Difference between revisions of "Part:BBa K4585011"

 
Line 1: Line 1:
 +
__NOTOC__
 +
<partinfo>BBa_K4585011 short</partinfo>
  
 +
We obtained this plasmid by enzyme cutting and linking for intracellular calcium flow assay. This plasmid can be transformed into and amplified in <i>E. coli</i> and then used as a template for amplification. This plasmid was detected to be able to normally express GnRH-C protein in HEK 293T cells.
 +
<html>
 +
 +
<head>
 +
           
 +
</head>
 +
       
 +
<body>
 +
<h2 class="pageContent-main__title">
 +
                <!--put tile here, <h2>title</h2>, class="pageContent-main__title" means it is the main title-->
 +
                pcDNA3.1(+)-3×HA-GAL4-VP64-NLS
 +
            </h2>
 +
            <div class="pageContent-main__textBox">
 +
                <!--all the content must included in this div-->
 +
                <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>
 +
                <!--put text here, <p>content</p>-->
 +
            </div>
 +
            <h2 class="pageContent-main__title pageContent-main__subtitle">
 +
                <!--class="pageContent-main__title" means it is the sub title-->
 +
                1 Pattern Diagram
 +
            </h2>
 +
            <div class="pageContent-main__textBox">
 +
                <!--all the content must included in this div-->
 +
                <p style="text-align: center;">
 +
                    <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-->
 +
                <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>
 +
            <h2 class="pageContent-main__title pageContent-main__subtitle">
 +
                <!--class="pageContent-main__title" means it is the sub title-->
 +
                2 Experiment
 +
            </h2>
 +
            <h2 class="pageContent-main__title pageContent-main__subtitle">
 +
                <!--class="pageContent-main__title" means it is the sub title-->
 +
                2.1 Method
 +
            </h2>
 +
            <div class="pageContent-main__textBox">
 +
                <!--all the content must included in this div-->
 +
                <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>
 +
                <!--put text here, <p>content</p>-->
 +
            </div>
 +
            <h2 class="pageContent-main__title pageContent-main__subtitle">
 +
                <!--class="pageContent-main__title" means it is the sub title-->
 +
                2.2 Results
 +
            </h2>
 +
            <div class="pageContent-main__textBox">
 +
                <!--all the content must included in this div-->
 +
                <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 style="text-align: center;">
 +
                    <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-->
 +
                <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>
 +
            </div>
 +
            <h2 class="pageContent-main__title pageContent-main__subtitle">
 +
                <!--class="pageContent-main__title" means it is the sub title-->
 +
                3 Caution
 +
            </h2>
 +
            <div class="pageContent-main__textBox">
 +
                <!--all the content must included in this div-->
 +
                <p>After sequencing and ensuring the sequence was correct, we applied it to the experiments. Store at 4℃.
 +
                </p>
 +
            </div>
 +
</body>
 +
</html>
 +
<br />
 +
<!-- -->
 +
<span class='h3bb'>Sequence and Features</span>
 +
<partinfo>BBa_K4585012 SequenceAndFeatures</partinfo>
 +
 +
 +
<!-- Uncomment this to enable Functional Parameter display
 +
===Functional Parameters===
 +
<partinfo>BBa_K4585012 parameters</partinfo>
 +
<!-- -->

Revision as of 04:12, 9 October 2023

pcDNA3.1(+)-GnRH-C

We obtained this plasmid by enzyme cutting and linking for intracellular calcium flow assay. This plasmid can be transformed into and amplified in E. coli and then used as a template for amplification. This plasmid was detected to be able to normally express GnRH-C protein in HEK 293T cells.

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