Difference between revisions of "Part:BBa K5317004"
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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 (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317007 K5317007]</span>) for cell-based metal detection. | 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 (<span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317007 K5317007]</span>) for cell-based metal detection. | ||
| − | + | To increase the efficiency of the activated MTF-1-responsive promoter, we constructed a synthetic promoter with multiple MREa sites. Searle and colleagues described in 1985 that at least two MREa sites are necessary for the zinc-induced expression of the downstream gene, here herpes simplex virus thymidine kinase. They also showed that the positioning of the MREs in the promoter sequence had little effect on the promoter efficiency but was increased with more MREa sites inserted. | |
=Cloning= | =Cloning= | ||
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===Theoretical Part Design=== | ===Theoretical Part Design=== | ||
| − | The promoter sequence contains four MREa sites with respective core consensus sites for binding of heavy metal-activated MTF-1, their positions and in-between DNA sequence based on our MREwt promoter, registry entry <span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317003 K5317003]</span>. | + | The promoter sequence contains four MREa sites with respective core consensus sites for binding of heavy metal-activated MTF-1, their positions, and in-between DNA sequence based on our MREwt promoter, registry entry <span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317003 K5317003]</span>. |
===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_K5317009 K5317009]</span> registry entry to view the results. |
=References= | =References= | ||
Latest revision as of 21:29, 1 October 2024
4xMREa promoter
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 (K5317007) for cell-based metal detection.
To increase the efficiency of the activated MTF-1-responsive promoter, we constructed a synthetic promoter with multiple MREa sites. Searle and colleagues described in 1985 that at least two MREa sites are necessary for the zinc-induced expression of the downstream gene, here herpes simplex virus thymidine kinase. They also showed that the positioning of the MREs in the promoter sequence had little effect on the promoter efficiency but was increased with more MREa sites inserted.
Cloning
Theoretical Part Design
The promoter sequence contains four MREa sites with respective core consensus sites for binding of heavy metal-activated MTF-1, their positions, and in-between DNA sequence based on our MREwt promoter, registry entry K5317003.
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 K5317009 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