Composite

Part:BBa_K5317011

Designed by: Jan Gelhoet   Group: iGEM24_Hannover   (2024-09-14)
Revision as of 14:44, 27 September 2024 by Annaseidler (Talk | contribs) (→‎Characterization)


MREdada-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 integrate the findings of Searle and colleagues (1985) and Wang and colleagues (2004) regarding the metal inducibility of a promoter with two MREa sites and a high affinity between MREd and MTF-1, we designed a synthetic promoter with two MREa and two MREd sites that alternate. The aim is to enhance the sensitivity and efficiency of the metal-dependent promoter.

Cloning

Theoretical Part Design

Placing the MREdada 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

To test the MREdada promoter functionality the reporter gene EGFP (K3338006) was cloned downstream of the promoter by inserting the MREdada promoter into the AseI- and NheI-digested EGFP-C2 backbone (K3338020) using NEB Hifi Assembly.

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

Primer name Sequence
MREdada_fw CCGCCATGCATTAGTTATGCACACTGGCGCT
MREdada_rev TGGCGACCGGTAGCGGACGCTTAGAGGACAGC
The vector map of the assembled construct is shown in figure 1.

Figure 1: The vector map depicts the integration of the MREdada promoter into the pEGFP-C2 backbone, placing it upstream of the reporter gene EGFP.

Characterization

Figure 2: HEK293T cells single-transfected with the MREdada-EGFP-C2 plasmid exhibited no EGFP-signal under unstimulated condiotions. Scale bar = 20 µm.

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

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

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