Difference between revisions of "Part:BBa K4585007"
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The purpose of the linear vector is to perform homologous recombination with the KRAB homologous recombination fragment to obtain the target product pcDNA3.1 (+) -3xHA-Gal 4-KRAB-NLS plasmid.
 
The purpose of the linear vector is to perform homologous recombination with the KRAB homologous recombination fragment to obtain the target product pcDNA3.1 (+) -3xHA-Gal 4-KRAB-NLS plasmid.
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<head>
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4585007 SequenceAndFeatures</partinfo>
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                pcDNA3.1(+)-3×HA-GAL4-VP64-NLS
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            </h2>
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===Functional Parameters===
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<partinfo>BBa_K4585007 parameters</partinfo>
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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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                <!--put text here, <p>content</p>-->
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                1 Pattern Diagram
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                <p style="text-align: center;">
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                    <img width="400px" src="https://static.igem.wiki/teams/4585/wiki/the-model-diagram-of-pcdna3-1-3-ha-gal4-vp64-nls.png"></p>
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                <br />
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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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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                2 Experiment
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                2.1 Method
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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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                2.2 Results
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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>
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                <p style="text-align: center;">
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                    <img width="400px" src="https://static.igem.wiki/teams/4585/wiki/biofluorescence-intensity-when-gal4-vp64-gal4-krab-400-ng.png"></p>
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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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                3.Caution
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                <p>After sequencing and ensuring the sequence was correct, we applied it to the experiments. Store at 4℃.
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            </div>
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</body>
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</html>

Revision as of 15:09, 8 October 2023


pcDNA3.1(+)-3xHA-Gal4-KRAB-NLS linearized vector

The purpose of the linear vector is to perform homologous recombination with the KRAB homologous recombination fragment to obtain the target product pcDNA3.1 (+) -3xHA-Gal 4-KRAB-NLS plasmid.

pcDNA3.1(+)-3×HA-GAL4-VP64-NLS

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.

1 Pattern Diagram


Fig.1 The model diagram of pcDNA3.1(+)-3×HA-GAL4-VP64-NLS

2 Experiment

2.1 Method

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.

2.2 Results

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.


Fig 2. Bioluminescence intensity when GAL4-VP64=GAL4-KRAB=400 ng

3.Caution

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