Difference between revisions of "Part:BBa K4585008"
 
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<partinfo>BBa_K4585008 short</partinfo>
 
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The objective was to synthesize the pGL4.35-3 HA-9 GAL4UAS-KRAB-NLS plasmid by homologous recombination with the KRAB homologous recombination fragment .  
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The objective was to synthesize the pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS plasmid by homologous recombination with the KRAB homologous recombination fragment.  
  
 
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                 <p>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.
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                 <p>We based on the sequence of the pGL4.35 plasmid. Through homologous recombination experiments, the GAL4 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.
 
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                 1 Pattern Diagram
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                 1 Diagrams
 
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                     <img width="400px" src="https://static.igem.wiki/teams/4585/wiki/pgl4-35-3-ha-9-gal4uas-krab-nls-linearized-vector/pgl4-35-3-ha-9-gal4uas-krab-nls-linearized-vector/pgl4-35-3-ha-9-gal4uas-krab-nls-linearized-vector.png"></p>
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                     <img width="400px" src="https://static.igem.wiki/teams/4585/wiki/pgl4-35-3-ha-9-gal4uas-krab-nls-linearized-vector/9-gal4uas.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 agarose gel electrophoresis of pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS linearized vector</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 agarose gel electrophoresis of pGL4.35-3×HA-9×GAL4UAS-KRAB-NLS linearized vector</p>
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                 2 Experiment
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                 2 Caution
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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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                 <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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                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>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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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4585008 SequenceAndFeatures</partinfo>

Latest revision as of 03:47, 12 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 GAL4 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 Diagrams


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

2 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.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 4423
    Illegal XbaI site found at 151
    Illegal XbaI site found at 1933
    Illegal XbaI site found at 4260
    Illegal XbaI site found at 4495
    Illegal SpeI site found at 4019
    Illegal PstI site found at 3108
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 4423
    Illegal NheI site found at 4451
    Illegal SpeI site found at 4019
    Illegal PstI site found at 3108
    Illegal NotI site found at 3087
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 4423
    Illegal BglII site found at 4498
    Illegal BamHI site found at 413
    Illegal XhoI site found at 4303
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 4423
    Illegal XbaI site found at 151
    Illegal XbaI site found at 1933
    Illegal XbaI site found at 4260
    Illegal XbaI site found at 4495
    Illegal SpeI site found at 4019
    Illegal PstI site found at 3108
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 4423
    Illegal XbaI site found at 151
    Illegal XbaI site found at 1933
    Illegal XbaI site found at 4260
    Illegal XbaI site found at 4495
    Illegal SpeI site found at 4019
    Illegal PstI site found at 3108
    Illegal NgoMIV site found at 55
    Illegal NgoMIV site found at 166
    Illegal NgoMIV site found at 1644
    Illegal NgoMIV site found at 1661
    Illegal NgoMIV site found at 1793
    Illegal NgoMIV site found at 1884
    Illegal NgoMIV site found at 2136
    Illegal AgeI site found at 1939
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 434
    Illegal SapI site found at 2145