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From 2014.igem.org

Results / Heavy metals / Arabidopsis thaliana T4MBP

Labwork

Transformation of A. thaliana with our T4MBP:

1. Exchange the original promoter enTCUP2 of the binary vector pORE_E3 (AY562536.1) into a 2x35s promoter using resctriction site based cloning.
2. We integrated our T4MBP in our modified pORE_E3_2x35S.
3. Application of our floral dip method.
4. After 6 weeks we harvested transgenic seeds from A. thaliana and plated these seeds on selection MSO-media.
5. At last we potted transformed plants.

Results

The exchange of the promoter was checked by sequencing. We achieved to regenerate young A. thaliana after the transformation with our T4MBP. A PCR and a immunoblot would indicate if tested plant were positive or negative for our T4MBP. To receive a stabile F2 generation seeds of transformed plants would have to be harvested and grown. As a next step of this test series the plants would have been transfered on medium with heavy metals. In a following analysis it would be detected if these plants bind the heavy metal zinc, copper, cadmium and/or arsenic.

Fig. 1: Pictures showing young potentially transgenic A. thaliana before, during and after the transfer from medium to earth.

Below in figures 2 and 3 you can see the original vector pORE_E3 with an enTCUP2 promoter. For a better expression of our T4MBP protein we exchanged the enTCUP2 with the 2x35S promoter. Each step of this procedure is visualized in this histroy . Furthermore this history includes the insertion of our T4MBP (there termed CDS for coding sequence).

Fig. 2: Vector pORE_E3 with the original enTCUP2 promoter.	Fig. 3: Vector pORE-E3 with 2x35S promoter and our T4MBP which includes a sequence for expansin 4, cellulose-binding domain and domains for the binding of copper, arsenic, zinc, cadmium.