Difference between revisions of "Part:BBa K3132000"
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==Contribution==
 
==Contribution==
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Group: Valencia_UPV iGEM 2018
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Group: CSU-CHINA 2023
 
<br>
 
<br>
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Author: Adrián Requena Gutiérrez, Carolina Ropero
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__NOTOC__
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<br>
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<partinfo>BBa_K4585012 short</partinfo>
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Summary: We have adapted the part to be able to assemble transcriptional units with the Golden Gate method and we have done the characterization of this RBS.
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<br>
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The pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid, which could express GAL4-VP64, was used for Luciferase detection experiment. GAL4 is a protein that can find and bind UAS (upstream activation sequence). VP64 is a transcription factor that, when used in combination with GAL4, can activate UAS and initiate the expression of downstream genes.
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Documentation:
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<html>
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<br>
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BBa_K2656011 is the RBS [https://parts.igem.org/Part:BBa_B0034 B0034] standardized into the Golden Braid assembly method. It also includes the BioBrick equivalent scar in the 3' extreme, so the insertion of this supplementary bases ensure correct spacing for the CDS expression when assembled into a TU.  
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<head>
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</head>
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<body>
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<h2 class="pageContent-main__title">
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                <!--put tile here, <h2>title</h2>, class="pageContent-main__title" means it is the main title-->
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                pcDNA3.1(+)-3×HA-GAL4-VP64-NLS
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            </h2>
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            <div class="pageContent-main__textBox">
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                <!--all the content must included in this div-->
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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>
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                <!--put text here, <p>content</p>-->
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            </div>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                1 Pattern Diagram
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            </h2>
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            <div class="pageContent-main__textBox">
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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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                <!--put image's url here-->
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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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            </div>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                <!--class="pageContent-main__title" means it is the sub title-->
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                2 Experiment
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            </h2>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                <!--class="pageContent-main__title" means it is the sub title-->
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                2.1 Method
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            </h2>
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            <div class="pageContent-main__textBox">
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                <!--all the content must included in this div-->
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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>
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                <!--put text here, <p>content</p>-->
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            </div>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                2.2 Results
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            </h2>
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            <div class="pageContent-main__textBox">
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                <!--all the content must included in this div-->
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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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<br />
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                <!--put image's url here-->
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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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            </div>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                <!--class="pageContent-main__title" means it is the sub title-->
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                3 Caution
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            </h2>
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            <div class="pageContent-main__textBox">
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                <!--all the content must included in this div-->
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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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                </p>
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            </div>
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</body>
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</html>
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<br />
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<!-- -->
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4585012 SequenceAndFeatures</partinfo>
  
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Characterization of the this part was performed with the transcriptional unit [https://parts.igem.org/Part:BBa_K2656101 BBa_K2656101 ], which was used in a comparative RBS expression experiment with composite parts [https://parts.igem.org/Part:BBa_K2656101 BBa_K2656101] and [https://parts.igem.org/Part:BBa_K2656102 BBa_K2656102].
 
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They all were assembled in a Golden Braid alpha1 plasmid using the same promoter, CDS and terminator.
 
  
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By using this [http://2018.igem.org/Team:Valencia_UPV/Experiments#exp_protocol experimental protocol], we have obtained the parameters to valide our  [http://2018.igem.org/Team:Valencia_UPV/Modeling#models constitutive model]and rationale choose its [http://2018.igem.org/Team:Valencia_UPV/Modeling#optimization optimization values] based on each RBS tested.
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K4585012 parameters</partinfo>
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Revision as of 09:25, 12 October 2023


Gal4-VP64

This part is transformed from the part: Gal4-KRAB(BBa_K2446037) of Igem17_Fudan. To turn this TF into an activating one, we replaced the KRAB domain with a VP64. And this approach successfully reversed its effect as we had expected. Gal4-VP64 containing three core domains from N-terminal to C-terminal: GAL4 DNA binding domain, nuclear location sequence and VP64 transcription regulating domain. And a (G4S) linker was added between DBD and NLS for providing region flexibility. GAL4DBD enable binding to specific DNA sequences, so that we can use Gal4-VP64 as a specific transcription factors to activate the expression of our downstream synthetic promoter elements and minimal CMV.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 41
    Illegal NgoMIV site found at 176
  • 1000
    COMPATIBLE WITH RFC[1000]


Contribution

Group: CSU-CHINA 2023

pcDNA3.1(+)-3XHA-GAL4-VP64-NLS

The pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid, which could express GAL4-VP64, was used for Luciferase detection experiment. GAL4 is a protein that can find and bind UAS (upstream activation sequence). VP64 is a transcription factor that, when used in combination with GAL4, can activate UAS and initiate the expression of downstream genes.

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℃.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 623
    Illegal XbaI site found at 584
    Illegal XbaI site found at 662
    Illegal SpeI site found at 5426
    Illegal PstI site found at 628
    Illegal PstI site found at 2057
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 623
    Illegal SpeI site found at 5426
    Illegal PstI site found at 628
    Illegal PstI site found at 2057
    Illegal NotI site found at 649
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 623
    Illegal BglII site found at 5189
    Illegal XhoI site found at 215
    Illegal XhoI site found at 656
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 623
    Illegal XbaI site found at 584
    Illegal XbaI site found at 662
    Illegal SpeI site found at 5426
    Illegal PstI site found at 628
    Illegal PstI site found at 2057
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 623
    Illegal XbaI site found at 584
    Illegal XbaI site found at 662
    Illegal SpeI site found at 5426
    Illegal PstI site found at 628
    Illegal PstI site found at 2057
    Illegal NgoMIV site found at 1167
    Illegal NgoMIV site found at 2508
    Illegal NgoMIV site found at 2793
    Illegal AgeI site found at 720
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 134
    Illegal BsaI.rc site found at 4321
    Illegal BsaI.rc site found at 6059
    Illegal SapI site found at 3238
    Illegal SapI.rc site found at 2357
    Illegal SapI.rc site found at 2567