iGEM 2023 — Team sz-shd

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## Team calendar

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | March 1st | May 1st | May 31th | June 3th | June 12th | June 20th | September 7th | September 31th |
| Team recruitment, team member training, brainstorm ideas | ✓ | ✓ | ✓ |  |  |  |  |  |
| Construction of plasmid for Bio fertilizer |  |  |  | ✓ | ✓ |  |  |  |
| Test of Bio fertilizer |  |  |  |  | ✓ | ✓ | ✓ | ✓ |
| Construction of plasmid for Phytosensor |  |  |  |  |  | ✓ | ✓ |  |
| Test of phytosensor |  | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ |
| Human Practice activities |  |  |  | ✓ | ✓ | ✓ | ✓ | ✓ |

### Lab notebooks scanning version - Click to open

[Note 1](https://static.igem.wiki/teams/4844/wiki/files/2023-10-2-annie-protocol.pdf)
[Note 2](https://static.igem.wiki/teams/4844/wiki/files/irene.pdf)
[Note 3](https://static.igem.wiki/teams/4844/wiki/files/irene.pdf)

For protocols and other supplementary materials, see [Supplementary materials](https://2023.igem.wiki/sz-shd/experiments)

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[1. Characterization of the next-generation Plant report gene: eyGFP\_UV](#characterization)

[Background](#characterization-1)
[Our design](#characterization-2)
[Conclusion](#characterization-3)
[2. Construction and test of low phosphate phytosensor](#construction)

[Background](#construction-1)
[Design of Low phosphate sensor (Basic)](#construction-2)
[Design of Low phosphate sensor (with Low noise amplifier)](#construction-3)
[Results](#construction-4)
[3. Design of intelligent phosphorus fertilizer system](#design)
[References](#references)

# Engineering success

---

## 1. Characterization of the next-generation Plant report gene: eyGFP\_UV

#### Background

Green fluorescent protein (GFP) has been widely used for monitoring gene expression and protein localization in diverse organisms. However, highly sensitive imaging equipment, like a fluorescence microscope, is usually required for the visualization of GFP, limiting its application to fixed locations in samples. This is due to the wave Excitation length and emission wavelength being too close, therefore an emission filter is needed. Secondly, the expression strength of normal GFP or enhanced GFP is relatively weaker in plants, therefore it was hard to observe the fluorescence with the naked eye.
![](https://static.igem.wiki/teams/4844/wiki/engineering/img1.png)
The traditional equipment is not only highly expensive but also requires lots of training for operation. This requirement poses practical constraints, as it confines the use of GFP to controlled laboratory settings, making it challenging to observe fluorescence in real-world scenarios or uncontrolled environments. Consequently, researchers often face limitations in their ability to study GFP-expressing organisms or tissues outside the lab.
![](https://static.igem.wiki/teams/4844/wiki/engineering/img2.png)
Thus, we are excited to introduce a new plant report gene, called eYGFP-uv (BBa\_K4844000), in transient expression we observed bright fluorescence under UV light on tobacco leaves. GFPuv (a GFP variant) was optimized for maximal fluorescence to be observed by naked eyes under UV light instead of using a fluorescence microscope.
![](https://static.igem.wiki/teams/4844/wiki/engineering/img3.png)

#### Our design

To transient express our eyGFP, a patented carbon nanodot-based tracked, transformation, translation, and trans regulation(TTTT) technique invented by our team members(Yingjie Lei; Jixiao Wu; Leyi Xu; Yujie Tao) and Jianhuang's lab at Soochow University (attribution table2) was used to deliver our vector into plants. (More about TTTT can be found at https://2023.igem.wiki/sz-shd/plant )
![](https://static.igem.wiki/teams/4844/wiki/engineering/img4.png)
The transient expression vector was built up with a plant-strong promoter CAMV 35S, a 5'UTR on the upstream of eygfp(uv) to enhance the translation efficiency in plants and fused with a 3\*flag tag downstream for western blot detection of the protein. (Our vector sequence can be downloaded at the supplementary material page https://2023.igem.wiki/sz-shd/experiments ).
![](https://static.igem.wiki/teams/4844/wiki/engineering/img5.png)
Once the tobacco plant has been transformed with carbon nanodots for three days, bright florescent can be visualized with the naked eye under the light source of a 398nm UV flashlight. We also ran a western blot using the protein extraction of tobacco tissue samples and anti-flag-tag antibodies. Clear band can be observed on the membrane (27.9 kDa).
![](https://static.igem.wiki/teams/4844/wiki/engineering/img6.png)

