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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 an immunoblot would indicate, if the tested plants 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 soil.

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 history. 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. (plasmid maps originally taken from www.snapgene.com/resources)	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. (plasmid maps originally taken from www.snapgene.com/resources)