Difference between revisions of "Part:BBa K5317005"

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===Usage and Biology===
 
===Usage and Biology===
  
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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.
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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 (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317003 K5317003]</span>) to exclude possible disruption of the MTF-1 and MRE interaction.
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To achieve a cell-based heavy metal sensor, our group generated multiple synthetic MTF1 responsive promoter constructs and tested them in regard to their efficiency. 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 [https://parts.igem.org/Part:BBa_K5317003]to exclude possible disruption of the MTF-1 and MRE interaction.
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=Cloning=
 
=Cloning=
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===Theoretical Part Design===
 
===Theoretical Part Design===
  
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The sequence of the MREd site was obtained from Seguin and colleagues (1987), as well as the positioning and spacer sequences (Searle ''et al.'' 1985). The construct was synthesized.
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The sequence of the MREd site was obtained from Seguin and colleagues (1987), as well as the positioning and spacer sequences (Searle ''et al.'', 1985). The construct was synthesized.
  
 
===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K5317005 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5317005 SequenceAndFeatures</partinfo>
  
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=Characterization=
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The promoter was analyzed by composing a gene cassette where its placed upstream of the reporter gene EGFP to assess the promoter's functionality and metal-dependent efficiency based on the fluorescent signal. Please visit the <span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317010 K5317010]</span> registry entry to view the results.
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=References=
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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
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Seguin, C., & Hamer, D. H. (1987). Regulation in vitro of metallothionein gene binding factors. Science (New York, N.Y.), 235(4794), 1383–1387. https://doi.org/10.1126/science.3103216
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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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Latest revision as of 21:31, 1 October 2024


4xMREd promoter

Usage and Biology

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

The sequence of the MREd site was obtained from Seguin and colleagues (1987), as well as the positioning and spacer sequences (Searle et al., 1985). The construct was synthesized.

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]

Characterization

The promoter was analyzed by composing a gene cassette where its placed upstream of the reporter gene EGFP to assess the promoter's functionality and metal-dependent efficiency based on the fluorescent signal. Please visit the K5317010 registry entry to view the results.

References

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

Seguin, C., & Hamer, D. H. (1987). Regulation in vitro of metallothionein gene binding factors. Science (New York, N.Y.), 235(4794), 1383–1387. https://doi.org/10.1126/science.3103216

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