Difference between revisions of "Part:BBa K5375001"
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===Usage and Biology===
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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We performed pA7-GFP plasmid linearization by enzyme digest and electrophoresis. We utilized this digest DNA fragment for ligation with the target gene.
 
We performed pA7-GFP plasmid linearization by enzyme digest and electrophoresis. We utilized this digest DNA fragment for ligation with the target gene.
  
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![pA7-GFP vector digested](https://static.igem.wiki/teams/5375/parts/1.png)
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<div style="text-align:center;">
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**Figure 1.** The vector of pA7-GFP was enzyme digested.
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    <img src="https://static.igem.wiki/teams/5375/parts/1.png" width="70%" style="display:block; margin:auto;"
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        alt="pA7-GFP vector digested" >
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        <caption>
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        Figure 1. The vector of pA7-GFP was enzyme digested.
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        </caption>
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    </div>
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</div>
  
 
<span id="measurement-characterization"></span>
 
<span id="measurement-characterization"></span>
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To obtain the fusion protein and expression, we constructed plasmids **pA7-HSP70-GFP** and **pA7-PFN3-GFP** and validated them by Sanger sequencing as shown in the figures.
 
To obtain the fusion protein and expression, we constructed plasmids **pA7-HSP70-GFP** and **pA7-PFN3-GFP** and validated them by Sanger sequencing as shown in the figures.
  
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A. **pA7-GFP-PFN3** 
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<div style="text-align:center;">
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![pA7-GFP-PFN3 Sanger sequencing](https://static.igem.wiki/teams/5375/parts/2a.png)
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    <img src="https://static.igem.wiki/teams/5375/parts/2a.png" width="50%" style="display:block; margin:auto;"
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        alt="pA7-GFP-PFN3 Sanger sequencing" >
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    <div style="text-align:center;">
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        <caption>
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        A. pA7-GFP-PFN3
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        </caption>
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    </div>
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</div>
  
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B. **PA7-GFP-HSP70** 
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<div style="text-align:center;">
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![pA7-GFP-HSP70 Sanger sequencing](https://static.igem.wiki/teams/5375/parts/2b.png)
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    <img src="https://static.igem.wiki/teams/5375/parts/2b.png" width="50%" style="display:block; margin:auto;"
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        alt="pA7-GFP-HSP70 Sanger sequencing" >
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    <div style="text-align:center;">
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        <caption>
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        B. PA7-GFP-HSP70
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        </caption>
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    </div>
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</div>
  
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**Figure 2.** Sanger sequencing map of PA7-HSP70 and PA7-PFN3.
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<div style="text-align:center;">
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    <caption>
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    Figure 2. Sanger sequencing map of PA7-HSP70 and PA7-PFN3.
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    </caption>
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</div>
  
 
<span id="reference"></span>
 
<span id="reference"></span>

Revision as of 08:57, 25 September 2024

pA7-GFP

pA7-GFP

Usage and Biology

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BamHI site found at 4127
    Illegal XhoI site found at 3302
  • 23
    INCOMPATIBLE WITH RFC[23]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
  • 25
    INCOMPATIBLE WITH RFC[25]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 4091


Origin

Synthesized by company

Properties

Expression of protein GFP

Usage and Biology

Green Fluorescent Protein (GFP) is a bioluminescent protein initially isolated from the jellyfish *Aequorea victoria*, characterized by its distinctive biological properties and extensive applications. GFP exhibits spontaneous green fluorescence without the necessity for any substrates or cofactors, rendering it an invaluable tool in biological research.

In cell biology and molecular biology, GFP is frequently employed as a reporter gene through fusion with the coding sequence of target proteins, enabling real-time monitoring of their localization, dynamic alterations, and expression patterns within live cells. Furthermore, GFP can be utilized to label specific cellular organelles or structures, thereby assisting researchers in observing cellular processes such as protein transport, signal transduction pathways, and cell division. Owing to its stability and ease of detection, GFP has become an essential component of contemporary life sciences research and has significantly advanced our understanding of cellular biological mechanisms.

Cultivation, Purification and SDS-PAGE

We performed pA7-GFP plasmid linearization by enzyme digest and electrophoresis. We utilized this digest DNA fragment for ligation with the target gene. 

   <img src="1.png" width="70%" style="display:block; margin:auto;"
       alt="pA7-GFP vector digested" >

       
       Figure 1. The vector of pA7-GFP was enzyme digested.

Measurement and Characterization

To obtain the fusion protein and expression, we constructed plasmids **pA7-HSP70-GFP** and **pA7-PFN3-GFP** and validated them by Sanger sequencing as shown in the figures. 

   <img src="2a.png" width="50%" style="display:block; margin:auto;"
       alt="pA7-GFP-PFN3 Sanger sequencing" >

       
       A. pA7-GFP-PFN3
       

   <img src="2b.png" width="50%" style="display:block; margin:auto;"
       alt="pA7-GFP-HSP70 Sanger sequencing" >

       
       B. PA7-GFP-HSP70
       

   
   Figure 2. Sanger sequencing map of PA7-HSP70 and PA7-PFN3.

Reference

Chalfie M., Tu Y., Euskirchen G., Ward W. W., & Prasher D. C. (1994). Green fluorescent protein as a marker for gene expression. *Science*, 263(5153), 802-805. https://doi.org/10.1126/science.8333281

Cava F., de Pedro M. A., Blas-Galindo E., Waldo G. S., Westblade L. F., & Berenguer J. (2008). Expression and use of superfolder green fluorescent protein at high temperatures in vivo: a tool to study extreme thermophile biology. *Environmental microbiology*, 10(3), 605–613. https://doi.org/10.1111/j.1462-2920.2007.01482.x