed GV3101 colony. Their were in
expected positions. (C) S1 and S2 represent the isolated Snakin 1 transformed GV3101 colony. Their were in expected positions.**

These gel results showed that the recombinant plasmids had already been transformed into agrobacterium GV3101 correctly.

Then we used positive agrobacterium GV3101 to transform T.atroviride. After several attempts and having got advice
from our PI, we finally obtained the transformed T.atroviride. We selected the recombinant T.atroviride by 50ug/ml
Hygromycin-B and PCR after extracting its genome. Each potential transformant was selected by 50ug/ml Hygromycin-B
4 times in case of unstable genetic inheritance caused by gene fragment inserting in cytoplasmic genome.

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-1-4.png)

**Figure 1-4. Agarose gel stained with epl 1, prb 1, snakin 1 integration of T.atroviride
genome was checked by PCR. (A) 1% agarose gel stained with Prb 1 integration PCR results. P+ is positive control
which is Prb 1 plasmid checked by PCR and P1 and P2 represent recombinants we chose. It is shown on the picture
that P2 is consist with P+, which means P2 is positive.(B) 2% agarose gel stained with Snakin 1 integration PCR
results. S+ is positive control which is Snakin 1 plasmid checked by PCR and S1 represents recombinant we chose.**

According to our PCR results, we can initially confirm that we have transformed Prb 1 and Snakin 1 into T.atroviride
successfully. Epl 1 transformant failed to grow up in the second time of selecting.

## **4.SDS-PAGE**

After verifying that our plasmid was successfully transferred into T. atroviride, we need to further verify its
expression activity in T. atroviride. We first used PRB 1 transformants as an example, Prb-1 transformants were
expanded in Mini medium, and R. solani fungus powder obtained by grinding in liquid nitrogen was used as an inducer
to induce Prb-1 expression in T.atroviride. After two days of culture, we extracted the whole protein of T.atroviride
using a fungal protein extraction kit and verified it by SDS-PAGE. As shown in Figure 1-5, lane 1 is the protein
extract of the Prb 1 transformant that was successfully transferred into the plasmid, lane 3 is the protein
extract of the wild-type T.atroviride, and the total protein concentration was adjusted to the same for both
groups of samples during loading. It can be seen that they have a relatively obvious band at the position of
about 42.3kD, and from the perspective of depth, the band corresponding to the Prb-1 transformant is darker than
that of the wild type, that is, the content is higher.

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-1-5.png)

**Figure 1-5. Result of SDS-PAGE（The picture on the right is the result of grayscale processing
of the picture on the left to facilitate observation and comparison）**

Due to the difficulty of selecting and purchasing antibodies derived from fungi, we did not use Western-blot for
further verification. Instead, we determined the **alkaline protease activity** of the prb-1 transformants and
compared it with that of the wild-type extract. The results of this experiment can be seen on the
[Proof of Concept](%7B%7Burl_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%7D%7D) page.

## **5. Suicide switch**

### **5.1 Plasmid production and preliminary validation**

We constructed the recombinant vector [Pcdh1-cdh1ls-MazEF-Tcdh1]-pCAMBIA1302. In order to produce the recombinant
vector in larger scale for subsequent experiments, we transferred the recombinant vector into E.coli DH5`\alpha`.
Transformants were clearly visible on the culture medium after 16 hours of incubation at 37o C.

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-1-6.png)

**Figure 1-6. Transformation result.**

We constructed the recombinant vector [Pcdh1-cdh1ls-MazEF-Tcdh1]-pCAMBIA1302. In order to produce the recombinant
vector in larger scale for subsequent experiments, we transferred the recombinant vector into E.coli DH5`\alpha`.
Transformants were clearly visible on the culture medium after 16 hours of incubation at 37o C.

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-1-7.png)

**Figure 1-7. 1% agarose gel stained with suicide switch (Pcdh1-cdh1ls-MazEF-Tcdh1) integration
of pCAMCIA1302 in E.coli DH5`\alpha` was checked by PCR. The capitalized word with a number represents the sample we choose.**

We further cultured the transformants and extracted the plasmids from them. Restriction enzyme XhoI was used for
single digestion. There are two restriction sites of XhoI on the plasmid [Pcdh1-cdh1ls-MazEF-Tcdh1]-pCAMBIA1302,
the theoretical sizes of bands after digestion are 1094bp and 10268bp. Electrophoresis was performed in a 1%
agarose gel. The results showed successful single-enzyme digestion and correct plasmid extraction. However, a
band of 1094bp size was not shown because a few plasmids were simultaneously cleaved twice by the enzyme Xhol.

