Difference between revisions of "Part:BBa K4585008"
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                 <p>Based on the sequence of the pGL4.35-3 plasmid, we designed the pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS linearized vectror by using the GAL 4 homologous recombination fragment, 9×UAS homologous recombination fragment and homologous recombination experiments.
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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>The two ends of the pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS linearized vectror need to be complementary to the two ends of the corresponding recombinant fragments to ensure the success of homologous recombination ligation.
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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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          3.Caution
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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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                 <p>The two ends of the pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS linearized vectror need to be complementary to the two ends of the corresponding recombinant fragments to ensure the success of homologous recombination ligation.
 
                 <p>The two ends of the pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS linearized vectror need to be complementary to the two ends of the corresponding recombinant fragments to ensure the success of homologous recombination ligation.
 
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Revision as of 16:03, 8 October 2023


pGL4.35-3xHA-9xGAL4UAS-KRAB-NLS linearized vector

The objective was to synthesize the pGL4.35-3 HA-9 GAL4UAS-KRAB-NLS plasmid by homologous recombination with the KRAB homologous recombination fragment .

pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS linearized vector

We based on the sequence of the pGL4.35 plasmid. Through homologous recombination experiments, the Gal 4 sequence, 3× HA sequence were ligated to the pGL4.35 plasmid, and nine UAS sequences were inserted simultaneously. Special primers were designed, and then the plasmid was cut into linear sequences of a length of about 4500 bp by PCR. The two ends of the linear sequence can be complementary to both ends of the KRAB homologous recombination fragment, and the purpose is to perform homologous recombination with the KRAB homologous recombination fragment to obtain the target product pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid.

1 Pattern Diagram


Fig.1 The agarose gel electrophoresis of pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS linearized vector

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

The two ends of the pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS linearized vectror need to be complementary to the two ends of the corresponding recombinant fragments to ensure the success of homologous recombination ligation.