Difference between revisions of "Part:BBa K5375001"
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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= Properties =
 
= Properties =
  
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Expression of protein GFP
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Expression of Green Fluorescent Protein (GFP)
  
 
<span id="usage-and-biology"></span>
 
<span id="usage-and-biology"></span>
 
= Usage and Biology =
 
= Usage and Biology =
  
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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.  
+
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, 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.
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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.
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<span id="cultivation-purification-sds-page"></span>
 
<span id="cultivation-purification-sds-page"></span>
 
= Cultivation, Purification and SDS-PAGE =
 
= Cultivation, Purification and SDS-PAGE =
  
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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.
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We performed pA7-GFP plasmid linearization by enzyme digest and electrophoresis. This digest DNA fragment was utilized for ligation with the target gene.
  
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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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     <img src="https://static.igem.wiki/teams/5375/bba-k5375001/1.png" width="70%" style="display:block; margin:auto;"
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         alt="pA7-GFP vector digested" >
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         alt="The vector of pA7-GFP was enzyme digested" >
 
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<span id="measurement-characterization"></span>
 
<span id="measurement-characterization"></span>
 
= Measurement and Characterization =
 
= Measurement and Characterization =
  
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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.
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To obtain the fusion protein and its expression, we constructed plasmids pA7-HSP70-GFP and pA7-PFN3-GFP. Validation was conducted by Sanger sequencing, as illustrated in the following figures:
  
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         alt="pA7-GFP-PFN3 Sanger sequencing" >
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         alt="A. pA7-GFP-PFN3 and B. pA7-GFP-HSP70" >
 
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         A. pA7-GFP-PFN3
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         A. pA7-GFP-PFN3 and B. pA7-GFP-HSP70
 
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         alt="pA7-GFP-HSP70 Sanger sequencing" >
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         alt="Sanger sequencing map of pA7-HSP70 and pA7-PFN3" >
 
     <div style="text-align:center;">
 
     <div style="text-align:center;">
 
         <caption>
 
         <caption>
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         B. PA7-GFP-HSP70
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         Figure 2. Sanger sequencing map of pA7-HSP70 and pA7-PFN3.
 
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    <caption>
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    Figure 2. Sanger sequencing map of PA7-HSP70 and PA7-PFN3.
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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
 
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
  
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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
+
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

Revision as of 10:16, 25 September 2024

pA7-GFP

pA7-GFP


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 Green Fluorescent 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, 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. This digest DNA fragment was utilized for ligation with the target gene.

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

Measurement and Characterization

To obtain the fusion protein and its expression, we constructed plasmids pA7-HSP70-GFP and pA7-PFN3-GFP. Validation was conducted by Sanger sequencing, as illustrated in the following figures:

A. pA7-GFP-PFN3 and B. pA7-GFP-HSP70
A. pA7-GFP-PFN3 and B. pA7-GFP-HSP70
Sanger sequencing map of pA7-HSP70 and pA7-PFN3
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