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-1-8.png)

**Figure 1-8. 1% agarose gel stained with suicide switch (Pcdh1-cdh1ls-MazEF-Tcdh1) integration
of pCAMCIA1302 in E.coli DH5`\alpha` was checked by single restriction enzyme digestion. M: 10000bp Marker. SS1: the
plasmid [Pcdh1-cdh1ls-MazEF-Tcdh1]-pCAMBIA1302, which showed closed circular plasmid DNA (cc DNA) and open circular
plasmid DNA (oc DNA). SS2: plasmid digested by restriction enzyme XhoI, which showed linear plasmid DNA (Linear DNA).**

### **5.2 Genetic transformation of Trichoderma atroviride**

To transfer recombinant plasmids into Trichoderma, we used Agrobacterium-mediated transformation (AMT) to
integrate the circuit of Trichoderma suicide switch (on T-DNA fragment of plasmid pCAMBIA1302) into the genome
of Trichoderma atroviride. Then we used positive Agrobacterium GV3101 to transform T.atroviride. We selected the
recombinant T.atroviride by 50ug/ml Hygromycin B and PCR after extracting its genome. Unfortunately, due to lack
of time, we did not carry out the subsequent characterization experiments.

## **6. Induction of Trichoderma atroviride conidiation**

In order to explore the optimal conditions for inducing Trichoderma atroviride conidiation in commercial production,
and to provide more spores for our subsequent experiments of engineered Trichoderma and TACE（Trichoderma’s acest
carrier ever）, we conducted corresponding literature research and experimental exploration. Through literature,
we found the following ways to induce Trichoderma conidiation:

* 1. During the growth of Trichoderma, white fluorescent lamps with light intensity of 600 LX were irradiated for a short time.
* 2. Cultivate Trichoderma in the dark for 1~2 days, which makes them grow more rapidly, and then expose them to white light to induce conidiation.
* 3. Mechanical cutting of Trichoderma on the culture medium causes mechanical injury to the mycelia, thus accelerating conidiation.

The above are general methods of Trichoderma SPP. For Trichoderma atroviride, it is necessary to try these methods
to explore the best method for conidiation induction. Therefore, we set up the following groups to induce
Trichoderma atroviride conidiation with different treatments.

**Table 1-1. Experimental groups**

![](https://static.igem.wiki/teams/4286/wiki/szu-results-table-1-1.jpg)

The sporulation rate and sporulation quantity of each group were different by different treatments.

* 1. Light induction: Compared with groups A5/A6 and A1/A2/A3/A4, spores could be observed on the 4th day of
  consistent exposure to white light. Compared with A1/A2 and A3/A4, it had no effect on conidiation through
  short-term exposure of strong white light during culture in dark. Consistent exposure to white light is the
  best choice for conidiation.
* 2. Injury induction: Compared with groups A1/A3/A5 and A2/A4/A6, mechanical damage significantly promoted
  conidiation, and most spores were produced along cracks. Mechanical damage is more conducive to conidiation.
* 3. Conidiation in different inoculation methods: compared with group A and group B, conidiation is more
  significant in the groups using germinated spore solution to inoculate. In addition, it was easier to
  observe the growth of fungus in the groups which were inoculated by Trichoderma clumps.

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-1-9.jpg)

**Figure 1-9. Morphological changes of Trichoderma atroviride in groups A1, B1, A2 and B2 within 4 to 10 days.**

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-1-10.jpg)

**Figure 1-10. Morphological changes of Trichoderma atroviride in groups A3, B3, A4 and B4 within 4 to 10 days.**

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-1-11.jpg)

**Figure 1-11. Morphological changes of Trichoderma atroviride in groups A5, B5, A6 and B6 within 4 to 10 days.**

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-1-12.png)

**Figure 1-12. Number of eluted spores in group A and group B on day 10.**

![](https://static.igem.wiki/teams/4286/wiki/szu-results-icon-2.png)

