Difference between revisions of "Part:BBa K5317010"
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The cassette composed of 4xMREd-EGFP was assembled by amplifying the 4xMREd (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317005 K5317005]</span>) promoter using the primers in table 1 and assembling the promoter in the AseI- and NheI-digested EGFP-C2 backbone (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K3338020 K3338020]</span>) using the NEB Hifi Assembly Kit.
 
The cassette composed of 4xMREd-EGFP was assembled by amplifying the 4xMREd (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317005 K5317005]</span>) promoter using the primers in table 1 and assembling the promoter in the AseI- and NheI-digested EGFP-C2 backbone (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K3338020 K3338020]</span>) using the NEB Hifi Assembly Kit.
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Revision as of 18:57, 14 September 2024


4xMREd-EGFP

Usage and Biology

The MRE-sites containing promoter enables the metal-dependent expression of a downstream positioned reporter gene 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 MTF1-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


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 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.

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

Primer name Sequence
4xMREd_fw CCGCCATGCATTAGTTATGCACACTGGCGCT
4xMREd_rev TGGCGACCGGTAGCGGACGCTTAGAGGACAGC

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