#### Conclusion

Therefore, the successful design and construction of our report gene is the solidary part of our low phosphate phytosensor. More potential applications of this report gene are waiting for us to explore.
![](https://static.igem.wiki/teams/4844/wiki/engineering/img7.png)

## 2. Construction and test of low phosphate phytosensor

#### Background

Plants usually have severe responses under the pressure of low phosphate. Some genes will be turned on to allow plants to better adapt to this situation. Therefore, in order to build a low-cost, real-time soil phosphate sensor, we first found a low phosphate response promoter in Maize which was reported by Jianrong Bai in 2018.
![](https://static.igem.wiki/teams/4844/wiki/engineering/img8.png)
Based on Bai's result, a 1502bp length promoter shows the strongest promoting strength under low phosphate pressure. Therefore, we decided to choose it as our low phosphate detection part. The sensor vector will be transformed into plants later with our "TTTT" system.
![](https://static.igem.wiki/teams/4844/wiki/engineering/img9.png)

#### Design of Low phosphate sensor (Basic)

![](https://static.igem.wiki/teams/4844/wiki/engineering/img10.png)
By combining the P1502-ZmPHR1 promoter with the eyGFP report system we already introduced. Also, a normally open GUS gene as the internal reference for downstream experiments (to calibrate the result). We get the basic version of the low phosphate sensor.

#### Design of Low phosphate sensor (with Low noise amplifier)

Although the P1502-ZmPHR1 promoter in the basic version up-regulates the expression when the plant is under low phosphate pressure. However, we found that the leaking expression (background expression without low phosphate pressure) of the original promoter was high and the promoting strength wasn't as high as we expected. Therefore, we decided to design a signal amplification system.

The major feature of synthetic biology was its' combination with engineering. In electrical engineering, amplifier circuits were used to amplify a weak input signal into a strong output. We also want to reduce the "noise" of our signal to make our sensor more precise.
![](https://static.igem.wiki/teams/4844/wiki/engineering/img11.png)
Therefore, since we were inspired by the article published by Müller, K. etal 2014, we designed an artificial transcription factor to regulate artificial promoters, aiming to enhance the low-phosphate response in plants. In this artificial transcription factor, we split the DNA binding domain (DBD) and the transcription activation domain into two separate proteins, placing them downstream of two relatively weaker promoters. We then used the N-terminal (N) and C-terminal (C) protein-protein interaction domains (PID) to guide the recombination and activation of the split transcription factors with their respective artificial promoters.
![](https://static.igem.wiki/teams/4844/wiki/engineering/img12.png)
In Müller, K. et al.'s work, the binding of their designed artificial transcription factor's PID depended on the Pif-PhyB protein interaction induced by red light. Given that our project did not require the use of a light control pathway, we adopted the PDZ-TP interaction system designed by Park, S., & Ryu, for the automatic binding and activation of our artificial transcription factor. PDZ is a peptide binding domain for specific target peptides (TP) in many proteins, and the second PDZ domain of mouse neuronal synaptic protein psd95 has been extensively studied. The binding strength of this PDZ to the TP sequence SIESDV was calculated to be Kd = 1.8uM, and due to its high specificity, this protein interaction system can be used as a good orthogonal component.

The use of VP16 as a component of the Hepes herpesvirus transcription activator (TA) has been validated through years of experimental applications. Sadowski, I. et al. were the first to design and use the GAL4-VP16 fusion protein, and they reported that VP16 had a strong promoter activation effect when this protein bound to the core promoter sequence upstream of the minimal promoter. Sadowski and colleagues also confirmed that in the absence of GAL4-VP16 binding, their artificially designed promoters had low leaky expression. Subsequent experimental studies, including those using artificial transcription factors by Müller, K. and others, also employed VP16 as a TA. By replacing the DBD, more artificial transcription factors can recognize different promoter sequences, achieving diverse and mutually orthogonal gene expression regulation. Similar to Müller, K. and colleagues' design, we also selected a protein structure with high DNA binding strength for our DBD and replaced TetR with LacI to reduce the possibility of sequence overlap in future experiments.
![](https://static.igem.wiki/teams/4844/wiki/engineering/img13.png)
The complete low-phosphate amplification circuit design is shown in the figure below, where LacI-PDZ and TP-VP16 are placed under the control of the pZmPHR1 and pZmSO promoters. When both promoters are simultaneously activated, LacI-PDZ and TP-VP16 proteins bind to the lacO sequence on the artificial promoter 35SE-lacO-mini35p, activating the downstream fluorescent protein gene transcription. Additionally, when LacI-PDZ is expressed alone, it's binding to DNA can block certain promoter leaky expressions, making this system more controllable.

