Difference between revisions of "Part:BBa K4585013"

 
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<partinfo>BBa_K4585013 short</partinfo>
  
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The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid, which could express GAL4-KRAB, was used for Luciferase detection experiment. GAL4 is a protein that can find and bind UAS (upstream activation sequence). KRAB is a transcription factor that represses downstream gene expression when combined with GAL4.
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                pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS
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                <p>The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid was obtained through homologous recombination of the KRAB homologous recombination insert (BBa_K4585003) with pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS linearized vector (BBa_K4585008).  The homologous recombination plasmid product was identified as the target product by sequencing and enzyme cutting and agarose gel electrophoresis.
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                1 Pattern Diagram
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                    <img width="400px" src="https://static.igem.wiki/teams/4585/wiki/the-model-diagram-of-pgl4-35-3-ha-9-gal4uas-krab-nls.png"></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 pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS</p>
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                2 Experiment
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                2.1 Method
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                <p>The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid could express GAL4-KRAB, and GAL4-KRAB could bind 9×UAS and inhibit downstream gene expression, therefore GAL4-KRAB could inhibit GnRH expression. Considering that the plasmid itself contains a 9×UAS sequence, GAL4-KRAB can also suppress its own expression.
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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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                    <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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Revision as of 16:18, 6 October 2023

pGL4.35-3XHA-9XGAL4UAS-KRAB-NLS

The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid, which could express GAL4-KRAB, was used for Luciferase detection experiment. GAL4 is a protein that can find and bind UAS (upstream activation sequence). KRAB is a transcription factor that represses downstream gene expression when combined with GAL4.

pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS

The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid was obtained through homologous recombination of the KRAB homologous recombination insert (BBa_K4585003) with pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS linearized vector (BBa_K4585008). 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 pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS

2 Experiment

2.1 Method

The pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid could express GAL4-KRAB, and GAL4-KRAB could bind 9×UAS and inhibit downstream gene expression, therefore GAL4-KRAB could inhibit GnRH expression. Considering that the plasmid itself contains a 9×UAS sequence, GAL4-KRAB can also suppress its own expression.

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℃.