Difference between revisions of "Part:BBa K4890014"
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     <div class="unterschrift"><b>Figure 2 Gel electrophoresis of Hid</b>
 
     <div class="unterschrift"><b>Figure 2 Gel electrophoresis of Hid</b>
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    <div class="unterschrift"><b>Figure 3 Gene sequencing of pMRE</b>
 
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Revision as of 06:55, 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


Assembly Compatibility:
  • 10
    INCOMPATIBLE 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
  • 12
    INCOMPATIBLE 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
  • 21
    INCOMPATIBLE 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
  • 23
    INCOMPATIBLE 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
  • 25
    INCOMPATIBLE 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
  • 1000
    COMPATIBLE 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.

Figure 1 Gel electrophoresis of MRE

Figure 2 Gel electrophoresis of Hid

Figure 3 Gene sequencing of pMRE