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1.Overview of the project background

Overview of a program to monitor soil heavy metals with luminescent plants:

In nature, when plants encounter heavy metals, there will be some specialized genes initiating expression in response to heavy metals; our goal is to find the genes in plants that respond to heavy metals, and use the promoters of the genes, that is, the switches that regulate the expression of the genes, for controlling the luminescence pathway in the luminescent plants, to realize that the luminescent plants respond to the heavy metal cadmium.

We expect to achieve the experimental goal is, when there is no heavy metal pollution in the soil, the plant grows normally; when cadmium pollution in the soil, the plant luminescence, the light emitted is visible to the naked eye, so as to realize the cadmium contamination of the soil with luminescent plants.

Currently lead to soil heavy metal pollution of heavy metal species, which is widely distributed, the consequences of serious, is cadmium pollution, molecular biology level evidence is also relatively sufficient, we plan to start with cadmium pollution, to find a general solution to monitor heavy metal pollution with plants, to be the program to go through, and then to the detection of heavy metal categories to expand./p>

In the scientific research community, researchers have successfully engineered the luminescent gene pathway (comprising a total of five genes) of the mushroom, a self-luminous eukaryote in nature, into Ben's tobacco, which allows the whole plant to emit light in a stable manner.

Our team built on their work by using the cadmium-responsive promoter we found to modify to produce the luminescent plants we use for cadmium pollution monitoring.

2.Introduction to the experimental framework (experimental planning pdf-Xmind)

It took us 4 months, from design, to constructing models, to experimental testing, 15 key experimental parts, through the relay of the team members, each experimental part went through 3~5 rounds of iteration, we constantly learnt lessons from failure, and from more than 30 candidate genes potentially responsive to cadmium, we found 2 promoters that can clearly respond to cadmium, and realized the engineering closed loop of the cadmium-responsive promoter function validation experiments.

The main part of our experiment is to link the designed target promoter with two relatively simpler and easy-to-verify functional genes, GUS and GFP, to see if these genes can be expressed in the presence of cadmium, and to verify that the promoter we found can respond to cadmium.

The experiment we designed is divided into a total of 15 steps, of which 6 main steps are selected to be introduced as follows: (Expand the following outline to add detailed experimental principles, four experimental materials, experimental results, experimental analysis data, etc.)

  • Promoter research: first of all, we need to find the known genes that may respond to cadmium through literature research.
  • Promoter amplification: through the design of primers, PCR experiments, the promoter we need, fishing out from the DNA template
  • Vector cleavage: Prepare the vector containing GUS and GFP genes, and excise the strong promoter, the original switch of these genes.
  • Homologous recombination: connect the promoter we found that can respond to cadmium with the linear vector of GUS and GFP genes after removing the promoter.
  • Agrobacterium soil transformation: transfer the successfully ligated recombinant vector from E. coli to Agrobacterium soil.
  • Transient transformation and staining: infiltrate plant leaves with Agrobacterium soilum, and then stain the leaves with staining reagents

Leaves of cadmium-treated, infested plants showed blue color, indicating that our promoter can respond to cadmium in plants.

Figure:Experiment Design(Drawing by student Gu Yuhan)