# Detection

#

# **LAMP-LFD detection system**

## **1. LAMP Amplification**

### **1.1 DNA extraction of Rhizoctonia solani (Evaluation of common laboratory extraction methods)**

We compared different labortory methods including crude extraction, protocols of three easy methods, CTAB
protocol, TPS protocol, which were used to extract DNA from R.solani in our experiments. Fungal universal primers
ITS5, ITS4 and ITS6 and ITS4 were used to amplify ITS sequences of R.solani, respectively (Fig. 2-1A, 2-1B), and
each amplification product was running in 3% agarose gel. Using each method, R.solani ITS sequences (650~750bp)
were amplified, with bands in the expected position (Fig. 2-1). Protocols of three easy methods could be used to
extract DNA rapidly. The corresponding bands of methods III and IV were bright. CTAB and TPS bands were brighter.
However, crude extraction is not ideal compared with other methods. ITS sequences of R.solani are unable to be
amplified in some samples (Fig. 2-1C).

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-2-1.png)

**Figure 2-1. ITS sequence of R.solani AG-3 DNA samples extracted by different laboratory
extraction methods. (A). DNA templates amplified by fungal universal primers ITS5 & ITS4. (B). DNA templates
amplified by fungal universal primers ITS6 & ITS4. (C). DNA templates amplified by fungal universal primers ITS6 & ITS4.
EMII: DNA extracted by easy method II. EMIII: DNA extracted by easy method III. EMIV: DNA extracted by easy method IV.
CTAB1, CTAB2: DNA extracted by CTAB protocol. TPS1, TPS2, TPS3, TPS4, TPS5: DNA extracted by TPS protocol. CE1~CE5:
DNA extracted by crude extraction. +CE control: a positive control group of DNA extracted by crude extraction.**

### **1.2 ITS sequence of R.solani and related PCR system**

We use the following universal primers and related PCR system for fungi to amplify ITS sequences of R.solani
though PCR reaction (Table 2-1). In our experiment, primers pair ITS1 & ITS4 was more suitable for R.solani AG-1,
primers pairs ITS5 & ITS4 and ITS6 & ITS4 were more suitable for R.solani AG-3 (Figure 2-3,2-4).

**Table 2-1. Primers for R.solani ITS sequences in PCR reaction**

![](https://static.igem.wiki/teams/4286/wiki/szu-results-table-2-1.png)

### **1.3 LAMP system**

### **LAMP primers**

We use the following primers to amplify ITS sequences of R.solani though LAMP reaction (Table 2-2).

**Table 2-2. Primers for R.solani ITS sequences in LAMP reaction**

![](https://static.igem.wiki/teams/4286/wiki/szu-results-table-2-2.png)

### **Optimization of Mg(2+) concentration**

The effect of LAMP amplification reaction was affected by Mg(2+) concentration. We set a Mg(2+) concentration
gradient (5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM), selected R.solani AG-3 genome DNA as templates, and used primers
(AG-3: RSAGF3, RSAGB3, RSAG-3FIP, RSAGBIP) for LAMP reaction. The amplification was carried out at 65o C for 60 min.
Each amplification product was running in 1.3% agarose gel (Fig. 2-2). The bands were brightest at a Mg(2+)
concentration of 7 mM (Fig. 2-2B). DNA extracted by each method was successfully amplified to present a ladder-like
band within this concentration of Mg(2+). Therefore, LAMP Reactions were performed with 7 mM Mg(2+) in subsequent
experiments.

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-2-2.png)

**Figure 2-2. Optimization of Mg(2+) concentration. (A). DNA LAMP amplification products with
Mg(2+) concentration of 5mM (left) and 6mM (left). (B). DNA LAMP amplification products with Mg(2+) concentration
of 7mM and 8mM. (C). DNA LAMP amplification products with Mg(2+) concentration of 9mM and 10mM. Use Rhizoctonia
solani AG-3 genome DNA as template. II: DNA extracted by easy method II. III: DNA extracted by easy method III.
IV: DNA extracted by easy method IV. CTAB1, CTAB2: DNA extracted by CTAB protocol. TPS1, TPS2: DNA extracted by
TPS protocol. +Control: An amplification product that is able to run out of the correct band. -Control: sterilized
water was used instead of the DNA template.**

### **1.4 Detection limit and sensitivity of LAMP protocol**

We used R.solani AG-3 and AG-1 DNA extracted by different methods as templates, each template was serially
diluted in sterile water to amplify its ITS sequence. For PCR reaction, we used primers (AG-3: ITS6 and ITS4;
AG-1:ITS1 and ITS4) as well as corresponding reaction temperatures. For LAMP reaction, We used primers (AG-3:
RSAGF3, RSAGB3, RSAG-3FIP, RSAGBIP; AG-1:RSAGF3, RSAGB3, RSAG-1FIP, RSAGBIP) with Mg(2+) concentration of 7mM
and amplified for 60 mins at 65 o C.

