Difference between revisions of "Part:BBa K4890014"
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Start from pMRE as template, we used restrictive endonuclease (NotI and XbaI) digestion to obtain a linearized pMRE vector. Hid gene fragment was amplified from the cDNA of wildtype Drosophila melanogaster by PCR. DNA electrophoresis confirmed the length of the PCR product (1233bp). Hid gene fragment was ligated with pMRE linearized vector by T4 ligase. pMRE-Hid plasmid was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (1233bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmid extracted from the colonies was confirmed to be pMRE-Hid by gene sequencing. | Start from pMRE as template, we used restrictive endonuclease (NotI and XbaI) digestion to obtain a linearized pMRE vector. Hid gene fragment was amplified from the cDNA of wildtype Drosophila melanogaster by PCR. DNA electrophoresis confirmed the length of the PCR product (1233bp). Hid gene fragment was ligated with pMRE linearized vector by T4 ligase. pMRE-Hid plasmid was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (1233bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmid extracted from the colonies was confirmed to be pMRE-Hid by gene sequencing. | ||
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+ | <div class="unterschrift"><b>Figure 1 Gel electrophoresis of MRE</b> | ||
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Revision as of 06:51, 9 October 2023
MRE-Hsp70-Hid
This part is responsible to the expression of Hid gene driven by MRE in Drosophila. It consists of MRE sequence (BBa_K4890002), Hsp70 sequence (BBa_K4890004) and Hid gene (BBa_K4890003). Metal response element (MRE) is derived from Drosophila melanogaster. In the cell nucleus, activated MTF-1 binds to MRE. The MRE is typically located in the promoter regions associated with genes involved in the response to heavy metals. Binding of MTF-1 to MRE activates the transcription of the corresponding genes, thereby promoting heavy metal-related gene expression. Head involution defective (Hid) gene is derived from Drosophila melanogaster. Hid induces cell apoptosis in Drosophila. Hsp70 is derived from pUAST plasmid. Hsp70 is a promoter that can bind to RNA polymerase and start transcription.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 360
Illegal XbaI site found at 1616
Illegal PstI site found at 222
Illegal PstI site found at 964
Illegal PstI site found at 1193
Illegal PstI site found at 1337 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 360
Illegal PstI site found at 222
Illegal PstI site found at 964
Illegal PstI site found at 1193
Illegal PstI site found at 1337
Illegal NotI site found at 378 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 360
Illegal BglII site found at 372
Illegal XhoI site found at 487
Illegal XhoI site found at 1393 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 360
Illegal XbaI site found at 1616
Illegal PstI site found at 222
Illegal PstI site found at 964
Illegal PstI site found at 1193
Illegal PstI site found at 1337 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 360
Illegal XbaI site found at 1616
Illegal PstI site found at 222
Illegal PstI site found at 964
Illegal PstI site found at 1193
Illegal PstI site found at 1337
Illegal NgoMIV site found at 1322 - 1000COMPATIBLE WITH RFC[1000]
Results
1 Construction of pMRE-Hid plasmid
We also reformed the pUAST plasmid into pMRE plasmid by restrictive endonuclease (Pst1) digestion to obtain a linearized pUAST vector and then substitute UAS sequence for MRE sequence. MRE was obtained by DNA synthesis and T4 ligase was used to combine the linearized vector into complete plasmid. pMRE plasmid was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (600 bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmid extracted from the colonies was confirmed to be pMRE by gene sequencing.
Start from pMRE as template, we used restrictive endonuclease (NotI and XbaI) digestion to obtain a linearized pMRE vector. Hid gene fragment was amplified from the cDNA of wildtype Drosophila melanogaster by PCR. DNA electrophoresis confirmed the length of the PCR product (1233bp). Hid gene fragment was ligated with pMRE linearized vector by T4 ligase. pMRE-Hid plasmid was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (1233bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmid extracted from the colonies was confirmed to be pMRE-Hid by gene sequencing.