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Using tobacco plant chassis and transient transformation experimental techniques, we successfully constructed 12 recombinant vectors and verified the cadmium-responsive properties of three promoter elements, namely NtNRAMP2, NtNRAMP5, and NtNRAMP6, after screening by two different vector construction methods and GUS function validation experiments, which confirms the efficiency and Feasibility.

Based on the PCR2-GUS and PCR2-GFP vectors constructed by us, our mentors carried out 2 rounds of functional validation experiments based on the same plant chassis, transient transformation method and cadmium treatment conditions as in our experiments, confirming that the PCR2 promoter can emit light under cadmium conditions.
Plant-based biosensors offer huge advantages in soil and water pollution detection, absorbing chemicals from the surrounding environment as they grow naturally and creating morphological changes, making them more convenient and efficient than existing electronic detection instruments and providing a better response to low concentrations of pollution. Plant-based biosensors have the added benefit of detecting without impacting the natural environment. Once the appropriate indicators have been determined, the average person can see the shape and quickly detect the contaminant with the naked eye.

:We have successfully designed plant sensors that utilize luminescent signals to detect heavy metal contamination:

We have made important progress in the direction of plant-based biosensors by combining the fungal bioluminescence pathway (FBP) with the promoter of heavy metal-responsive genes, and successfully designing a plant sensor that can detect soil heavy metals. This is a simple, efficient and low-cost plant-based biosensor, which makes it possible to quickly recognize feedback signals with the naked eye.

We utilized tobacco and periwinkle as plant-based biosensors, Tobacco (Nicotiana tabacum) is an annual herbaceous plant of the Solanaceae family that originated in tropical and subtropical America and is a rich source of the alkaloid nicotine, which has been used for a long time as an herbal medicine and social stimulant. So far, tobacco has been cultivated worldwide and it is not only a major cash crop but also a model plant for the study of plant genetics, molecular biology and cell biology. However, tobacco can only carry out leaf transient transformation experiments. In order to enrich the types of tissue organs in the plant chassis and give full play to the unique advantages of plants, we successfully tried and reproduced Agrobacterium-mediated transient transformation experiments by using the floral organs of the horticultural ornamental plant, periwinkle (Catharanthus roseus).

Firstly, we chose cadmium as the metal pollutant of interest, screened and verified the promoters of cadmium-specific expression genes in plants using various technical means (transcriptome screening, GUS staining verification and GFP fluorescence verification), and then transformed them into plant cells through vector construction, E. coli and Agrobacterium transformation, and finally expressed them successfully and verified their functions.

clean water
Cadmium (50ng/μL)
blank comparison
positive control
Conclusion Strong response, strong specificity Strongly responsive, weakly specific,
this promoter may be responsive to
drought in addition to cadmium
Strong response, strong specificity

After conducting experiments in multiple stages, we believe that in a limited time, we have screened out many very promising plant cadmium response regulatory switches, such as NtNRAMP2, NtNRAMP6, and NtNRAMP5.

Finally, the luminescence expression module was designed, which realized the expression of the FBP pathway and generated bioluminescence when exposed to cadmium metal, and successfully developed a set of simple and efficient biosensors for detecting heavy metal (e.g., cadmium) contamination in soil (water).

While our first focus is on cadmium metals, ideally our system could be applied to a variety of soil (water body) heavy metal contaminants. The system is based on a biosensor that creates a self-luminous system in the plant as well as promoters for responding to various metal contaminants.Exposure of the plant to the contaminated soil (water body) results in the triggering of the FBP pathway, which ultimately causes the plant to emit light that is visible to the naked eye, meaning that the plant can be quickly detected at night when it has been exposed to the contaminant. This is obvious to any observer and can be detected in time to take the necessary precautions.

Due to time constraints, we cannot continue to conduct more functional verification experiments for promoters.We requested help from the teachers at BGI. As the experimental difficulty of reconstructing the luminescent carrier itself is greater, we hope that they can conduct subsequent luminescence tests based on our existing experimental results. Finnally, our promoter PCR2 can really respond to cadmium.

:We have successfully solved the problem of regulation in plant synthetic biology:

Heavy metal contamination is a global problem that has serious implications for plant growth, food safety and environmental health. In order to engineer plant-based biosensors to exhibit bioluminescent properties in the presence of heavy metals, a novel and efficient method to detect and monitor heavy metal content in soil (water) was generated. We attempted to alter gene promoters to deliberately respond to external changes and successfully realized that the intensity of plant luminescence varies with the external heavy metal content, providing a solution for controlling the expression module.

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:The plant parts/tools/protocols we have developed have provided significant support to other teams conducting related research:

The GUS gene is present in some bacteria and encodes β-glucuronidase (GUS), a hydrolase that catalyzes the hydrolysis of many β-glucosidic esters. Because endogenous GUS activity does not exist in the vast majority of plant cells, the GUS gene is widely used as a reporter gene in transgenic plants, and is especially widely used in the study of spatiotemporal expression of genes. We utilized this technique to successfully overcome the challenge of a long plant growth cycle and complete the engineering closure in a limited time, for which a complete validation system was constructed, in which the developed plant parts, tools and protocols are exemplary for carrying out plant-related research.

Including the construction of plant culture environment, the development of operation manuals for tobacco seed disinfection, germination, seedling raising and management and maintenance; figuring out and perfecting Agrobacterium-based plant transient transformation technology, verifying the promoter function by using GUS staining technology, and sorting out and perfecting the SOPs of related experimental operations.

The specific heavy metal-responsive gene promoters we identified and characterized provide a range of functional expression elements for future use in plant synthetic biology. In addition, the various protocols and methods employed in this project, including plant promoter cloning, homologous recombination and clonal transformation, were documented and shared on the iGEM wiki platform, further facilitating the accessibility and usefulness of these components, tools, and protocols to the broader scientific community. Our contribution to plant biology can be said that we have compiled a complete list of protocols commonly used in plant synthetic biology, especially plant seedling, Agrobacterium transformation, tobacco transient transformation, gus staining, and so on, which will provide a very good reference material for the later projects working on plant synthetic biology related projects!

Overall, we have made notable contributions and demonstrated success in engineering plant cells for heavy metal-responsive bioluminescent response modules, solving a pertinent problem in plant synthetic biology, efficiently utilizing plant chassis, and providing a valuable resource for other teams.

Appendix: List of Contributing Resources Table
(click on the detailed PDF to jump to the experimental section for download)

1.Disinfection and Germination of Tobacco Seeds.
2.Bacteria Culture
3.PCR Products Purification
4.Homologous Recombination and Clonal Transformation
5.Agrobacterium Transformation SOP
6.Transient Transfection of Tobacco Leaves
7.GUS Staining SOP.


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