**Table 2-3. Dilution gradient of DNA samples extracted by different methods and the quality of
DNA in the amplification reaction.**

![](https://static.igem.wiki/teams/4286/wiki/szu-results-table-2-3.png)
![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-2-3.png)

Figure 2-3. Detection limit and sensitivity of PCR protocol. A. DNA extracted by easy method III. B. DNA extracted
by easy method IV. C. DNA extracted by CTAB protocol. D. DNA extracted by TPS protocol. Use Rhizoctonia solani
AG-3 genome DNA as template. The number above the loading well indicates the order of magnitude of the corresponding
DNA template content in nanograms.

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-2-4.png)

**Figure 2-4. Detection limit and sensitivity of PCR protocol. Use DNA extracted by TPS protocol.
Use Rhizoctonia solani AG-1 genome DNA as template. The number above the loading well indicates the order of
magnitude of the corresponding DNA template content in nanograms.**

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-2-5.png)

**Figure 2-5. Detection limit and sensitivity of LAMP protocol. A. DNA extracted by easy method
and easy method IV. B. DNA extracted by CTAB protocol and TPS protocol. C. DNA extracted using the four methods,
corresponding to the lowest dilution. Use Rhizoctonia solani AG-3 genome DNA as template. The number above the
loading well indicates the order of magnitude of the corresponding DNA template content in nanograms.**

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-2-6.png)

**Figure 2-6. Detection limit and sensitivity of LAMP protocol. DNA extracted by CTAB protocol
and TPS protocol. Use Rhizoctonia solani AG-1 genome DNA as template. The number above the loading well indicates
the order of magnitude of the corresponding DNA template content in nanograms.**

For **more information** about the LAMP system and implementation of LAMP in LFD for the detection of R.solani,
please view more details on [Proof of Concept](%7B%7Burl_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%7D%7D).

![](https://static.igem.wiki/teams/4286/wiki/szu-results-icon-3.png)

# Treatment

#

# **RNAi therapy**

## **1. siRNA stage**

After deciding to use RNAi technology to biocide Rhizoctonia solani, we need to consider which small RNA(sRNA)
molecules can trigger the RNAi process. We first noticed that siRNA molecules can be double stranded RNA (dsRNA)
cleaved by RNAse III (such as Dicer) in cells into 21~25bp double stranded RNA.

siRNA fragments are loaded into RISC, which consists of Argonaute-2, Dicer and TRBP proteins. Then the two siRNA
chains are separated, one of which leaves the complex. The RISC complex carrying the antisense chain recognizes
the target mRNA, and finally completes the mRNA cleavage and degradation. Due to its extensive clinical application
and mature commercial system, we hope to realize our RNAi process through siRNA molecules.

So we found the first target gene, PG, which plays a key role in the process of starting infection of Rhizoctonia
solani. According to the sequence of this target gene, we designed two different siRNA molecules (Table 3-1) and
sent the sequence information to biological company for chemical synthesis. Based on the chemically synthesized
molecules, we can preliminarily verify whether RNAi is feasible in the treatment of rice sheath blight.

**Table 3-1. siRNA information targeting the PG gene of Rhizoctonia solani**

![](https://static.igem.wiki/teams/4286/wiki/szu-results-table-3-1.png)

## **2. shRNA stage**

Because of the high cost of siRNA synthesis and the poor effect of silencing target genes, we hope to use
fermentation technology to produce sRNA molecules in large quantities. We chose Escherichia coli, which is
commonly used in fermentation engineering, to produce our sRNA molecules. In our expectation, sRNA molecules
will be sprayed in farmland to play a role after production. The environmental factors of farmland are complex,
and our sRNA needs to ensure its stable existence in the natural environment to ensure its continuous function.
Therefore, we focus on shRNA, which is more stable than siRNA, in the selection of sRNA molecules, and expect it
to play a more stable and effective silent role。

