Revision as of 14:41, 27 September 2024

4xMREd-EGFP

Usage and Biology

The MRE-sites containing promoter enables the metal-dependent expression of the downstream positioned reporter gene EGFP via the metal ion-dependent transcription factor MTF-1 for cell-based metal detection.

In order to develop a cell-based heavy metal sensor, our research group generated a series of synthetic MTF-1-responsive promoter constructs and evaluated their efficacy. Since Wang and colleagues (2004) suggested a high affinity of MTF-1 towards MREd we synthesized a promoter sequence containing four MREd elements at the positioning of the MREs of the MREwt promoter (K5317003) to exclude possible disruption of the MTF-1 and MRE interaction.

Cloning

Theoretical Part Design

Placing the 4xMREd-containing promoter upstream of the reporter gene EGFP allows the visualization of primarily metal-dependent activation of MTF-1.

Sequence and Features

Cloning

The cassette composed of 4xMREd-EGFP was assembled by amplifying the 4xMREd (K5317005) promoter using the primers in table 1 and assembling the promoter in the AseI- and NheI-digested EGFP-C2 backbone (K3338020) using the NEB Hifi Assembly Kit.

Table1: Primers used to create matching overhangs on promoter amplicon to digested pEGFP-C2 backbone

The vector map of the assembled construct is shown in figure 1.

Figure 1: Vector map of the assembled plasmid 4xMREd-EGFP-C2 integrating the 4xMREd promoter upstream of the reporter gene EGFP in the pEGFP-C2 backbone.

Characterization

Figure 2: Transfection of HEK293T cells with the 4xMREd-EGFP-C2 plasmid showed no base signal of the promoter without metal ion stimulation. Scale bar = 20 µm.

Reference

Wang, Y., Lorenzi, I., Georgiev, O., & Schaffner, W. (2004). Metal-responsive transcription factor-1 (MTF-1) selects different types of metal response elements at low vs. high zinc concentration. Biological chemistry, 385(7), 623–632. https://doi.org/10.1515/BC.2004.077