Difference between revisions of "Part:BBa K4585012"
 
(23 intermediate revisions by the same user not shown)
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
−
<partinfo>BBa_K4585000 short</partinfo>
+
<partinfo>BBa_K4585012 short</partinfo>
  
−
A protein of truncated gonadotropin-releasing hormone.
+
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.
 
<html>
 
<html>
  
Line 13: Line 12:
 
<h2 class="pageContent-main__title">
 
<h2 class="pageContent-main__title">
 
                 <!--put tile here, <h2>title</h2>, class="pageContent-main__title" means it is the main title-->
 
                 <!--put tile here, <h2>title</h2>, class="pageContent-main__title" means it is the main title-->
−
                 Insulin
+
                 pcDNA3.1(+)-3×HA-GAL4-VP64-NLS
 
             </h2>
 
             </h2>
 
             <div class="pageContent-main__textBox">
 
             <div class="pageContent-main__textBox">
 
                 <!--all the content must included in this div-->
 
                 <!--all the content must included in this div-->
−
                 <p>We have proved that Insulin can be processed and secreted outside the cell in 293T cells.
+
                 <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.
 
                 </p>
 
                 </p>
 
                 <!--put text here, <p>content</p>-->
 
                 <!--put text here, <p>content</p>-->
Line 23: Line 22:
 
             <h2 class="pageContent-main__title pageContent-main__subtitle">
 
             <h2 class="pageContent-main__title pageContent-main__subtitle">
 
                 <!--class="pageContent-main__title" means it is the sub title-->
 
                 <!--class="pageContent-main__title" means it is the sub title-->
−
                 1 diagram
+
                 1 Pattern Diagram
 
             </h2>
 
             </h2>
 
             <div class="pageContent-main__textBox">
 
             <div class="pageContent-main__textBox">
 
                 <!--all the content must included in this div-->
 
                 <!--all the content must included in this div-->
 
                 <p style="text-align: center;">
 
                 <p style="text-align: center;">
−
                     <img width="400px" src="https://static.igem.wiki/teams/4585/wiki/gnrh.png"></p>
+
                     <img width="400px" src="https://static.igem.wiki/teams/4585/wiki/the-model-diagram-of-pcdna3-1-3-ha-gal4-vp64-nls.png"></p>
 +
                <br />
 
                 <!--put image's url here-->
 
                 <!--put image's url here-->
−
                 <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.1 The model diagram of 9XUAS-Insulin</p>
+
                 <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>
 
             </div>
 
             </div>
 
             <h2 class="pageContent-main__title pageContent-main__subtitle">
 
             <h2 class="pageContent-main__title pageContent-main__subtitle">
 
                 <!--class="pageContent-main__title" means it is the sub title-->
 
                 <!--class="pageContent-main__title" means it is the sub title-->
−
                 2.Experiment
+
                 2 Experiment
 
             </h2>
 
             </h2>
 
             <h2 class="pageContent-main__title pageContent-main__subtitle">
 
             <h2 class="pageContent-main__title pageContent-main__subtitle">
Line 42: Line 42:
 
             <div class="pageContent-main__textBox">
 
             <div class="pageContent-main__textBox">
 
                 <!--all the content must included in this div-->
 
                 <!--all the content must included in this div-->
−
                 <p>In the experiment, we used an ELIAS kit specially designed to detect human mature insulin to detect cell eliquidation.
+
                 <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.
 
                 </p>
 
                 </p>
 
                 <!--put text here, <p>content</p>-->
 
                 <!--put text here, <p>content</p>-->
Line 52: Line 52:
 
             <div class="pageContent-main__textBox">
 
             <div class="pageContent-main__textBox">
 
                 <!--all the content must included in this div-->
 
                 <!--all the content must included in this div-->
−
                 <p>Insulin can be processed and secreted outside the cell in 293T cells.
+
                 <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.
 
                 </p>
 
                 </p>
 
                 <p style="text-align: center;">
 
                 <p style="text-align: center;">
−
                     <img width="400px" src="https://2021.igem.org/wiki/images/5/58/T--CSU_CHINA--tupian30.png"></p>
+
                     <img width="400px" src="https://static.igem.wiki/teams/4585/wiki/biofluorescence-intensity-when-gal4-vp64-gal4-krab-400-ng.png"></p>
 +
<br />
 
                 <!--put image's url here-->
 
                 <!--put image's url here-->
−
                 <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.2 Standard curve of absorbance and insulin concentration</p>
+
                 <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>
−
                <p style="text-align: center;">
+
−
                    <img width="400px" src="https://2021.igem.org/wiki/images/5/57/T--CSU_CHINA--tupian31.png"></p>
+
−
                <!--put image's url here-->
+
−
                <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.3 Insulin concentration in supernatant after blue light irradiation and dark treatment respectively</p>
+
−
                <p style="text-align: center;">
+
−
                    <img width="400px" src="https://2021.igem.org/wiki/images/6/6c/T--CSU_CHINA--tupian32.png"></p>
+
−
                <!--put image's url here-->
+
−
                <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.4 Insulin concentration in supernatant (excluding insulin in medium) after blue light irradiation and dark treatment respectively</p>
+
 
             </div>
 
             </div>
 
             <h2 class="pageContent-main__title pageContent-main__subtitle">
 
             <h2 class="pageContent-main__title pageContent-main__subtitle">
 
                 <!--class="pageContent-main__title" means it is the sub title-->
 
                 <!--class="pageContent-main__title" means it is the sub title-->
−
                 3.Caution
+
                 3 Caution
 
             </h2>
 
             </h2>
 
             <div class="pageContent-main__textBox">
 
             <div class="pageContent-main__textBox">
 
                 <!--all the content must included in this div-->
 
                 <!--all the content must included in this div-->
−
                 <p>Insulin is not suitable for long time preservation under the best culture temperature of cells (37℃). In the mean time, cells would also consume some of the expressed insulin. So the best tst time for Insulin is approximately 24 hours
+
                 <p>After sequencing and ensuring the sequence was correct, we applied it to the experiments. Store at 4℃.
−
                    after transfection, it is relatively shorter than the test interval of LUC.
+
−
                </p>
+
−
            </div>
+
−
            <h2 class="pageContent-main__title pageContent-main__subtitle">
+
−
                <!--class="pageContent-main__title" means it is the sub title-->
+
−
                Reference：
+
−
            </h2>
+
−
            <div class="pageContent-main__textBox">
+
−
                <!--all the content must included in this div-->
+
−
                <p>[1]Mingqi Xie, Haifeng Ye, Hui Wang.β-cell-mimetic designer cells provide closed-loop glycemic control[J].Science.2016 Dec 9;354(6317):1296-1301.
+
 
                 </p>
 
                 </p>
 
             </div>
 
             </div>
 
</body>
 
</body>
 
</html>
 
</html>
−
 
+
<br />
−
<!-- Add more about the biology of this part here
+
−
===Usage and Biology===
+
−
 
+
 
<!-- -->
 
<!-- -->
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
−
<partinfo>BBa_K4585000 SequenceAndFeatures</partinfo>
+
<partinfo>BBa_K4585012 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
−
<partinfo>BBa_K4585000 parameters</partinfo>
+
<partinfo>BBa_K4585012 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Latest revision as of 03:39, 9 October 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