### **2.1 Selection of shRNA Molecular Target Genes**

After screening, we finally selected several target genes as the silencing targets of our RNAi molecules. The
information of these target genes is shown in Table 3-2. PG, RPMK1-1 and RPMK1-2 are key genes of Rhizoctonia solani
in the process of infecting rice. Cohesin complex subunit Psm1 (Psm1), Importin beta-1 subunit(`\beta`-1), RNA pol III
subunit C6 (C6) and Ubiquitin ligase E3 (E3) are essential genes for the survival of Rhizoctonia solani.

**Table 3-2.Target genes of shRNA and their target sequence**

![](https://static.igem.wiki/teams/4286/wiki/szu-results-table-3-2.png)

After selecting the above target genes, we designed the corresponding shRNA molecule through the CDS sequence of
the target gene. After BLAST and confirmation of its specificity in the total nucleic acid library, we assembled
it through the sequence of XbaI restriction site - RNAi fragment - loop - reverse complementary fragment of RNAi
fragment - XhoI restriction site, and connected it to the pET-28a (+) plasmid to obtain our shRNA expression vector.

### **2.2 IPTG induced production**

We transformed the constructed shRNA expression vector into E. coli HT115 (DE3), and PCR after extracting the
plasmid. Our specific primer can amplify a 260 bp band on the plasmid, and the results verify the successful
transformation of the plasmid (Fig. 3-1).

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-3-1.png)

**Figure 3-1 Agarose gel electrophoresis after plasmid PCR
1-7: Plasmids extracted from Escherichia coli; 8: Plasmid control; M：DL500 marker
After IPTG induction, its RNA was extracted by Trizol method, and the results in Figure 2 were obtained after
electrophoresis.**

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-3-2.png)

**Figure 3-2 Electrophoresis of RNA extracted from HT115 (DE3)**

It can be seen from the figure 3-2 that there is a brighter band between 50-100 bp in the induced sample lane than
in the uninduced sample. The size of our shRNA is 59 bp, which proves that our shRNA extraction is successful
and the shRNA produced meets the expected size.

### **2.3 shRNA can be absorbed by R.solani**

After inoculated with R.solani, its hyphae will be on the leaves and invade the leaves. We can observe the
extension of hyphae in leaves under different times of microscope (Fig. 3-3).

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-3-3.png)

**Figure 3-3 Infection of hyphae in leaves.
(a) The mycelium is in the leaf (40×). (b) The
mycelium is in the leaf(100×). (c) The mycelium is in the leaf (400×).**

The infected leaves were sprayed with Cy5 dye labeled gfp-shRNA and observed under laser confocal microscope.
As shown in Figure 3-4, shRNA can effectively enter the mycelia during the process of infection of leaves by mycelia,
which provides basic evidence for shRNA silencing in R.solani.

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-3-4.png)

**Figure 3-4 Adding GFP shRNA of Cy5 labeled bundled CNT to R.solani on leaves**

## **3. Nanomaterials-shRNA stage**

However, the instability of shRNA in the field environment cannot make our products play a better role. After
understanding our expectations, our PI proposed that we could try to bind shRNA with nanomaterials for spraying,
and provided CNT and LDH nanomaterials and their fabrication methods.

### **3.1 shRNA can enter R.solani after binding CNT**

Scratch micro mycelia on the plate, add the gfp shRNA labeled with Cy5 dye after binding CNT to the mycelia,
and observe the distribution of shRNA in the mycelia through laser confocal microscope after treatment. The results
showed that shRNA could also effectively enter the cells of Rhizoctonia solani (Fig. 3-5).

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-3-5.png)

**Figure 3-5. Adding GFP shRNA of Cy5 labeled bundled CNT to R.solani**

### **3.2 Research on LDH binding conditions**

At the same time, in order to explore the optimal conditions for binding with LDH, we explored the effect of co
incubation with LDH at different ratios, and confirmed the reasonable packing concentration by setting gradient
in the range of 1:1-1:10. The binding mode of LDH and DNA/RNA is mainly electrostatic binding. The interaction
between the two and the large molecular volume of LDH cause it to stay at the sample hole on the gel, resulting
in the RNA bound with it to stay here but enter the edge gel. Therefore, a brighter fluorescence will appear
around the sample hole. From the results in Figure 3-6, the groups wrapped in 1:1 and 1:3 ratios are not complete,
and there are still obvious bands in the target region. Due to insufficient LDH concentration, some RNA cannot
bind to LDH, so bright fluorescence appears at the target band. It can be inferred that the concentration of
complete encapsulation is between 1:3 and 1:5.

