Difference between revisions of "Part:BBa K5317005"
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===Usage and Biology=== | ===Usage and Biology=== | ||
| − | To develop a cell-based heavy metal sensor, our research group generated a series of synthetic | + | 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. |
=Cloning= | =Cloning= | ||
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===Theoretical Part Design=== | ===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. | + | 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=== | ||
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=Characterization= | =Characterization= | ||
| − | The promoter was analyzed by composing a gene cassette where its placed upstream of the reporter gene EGFP to assess the | + | 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. |
=References= | =References= | ||
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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