#### Results

Hence, we validate our design through different ways. ( Detailed protocols can be found on the supplementary material page https://2023.igem.wiki/sz-shd/experiments ).
1. Successful in vitro validation of LacI-LacO binding with electrophoretic mobility shift assay (EMSA)
![](https://static.igem.wiki/teams/4844/wiki/engineering/img14.png)
Our result proved that the LacI protein can bind to the LacO DNA sequence as we designed.

2. qPCR results indicate the function of our gene circuit at the transcription level
To verify the function of the amplification gene circuit, we decided to use qPCR- a semi-quantitative strategy to measure transcription efficiency. We extract RNA from tobacco leaves and run an RT-PCR. The cDNA was then used as the template for qPCR. The GUS tag in all vectors was used as an internal reference to calibrate the result and obtain a value for comparison.
![](https://static.igem.wiki/teams/4844/wiki/engineering/img15.png)
The qPCR result indicates that our low noise amplifier part can not only increase the expression strength but also reduce leaky expression.

3. Live visualization of eyGFP(UV) under UV flashlight
To test the real-world application of our product, we use a UV flashlight to visualize the eyGFP(UV) of the carbon dots transformed tobacco leaves after 5 days of low phosphate treatment.
![](https://static.igem.wiki/teams/4844/wiki/engineering/img16.png)
Therefore, our phytosensor design showed engineering success and has the potential to turn into a product and application in agricultural production.

## 3. Design of intelligent phosphorus fertilizer system

Our inspiration came from the team Nanjing\_high\_school 2020. In our design, the system harnesses the power of specially engineered bacteria that respond to low-phosphate stress signals emanating from plant roots. When activated, these bacteria produce and secrete gluconic acid, a naturally occurring compound renowned for its ability to enhance the solubility of calcium salts in the soil. This ingenious mechanism effectively mobilizes otherwise insoluble phosphate salts, rendering them accessible to plants in a form they can readily absorb. By dramatically enhancing phosphate utilization in the soil, this system serves as a game-changer, not only significantly boosting crop productivity but also mitigating the environmental repercussions associated with excessive fertilizer application.
![](https://static.igem.wiki/teams/4844/wiki/engineering/img17.png)

Details for our design and results can be seen at [Contribution](https://2023.igem.wiki/sz-shd/contribution)

## References

1. Sears, R.G., Rigoulot, S.B., Occhialini, A., Morgan, B., Kakeshpour, T., Brabazon, H., Barnes, C.N., Seaberry, E.M., Jacobs, B., Brown, C., Yang, Y., Schimel, T.M., Lenaghan, S.C. and Neal Stewart, C., Jr. (2023), Engineered gamma radiation phytosensors for environmental monitoring. Plant Biotechnol. J, 21: 1745-1756.

2. Guoliang Yuan, Haiwei Lu, Dan Tang, Md Mahmudul Hassan, Yi Li, Jin-Gui Chen, Gerald A Tuskan, Xiaohan Yang, Expanding the application of a UV-visible reporter for transient gene expression and stable transformation in plants, Horticulture Research, Volume 8, 2021, 234,

3. Wang, J., & Li, Y. (2018). Design of an artificial transcription factor to regulate artificial promoters for enhancing the low-phosphate response in plants. Journal of Northwest A&F University (Natural Science Edition), 46(7), 1-9.

4. Müller, K., Fedosov, D. & Gompper, G. Margination of micro- and nano-particles in blood flow and its effect on drug delivery. Sci Rep 4, 4871 (2014).

