Difference between revisions of "Part:BBa K4585011"
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                 pcDNA3.1(+)-3×HA-GAL4-VP64-NLS
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                 pcDNA3.1(+)-GnRH-C
 
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                 <p>The pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid was obtained through homologous recombination of the VP64 homologous recombination insert (BBa_K4585002) with pcDNA3.1(+)-3×HA-GAL4-VP64-NLS linearized vector (BBa_K4585006). The homologous recombination plasmid product was identified as the target product by sequencing and enzyme cutting and agarose gel electrophoresis.
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                 <p>The full-length GnRH gene sequence was purchased and the DNA sequence corresponding to the GnRH-C GAP (GnRH-associated peptide) was excised with restriction enzyme to obtain a truncated GnRH-C. Then we obtained the pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid through homologous recombination of the GnRH-C recombination insert (BBa_K4585000) with pcDNA3.1(+)-GnRH-C linearized vector (BBa_K4585016). The homologous recombination plasmid product was identified as the target product by sequencing and enzyme cutting and agarose gel electrophoresis.
 
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                 <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.1 The model diagram of pcDNA3.1(+)-3×HA-GAL4-VP64-NLS</p>
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                 <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.1 The model diagram of pcDNA3.1(+)-GnRH-C</p>
 
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                 <p>The pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid could express GAL4-VP64, thereby activating 9×UAS, which could activate the expression of its downstream gene, GAL4-KRAB or Luciferase.
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                 <p>We transfected pcDNA3.1 (+) -GnRH-C plasmid into HEK 293T cells and cultured for sufficient time, after the transfected HEK 293T cells secreted the corresponding protein into the supernatant, took the supernatant of the cell culture medium for the detection of intracellular calcium flow changes, so as to obtain the secretion of GnRH protein in HEK 293T cells.
 
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                 <p>HEK 293T cells were transiently transfected with GAL-VP64 and GAL-KRAB plasmids, and an appropriate amount of Luciferase plasmids were transfected to simulate GnRH. The experiment showed that the GAL-VP64 plasmid could initiate the expression of GAL4-KRAB and Luciferase.
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                 <p>The results of the detection of changes in intracellular calcium flow corresponding to the supernatant were compared with the standard curve to obtain the corresponding data on the curve to successfully prove the successful expression of pcDNA3.1 (+) -GnRH-C plasmid in HEK 293T cells.
 
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                <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig 2. Bioluminescence intensity when GAL4-VP64=GAL4-KRAB=400 ng</p>
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<span class='h3bb'>Sequence and Features</span>
 
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===Functional Parameters===
 
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Revision as of 04:28, 9 October 2023

pcDNA3.1(+)-GnRH-C

We obtained this plasmid by enzyme cutting and linking for intracellular calcium flow assay. This plasmid can be transformed into and amplified in E. coli and then used as a template for amplification. This plasmid was detected to be able to normally express GnRH-C protein in HEK 293T cells.

pcDNA3.1(+)-GnRH-C

The full-length GnRH gene sequence was purchased and the DNA sequence corresponding to the GnRH-C GAP (GnRH-associated peptide) was excised with restriction enzyme to obtain a truncated GnRH-C. Then we obtained the pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid through homologous recombination of the GnRH-C recombination insert (BBa_K4585000) with pcDNA3.1(+)-GnRH-C linearized vector (BBa_K4585016). The homologous recombination plasmid product was identified as the target product by sequencing and enzyme cutting and agarose gel electrophoresis.

1 Pattern Diagram


Fig.1 The model diagram of pcDNA3.1(+)-GnRH-C

2 Experiment

2.1 Method

We transfected pcDNA3.1 (+) -GnRH-C plasmid into HEK 293T cells and cultured for sufficient time, after the transfected HEK 293T cells secreted the corresponding protein into the supernatant, took the supernatant of the cell culture medium for the detection of intracellular calcium flow changes, so as to obtain the secretion of GnRH protein in HEK 293T cells.

2.2 Results

The results of the detection of changes in intracellular calcium flow corresponding to the supernatant were compared with the standard curve to obtain the corresponding data on the curve to successfully prove the successful expression of pcDNA3.1 (+) -GnRH-C plasmid in HEK 293T cells.

3 Caution

After sequencing and ensuring the sequence was correct, we applied it to the experiments. Store at 4℃.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 249
    Illegal PstI site found at 2330
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 895
    Illegal SpeI site found at 249
    Illegal PstI site found at 2330
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 12
    Illegal XhoI site found at 1000
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 249
    Illegal PstI site found at 2330
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 249
    Illegal PstI site found at 2330
    Illegal NgoMIV site found at 1440
    Illegal NgoMIV site found at 2781
    Illegal NgoMIV site found at 3064
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 882
    Illegal BsaI.rc site found at 4589
    Illegal SapI site found at 3509
    Illegal SapI.rc site found at 2630
    Illegal SapI.rc site found at 2840