Difference between revisions of "Part:BBa K2986004"
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2986004 short</partinfo>
 
<partinfo>BBa_K2986004 short</partinfo>
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<h2>Usage and Biology</h2>
 
<h2>Usage and Biology</h2>
  
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The luciferase from the marine copepod Gaussia princeps (Gluc) is the smallest coelenterazine (CTZ)-utilizing luciferase known thus far. It can emit green light and this property makes it to be used as a reporter gene. Gaussia luciferase exhibits an activity up to 1,000-fold higher than to Renilla reniformis luciferase (Rluc), firefly luciferase (Fluc), or bacterial luciferases (LuxAB). The outstanding sensitivity of Gluc-based assays was previously demonstrated detecting as low as 10−18 mol purified Gluc or one eukaryotic cell transiently expressing Gluc.
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The luciferase from the marine copepod Gaussia princeps (Gluc) is the smallest coelenterazine (CTZ)-utilizing luciferase known thus far. It can emit green light and this property makes it to be used as a reporter gene. Gaussia luciferase exhibits an activity up to 1,000-fold higher than to Renilla reniformis luciferase (Rluc), firefly luciferase (Fluc), or bacterial luciferases (LuxAB). The outstanding sensitivity of Gluc-based assays was previously demonstrated detecting as low as 10−18 mol purified Gluc or one eukaryotic cell transiently expressing Gluc.<br/>
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We decided to modify the [https://parts.igem.org/Part:BBa_K509005 Biobrick BBa_K509005]from iGEM11_UEA-JIC_Norwich 2011 team, this team optimized Gluc for use in the algal species Chlamydomonas reinhardtii. We designed to improve the Gluc to the humanized Gaussia princeps which can be expressed in mammalian cells. This made the visualization of the target gene expression possible if we ligated the hGluc with target gene.
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We decided to modify the <h4>[https://parts.igem.org/Part:BBa_K509005 Biobrick BBa_K509005]from iGEM11_UEA-JIC_Norwich 2011 team</h4> this team optimized Gluc for use in the algal species Chlamydomonas reinhardtii. We designed to improve the Gluc to the humanized Gaussia princeps which can be expressed in human cell lines.
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<h2>Design</h2>
 
<h2>Design</h2>
 
We wanted to design a gene expression system with hGluc as reporter, so that we can simplify the production detection process into fluorescent observation. We designed a plasmid ligate our target gene with hGluc using mammalian lentivirus expression vector. We use a 45bp linker to connect the hGluc with the CD11b transmembrane domain and use P2A (2A peptide allows an open reading frame (ORF) to translate a peptide chain into several independent peptide chains) connect with Interleukin 8 CDS, in our assumption this allow the stable expression of both our target gene and the hGluc in Hela cells.
 
We wanted to design a gene expression system with hGluc as reporter, so that we can simplify the production detection process into fluorescent observation. We designed a plasmid ligate our target gene with hGluc using mammalian lentivirus expression vector. We use a 45bp linker to connect the hGluc with the CD11b transmembrane domain and use P2A (2A peptide allows an open reading frame (ORF) to translate a peptide chain into several independent peptide chains) connect with Interleukin 8 CDS, in our assumption this allow the stable expression of both our target gene and the hGluc in Hela cells.
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===Location of features===
 
===Location of features===
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P2A :3990-4055<br/>
 
P2A :3990-4055<br/>
 
Interleukin 8 CDS :4056-4352<br/>
 
Interleukin 8 CDS :4056-4352<br/>
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Later we transfected this plasmid into Hela cells, we tested whether we successfully transfected. We use the hGluc kit to test the function of hGluc after assembled into the plasmid. The result showed that hGluc can normally paly its role.  
 
Later we transfected this plasmid into Hela cells, we tested whether we successfully transfected. We use the hGluc kit to test the function of hGluc after assembled into the plasmid. The result showed that hGluc can normally paly its role.  
 
And we tested the fluorescent activity under different light exposure time, and analyzed data is shown here.
 