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[Protocols](#protocol)

[Culture of the Tobacco plant](#protocol-1)
[Low phosphate treatment](#protocol-2)
[Electrophoretic mobility shift assay (EMSA)](#protocol-3)
[Extraction of Plant tissue protein](#protocol-4)
[Detection of protein through western blot](#protocol-5)
[Transformation of tobacco leaf using carbon nanodots](#protocol-6)
[Other minor protocols](#protocol-7)
[Plasmid and Primer .DNA files](#files)

## Protocols

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#### Culture of the Tobacco plant (strain Yun yan 87)

1. Sterilize the seeds

* Put 50-100 seeds in a 2ml sterilized EP tube
* Wash the seed with 1ml 70% ethanol for 30s, gently flip the EP tube
* Wash the seed with 1ml sterilized water for 2 time
* Wash the seeds with 1ml 3% Sodium hypochlorite solution for 5min, gently flip the ep tube
* Resine the seeds with 1ml sterilized water for 5 time

2. Use a pipet tip to plant seed on 1/2 N6 medium(3g/L N6 salt, 10g/L sucrose, 7g/L agar; autoclave 121℃ 20min) in a Petri dish, use a sterilized filter paper to soak excess water on the dish surface.
3. Place the Petri dish in 4℃ for 48h
4. Place the Petri dish in 21℃, 16h light 8h dark cycle light incubator for 14 days
5. Transfer the tobacco plant into a hydroponic cell for further experiment

#### Low phosphate treatment of tobacco plants

1. Prepare the following solutions:- Prepare the following solutions:
     * Culture solution A:
     + 2mMCa(NO3)2
     + 0.65mMMgSO4
     + 25μM Fe·EDTA
     + 5μM MnSO4
     + 2μM ZnSO4
     + 0.5μM CuSO4
     + 0.005μM (NH4)6Mo24·4H2O
     + 25μM H3BO4
     + 50μM KCl* Normal culture solution: 1 mM KH2PO4 + Culture solution A
     * Low phosphate culture solution: 10 μM KH2PO4, 0.99 μM KCl+ Culture solution A
2. Grow the control group in normal culture solution and the low phosphate group in low phosphate culture solution, in a light incubator with 21℃, 16h light 8h dark cycle.

#### Electrophoretic mobility shift assay (EMSA) essay

1. Express the candidate protein in Ecoli and purify with His-tag
2. The Probe was synthesis by Tskingke Biotech by two single strain oligos with 5' Biotin modify
3. Mix the two strains with a 1:1 mole ratio in TE buffer, put them in a 95℃ water bath, turn off the water bath, and allow it to cool down to room temperature. This will anneal the two strains into one single strain.
4. Perform the Emsa experiment and transfer to a nylon membrane
5. Follow the instruction and use DIG DNA Labeling and Detection Kit (Roche, 11093657910) to detect the DNA probe on the membrane.
6. Use a CCD chemiluminescence image system to visualize the result.

#### Extraction of plant tissue protein

1. Weight 0.1g plant tissue sample and placed in a 2ml EP tube
2. Frozen with liquid nitrogen and grind into powder
3. Add 0.3ml RIPA buffer and place on ice for 30min
4. Centrifuge at 4℃ 12,000g for 10min and keep the supernatant

#### Detection of protein through western blot

1. The protein has been transformed from an SDS-PAGE gel to PVDF membrane (millipore, R1NB740V8) (soak in Methanol 3min before use) at 300V for 45min.(put the device in ice to cool down)
2. The membrane was blocked in 5% non-fat milk(dissolve in TBS buffer) for 1h at room temperature.
3. The membrane was washed in TSBT buffer for 10min
4. The membrane was incubated with anti-HA tag antibody(1:10000 in block buffer) and antiβ-actin antibody(internal reference) at 4℃ overnight.
5. The membrane was washed in TBST buffer for 15min \* 4 times
6. The membrane was incubated with Rabbit Anti-Mouse IgG H&L (HRP)(Abcam, ab6728) (1:5000in block buffer) for 1h at room temperature.
7. The membrane was washed in TBST buffer for 15min \* 4 times
8. The buffer A and buffer B of the ECL detection reagent(Beyotime,P0018S) was mixed and dropped on the membrane, signal was detected by the Tanon 5200 chemiluminescence system.

#### Transformation of tobacco leaf using carbon nanodots

1. Ligate the vector with carbon nanodots by mixing CDP 10ul, Plasmid vector (final concentration 10ng/ul), 50X MES buffer 10ul, 10% glycerol 25ul, ddH2O to total volume 500ul
2. Incubate the mixture in 37℃ for 30min
3. Brush on the leaf at 3:00pm every day for 3 or more days.

#### Other minor protocols - [Click to open PDF](https://static.igem.wiki/teams/4844/wiki/files/protocols-additional.pdf)

## Plasmid and Primer .DNA files - [Click to download](https://static.igem.wiki/teams/4844/wiki/files/sz-shd2023-dna-files.woff2) (change .woff2 suffix to .zip to get zip file)

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## What should this page contain?

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* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

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* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

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You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

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* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

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