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.
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 )
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.
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.
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.
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.
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)
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.
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.
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.
Details for our design and results can be seen at Contribution
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).