Difference between revisions of "Part:BBa K5317010"
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===Theoretical Part Design===
 
===Theoretical Part Design===
  
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Placing the 4xMREd-containing promoter upstream of the reporter gene EGFP allows the visualization of primarily metal-dependent activation of MTF-1.  
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Placing the 4xMREd-containing promoter upstream of the reporter gene EGFP allows the visualization of primarily metal-dependent activation of MTF-1.
  
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===Sequence and Features===
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<partinfo>BBa_K5317010 SequenceAndFeatures</partinfo>
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===Cloning===
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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.
 
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===Sequence and Features===
 
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<partinfo>BBa_K5317010 SequenceAndFeatures</partinfo>
 
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===Cloning===
 
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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 vector map of the assembled construct is shown in figure 1.
 
The vector map of the assembled construct is shown in figure 1.

Revision as of 18:54, 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.