Difference between revisions of "Part:BBa K5107002"

Latest revision as of 09:14, 30 September 2024

HRE5

HRE5 is the five tandem repeats of the HRE minimal:(BBa_K5107000) to increase the interaction surface of the hormone receptor with the DNA.

Usage and Biology

Previously, in-cell systems have successfully used five tandem repeats of the REs for different human hormonal receptors[1]. Inserted in plasmids such as those we used for our in cell-assay, it is shown efficient to express Lac-Z when the associated hormone-receptor complex is present in the cell. In this manner, hormone work as trancription activator.

**Figure 1:**Schematic overview of the in cell system which uses the Lac-Z as reporter gene when hormone/edc i spresent in the cell.

Assemply

These elements are part of the plasmid fist presented by (Miller et all,2010)[1]. For the list of the plasmid check BBa_K5107000.
We used specific primers to amplify the REs from two of these plasmids(pRR-ERalpha-5Z,pRR-MR-5Z). The plamsids were a gift from Charles Miller (Addgene plasmid # 23061, # 23059 ; http://n2t.net/addgene:23061,http://n2t.net/addgene:23059 ; RRID:Addgene_23061,RRID:Addgene_23059).

Primers for HRE5 and ERE5
	Forward Primer	Reverse Primer
HRE5	ataggtagaNCCGTCTGGTACAGGGTGTTCTT	atgtgggNCCCCGGGAAAAAGAACACCCTGTA
ERE5	ataggtagaNCCCAGGTCAGAGTGACCTG	atgtgggNCCGACCCCGGGAAAAAGC

After that, USER Cloning was performed so the final constucts are derived. BBa_K5107006 and BBa_K5107012

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]

References

Miller, C. A., Tan, X., Wilson, M., Bhattacharyya, S., & Ludwig, S. (2010). Single plasmids expressing human steroid hormone receptors and a reporter gene for use in yeast signaling assays. Plasmid, 63(2), 73–78. https://doi.org/10.1016/j.plasmid.2009.11.003

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-HRE5 is the five tandem repeats of the HRE minimal:[https://parts.igem.org/Part:BBa_K5107000 BBa_K5107000] to increase the interaction surface of the hormone receptor with the DNA.
+HRE5 is the five tandem repeats of the HRE minimal:([https://parts.igem.org/Part:BBa_K5107000 BBa_K5107000]) to increase the interaction surface of the hormone receptor with the DNA.
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 </html>
+===Assemply===
+*These elements are part of the plasmid fist presented by (Miller et all,2010)<html><a href="#ref1">[1]</a></html>. For the list of the plasmid check [https://parts.igem.org/Part:BBa_K5107000 BBa_K5107000].
+*We used specific primers to amplify the REs from two of these plasmids(<strong>pRR-ERalpha-5Z,pRR-MR-5Z</strong>). The plamsids were a gift from Charles Miller (Addgene plasmid # 23061, # 23059 ; http://n2t.net/addgene:23061,http://n2t.net/addgene:23059  ; RRID:Addgene_23061,RRID:Addgene_23059).
+{| class="wikitable"
+|+ Primers for HRE5 and ERE5
+|-
+! !! Forward Primer !! Reverse Primer
+|-
+| HRE5 || ataggtagaNCCGTCTGGTACAGGGTGTTCTT || atgtgggNCCCCGGGAAAAAGAACACCCTGTA
+|-
+| ERE5 || ataggtagaNCCCAGGTCAGAGTGACCTG || atgtgggNCCGACCCCGGGAAAAAGC
+|}
+*After that, USER Cloning was performed so the final constucts are derived. [https://parts.igem.org/Part:BBa_K5107006 BBa_K5107006] and [https://parts.igem.org/Part:BBa_K5107012 BBa_K5107012]