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-3-6.png)

**Figure 3-6. Research on binding conditions with LDH**

Due to the limited time, we only sprayed shRNA binding with CNT, but did not carry out the verification experiment
of shRNA after binding LDH. In the future, we plan to complete this part and compare the silencing effect of shRNA
after binding LDH and CNT respectively.

For **more information** about the verification of siRNA, shRNA, nanomaterials-shRNA in the RNAi process,
please view more details on [Proof of Concept](%7B%7Burl_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%7D%7D).

# **4. R-body**

## **4.1 Plasmid transformation and preliminary validation**

We constructed the recombinant vectors [RebABDC]-pRSFDuet1 and [RebBAC]-pRSFDuet1, through which we compared
the R-body production between the part of 2019 SZU-China and our improved part. We transferred the recombinant
vector into E.coli Ht115(DE3). Transformants were clearly visible on the culture medium after 16 hours of
incubation at 37o C.

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-3-7.png)

**Figure 3-7. Colonial morphology of E.coli transformants (left: [RebABDC]-pRSFDuet1
transformants; right: [RebBAC]-pRSFDuet1 transformants).**

We selected 8 single colonies on each culture medium and carried out colony PCR for plasmid amplification.
The theoretical length of the amplified product was 1730bp (RebABDC) and 1340bp (RebBAC). Electrophoresis was
performed in a 1% agarose gel. The results showed that all the colonies were positive transformants, which
indicated that the recombinant vector was successfully transformed

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-3-8.png)

**Figure 3-8. 1% agarose gel stained with RebABDC and RebBAC integration of pRSFDuet1 in E.coli
Ht115(DE3) was checked by PCR. The capitalized word with a number represents the sample we choose. M: 2000bp Marker.
RA1~8: RebABDC transformants. RB1~8: RebBAC transformants.**

After enlarged production of recombinant plasmids, we conducted double restriction enzyme digestion for further
verification. Restriction enzyme NdeI & HindIII was used for digestion. For [RebABDC]-pRSFDuet1, the theoretical
sizes of bands are 449bp and 4094bp; for [RebBAC]-pRSFDuet1, the theoretical sizes of bands are 98bp and 4196bp.
Electrophoresis was performed in a 1% agarose gel. The results showed successful double-enzyme digestion and
correct plasmid extraction.

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-3-9.png)

**Figure 3-9. 1% agarose gel stained with RebABDC and RebBAC integration of pRSFDuet1 in E.coli
Ht115(DE3) was checked by restriction enzyme digestion. M: 10000bp Marker. pRA1, pRA2, pRB1, pRB2: the plasmids
without enzyme digestion, which showed closed circular plasmid DNA (cc DNA) and open circular plasmid DNA (oc DNA).
s-pRA1, s-pRA2, s-pRB1, s-pRB2: plasmid digested by restriction enzyme HindIII, which showed linear plasmid DNA
(Linear DNA) with theoretical band size 4543bp and 4294bp for RA and RB, respectively. d-pRA1, d-pRA2, d-pRB1,
d-pRB2: plasmid digested by restriction enzyme NdeI and HindIII, which showed theoretical band size 449bp and
4094bp for RA, 98bp and 4196bp for RB.**

## **5. Rice cultivation**

Since we needed to test the effectiveness of our product, we cultured rice（ZH11） in our laboratory culture
room so that it can be used for infection by R.solani AG1-IA in subsequent experiments. We sowed the seeds under the soil after germination and selected
rice leaves and stems from the 6th to 8th week as the material. Below are the approximate changes in rice plant
height that we recorded during this time.

![](https://static.igem.wiki/teams/4286/wiki/szu-results-figure-3-10.png)

**Figure 3-10. Height of ZH11 every 7 days. Every Monday is Seeding Day and we set every Thursday
of the week to record the heights, including the first week which was the week we sowed the seeds.**

{% endblock %}