Difference between revisions of "Part:BBa K4890013"
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We took a commercialized recombinant plasmid pUAST as template, and used restrictive endonuclease (BglII and XhoI) digestion to obtain a linearized pUAST vector. MTF-1 gene fragment was amplified from the cDNA of wildtype Drosophila melanogaster by PCR. DNA electrophoresis confirmed the length of the PCR product (2376bp). MTF-1 gene fragment was ligated with the pUAST linearized vector by T4 ligase. pUAST-MTF-1 was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (2376bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmid extracted from the colonies was confirmed to be pUAST-MTF-1 plasmid by gene sequencing (Figure 1-2).
 
We took a commercialized recombinant plasmid pUAST as template, and used restrictive endonuclease (BglII and XhoI) digestion to obtain a linearized pUAST vector. MTF-1 gene fragment was amplified from the cDNA of wildtype Drosophila melanogaster by PCR. DNA electrophoresis confirmed the length of the PCR product (2376bp). MTF-1 gene fragment was ligated with the pUAST linearized vector by T4 ligase. pUAST-MTF-1 was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (2376bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmid extracted from the colonies was confirmed to be pUAST-MTF-1 plasmid by gene sequencing (Figure 1-2).
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    <div class="unterschrift"><b>Figure 1 Gel electrophoresis of MTF-1</b>
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    <div class="unterschrift"><b>Figure 2 Gel electrophoresis of pUAST-MTF-1 plasmid <br/>(From left to right: marker, pUAST-MTF-1, pMRE-Hid and pMRE-GFP)</b>
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Revision as of 11:00, 9 October 2023


UAS-Hsp70-MTF1

This part is responsible to the expression of MTF-1 gene driven by UAS in Drosophila. It consists of UAS sequence (BBa_K3281012), Hsp70 sequence (BBa_K4890004) and MTF-1 gene (BBa_K4890001). UAS and Hsp70 are derived from pUAST plasmid. Upstream activating sequence (UAS) is a cis-acting regulatory sequence. It increases the expression of a neighbouring gene when binds to GAL4. Hsp70 is a promoter that can bind to RNA polymerase and start transcription. MTF-1 gene is derived from Drosophila melanogaster. It encodes MTF-1 (metal-responsive transcription factor-1) which is a transcription factor. MTF-1 protein can be activated by heavy metals. In the cell nucleus of Drosophila cells, MTF-1 binds to MRE to recruit RNA polymerase, in turn increasing the expression of Mto/Mtn to detoxicate metals.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 382
    Illegal EcoRI site found at 2296
    Illegal PstI site found at 244
    Illegal PstI site found at 2054
    Illegal PstI site found at 2232
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 382
    Illegal EcoRI site found at 2296
    Illegal NheI site found at 509
    Illegal PstI site found at 244
    Illegal PstI site found at 2054
    Illegal PstI site found at 2232
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 382
    Illegal EcoRI site found at 2296
    Illegal BglII site found at 527
    Illegal BamHI site found at 479
    Illegal BamHI site found at 1468
    Illegal BamHI site found at 1864
    Illegal BamHI site found at 1891
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 382
    Illegal EcoRI site found at 2296
    Illegal PstI site found at 244
    Illegal PstI site found at 2054
    Illegal PstI site found at 2232
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 382
    Illegal EcoRI site found at 2296
    Illegal PstI site found at 244
    Illegal PstI site found at 2054
    Illegal PstI site found at 2232
    Illegal NgoMIV site found at 1730
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

1 Construction of pUAST-MTF-1 plasmid

We took a commercialized recombinant plasmid pUAST as template, and used restrictive endonuclease (BglII and XhoI) digestion to obtain a linearized pUAST vector. MTF-1 gene fragment was amplified from the cDNA of wildtype Drosophila melanogaster by PCR. DNA electrophoresis confirmed the length of the PCR product (2376bp). MTF-1 gene fragment was ligated with the pUAST linearized vector by T4 ligase. pUAST-MTF-1 was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (2376bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmid extracted from the colonies was confirmed to be pUAST-MTF-1 plasmid by gene sequencing (Figure 1-2).

Figure 1 Gel electrophoresis of MTF-1

Figure 2 Gel electrophoresis of pUAST-MTF-1 plasmid
(From left to right: marker, pUAST-MTF-1, pMRE-Hid and pMRE-GFP)