And we tested the fluorescent activity under different light exposure time, and analyzed data is shown here.
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<html>
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<figure>
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    <img src="https://2019.igem.org/wiki/images/c/cc/T--SUSTech_Shenzhen--cck8.jpg" "alt="" style="width:50%;" />
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    <figcaption>hGluc chemiluminescence test</figcaption>
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</figure>
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</html>
  
 
[[File:T--SUSTech hGluc.png|450px|thumb|center|Figure2.hgluc activity after light exposure ]]
 
[[File:T--SUSTech hGluc.png|450px|thumb|center|Figure2.hgluc activity after light exposure ]]
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We characterized the transcription process by testing the change of RNA through quantitative PCR. Next, we characterized the translation process by testing the dynamic change of mRuby through flow cytometer. The final step is to characterize the secretion process. Since we have chosen hGluc as our target product, we did it by measuring the chemiluminescence value.
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[[File:T--SUSTech--yong13.png|400px|thumb|center|Figure3. Result of hGluc chemiluminescence value on secretion characterization]]
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For the hGluc chemiluminescence test, we characterized the secretion process after protein translation. This set of data enable us to characterize he relationship between light exposure and Gene expression on multi-level (transcription level, translation level and secretion level), which is vital for further acquisition of experimental parameters and model constructions.
  
  

Latest revision as of 21:26, 21 October 2019


hGluc

Usage and Biology

The luciferase from the marine copepod Gaussia princeps (Gluc) is the smallest coelenterazine (CTZ)-utilizing luciferase known thus far. It can emit green light and this property makes it to be used as a reporter gene. Gaussia luciferase exhibits an activity up to 1,000-fold higher than to Renilla reniformis luciferase (Rluc), firefly luciferase (Fluc), or bacterial luciferases (LuxAB). The outstanding sensitivity of Gluc-based assays was previously demonstrated detecting as low as 10−18 mol purified Gluc or one eukaryotic cell transiently expressing Gluc.

We decided to modify the

Biobrick BBa_K509005from iGEM11_UEA-JIC_Norwich 2011 team

this team optimized Gluc for use in the algal species Chlamydomonas reinhardtii. We designed to improve the Gluc to the humanized Gaussia princeps which can be expressed in human cell lines.


Design

We wanted to design a gene expression system with hGluc as reporter, so that we can simplify the production detection process into fluorescent observation. We designed a plasmid ligate our target gene with hGluc using mammalian lentivirus expression vector. We use a 45bp linker to connect the hGluc with the CD11b transmembrane domain and use P2A (2A peptide allows an open reading frame (ORF) to translate a peptide chain into several independent peptide chains) connect with Interleukin 8 CDS, in our assumption this allow the stable expression of both our target gene and the hGluc in Hela cells.


Location of features

Figure1.the plasmid used with hgluc

hGluc: 3120-3710
linker: 3711-3755
CD11b transmembrane domain: 3756-3989
P2A :3990-4055
Interleukin 8 CDS :4056-4352


Properties

Later we transfected this plasmid into Hela cells, we tested whether we successfully transfected. We use the hGluc kit to test the function of hGluc after assembled into the plasmid. The result showed that hGluc can normally paly its role. And we tested the fluorescent activity under different light exposure time, and analyzed data is shown here.

hGluc chemiluminescence test

Figure2.hgluc activity after light exposure

We characterized the transcription process by testing the change of RNA through quantitative PCR. Next, we characterized the translation process by testing the dynamic change of mRuby through flow cytometer. The final step is to characterize the secretion process. Since we have chosen hGluc as our target product, we did it by measuring the chemiluminescence value.

Figure3. Result of hGluc chemiluminescence value on secretion characterization

For the hGluc chemiluminescence test, we characterized the secretion process after protein translation. This set of data enable us to characterize he relationship between light exposure and Gene expression on multi-level (transcription level, translation level and secretion level), which is vital for further acquisition of experimental parameters and model constructions.


References

Tannous B. A. (2009). Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. Nature protocols, 4(4), 582–591. doi:10.1038/nprot.2009.28.


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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 604