Part:BBa_K5317009

4xMREa-EGFP

Usage and Biology

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

In an effort to increase the efficiency of the activated MTF-1-responsive promoter, we constructed a synthetic promoter with multiple MREa sites. Searle and colleagues described 1985 that at least two MREa sites are necessary for the zinc-induced expression of the downstream gene, here herpes simplex virus thymidine kinase. They also showed that the positioning of the MREs in the promoter sequence had little effect on the promoter efficiency but was increased with more MREa sites inserted. Therefore, we put a promoter together with four MREa sites positioned at the sites of the MREwt promoter (K5317003).

Cloning

Theoretical Part Design

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

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Cloning

The promoter was synthesized and inserted by NEB HiFi Assembly into the pEGFP-C2 backbone plasmid (K3338020) after its restriction enzyme digestion with AseI and NheI, generating the MREwt-EGFP cassette. The assembled plasmid is shown in figure 1.

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

Primer name	Sequence
4xMREa_fw	CCGCCATGCATTAGTTATGCACACTGGCGCT
4xMREa_rev	TGGCGACCGGTAGCGGACGCTTAGAGGACAGC

Figure 1: Assembled vector map with 4xMREa-EGFP integrated into the pEGFP-C2 backbone.

Characterization

Transfection experiments in mammalian HEK293T cells assessed the promoter functionality, sensitivity and specifity. First, the composite part carrying plasmid was introduced via transfection to establish a baseline of endogenous promoter activity before performing co-transfection experiments with the CMV-MTF-1-mRuby2 carrying plasmid (composite part K5317012) under varying copper concentration for stimulation. The EGFP fluorescence signal was analyzed for localization by microscopy and intensity by FACS analysis.

Single-transfection experiments

Figure 2: Single-transfected HEK293T cells with the 4xMREa-EGFP-C2 plasmid depicted no EGFP-signal under unstimulated conditions. Scale bar = 20 µm.

The single transfection with the 4xMREa-EGFP-C2 plasmid in HEK293T cells showed no base signal without the co-transfection with the CMV-MTF1-mRuby2 plasmid and no metal ion stimulation. The experiments allow conclusions about sensitivity and specificity of the promoter under homeostatic conditions. Therefore, the generated promoter has no unspecific expression by for example other, under homeostatic conditions active, transcription factors. The possible endogenous expression of MTF-1 is also not enough to generate a fluorescent signal under unstimulated conditons.

Co-transfection experiments with MTF-1

Figure 3: Representative microscopy images of HEK293T cells co-transfected with MTF-1-mRuby2-C2 together with 4xMREa-EGFP-C2. MTF-1 depicts a nuclei-localized signal, the EGFP signal is cytoplasmatic. Shown are brightfield channels (left), fluorescence channels (images in the center) and an overlay of the channels (right).

FACS analysis

Figure 4: Quantitive validation by flow cytometry analysis. The percentage of cells expressing the fluorophore EGFP under the control of the 4xMREa promoter is displayed as a function of various concentrations of copper sulfate incubated for four hours.

Reference

Searle, P. F., Stuart, G. W., & Palmiter, R. D. (1985). Building a metal-responsive promoter with synthetic regulatory elements. Molecular and cellular biology, 5(6), 1480–1489. https://doi.org/10.1128/mcb.5.6.1480-1489.1985