Revision as of 06:34, 20 September 2024

MREwt 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.

The varying Metal Responsive Elements (MREs) upstream of the eukaryotic metallothionein (MT) gene were discovered in the early 80s (Carter et al. 1984, Stuart et al. 1985). All MREs a-d carry core consensus sites (TGCRCNC) to which the primary MRE-binding transcription factor MTF-1 can bind after binding to heavy metal ions and translocating into the nucleus (Wang et al. 2004). Physiologically, this leads to the expression of metallothionein, a protein capable of binding metals such as zinc, cadmium, copper and others for metal homeostasis and detoxification (Cousins 1983). The arrangement of the MREs in our promoter construct was inspired by publications from Glanville et al. (1981) and Searle et al. (1985), maintaining the order of MREs from the physiological murine MT-1 promoter.

Cloning

Theoretical Part Design

The MREwt promoter was synthesized, and the MREa-d were positioned similar to the physiological murine promoter upstream of the metallothionein gene.

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 promoters functionality and metal-dependent efficiency based on the fluorescent signal. Please visit the K5317008 registry entry to view the results.

References

Carter, A. D., Felber, B. K., Walling, M. J., Jubier, M. F., Schmidt, C. J., & Hamer, D. H. (1984). Duplicated heavy metal control sequences of the mouse metallothionein-I gene. Proceedings of the National Academy of Sciences of the United States of America, 81(23), 7392–7396. https://doi.org/10.1073/pnas.81.23.7392

Cousins R. J. (1983). Metallothionein--aspects related to copper and zinc metabolism. Journal of inherited metabolic disease, 6 Suppl 1, 15–21. https://doi.org/10.1007/BF01811318

Glanville, N., Durnam, D. M., & Palmiter, R. D. (1981). Structure of mouse metallothionein-I gene and its mRNA. Nature, 292(5820), 267–269. https://doi.org/10.1038/292267a0

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

Stuart, G. W., Searle, P. F., & Palmiter, R. D. (1985). Identification of multiple metal regulatory elements in mouse metallothionein-I promoter by assaying synthetic sequences. Nature, 317(6040), 828–831. https://doi.org/10.1038/317828a0#

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

@@ Line 17: / Line 17: @@
 ===Theoretical Part Design===
-The MREwt promoter was synthesized and please visit the <span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317008 K5317008]</span> registry entry for functionality analysis.
+The MREwt promoter was synthesized, and the MREa-d were positioned similar to the physiological murine promoter upstream of the metallothionein gene.
 ===Sequence and Features===
 <partinfo>BBa_K5317003 SequenceAndFeatures</partinfo>
+=Characterization=
+The promoter was analyzed by composing a gene cassette where its placed upstream of the reporter gene EGFP to assess the promoters functionality and metal-dependent efficiency based on the fluorescent signal. Please visit the <span class="plainlinks">[https://parts.igem.org/Part:BBa_K5317008 K5317008]</span> registry entry to view the results.
 =References=