Difference between revisions of "Part:BBa K1537016"

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2A peptide sequences were found in Picornaviruses to mediate "cleavage" between two proteins. We use 2A peptide-linked  
 
2A peptide sequences were found in Picornaviruses to mediate "cleavage" between two proteins. We use 2A peptide-linked  
 
multicistronic vectors to express multiple proteins from a single open reading frame (ORF) effectively. To minimize the risk of homologous recombination, it is important to use different 2A peptide sequences if more than two genes are being linked. The 2A peptide system has thus far worked successfully in all eukaryotic systems tested, from mammalian cells, yeast, and plants.(A El et al，2004)[[File:abc.jpg]]
 
multicistronic vectors to express multiple proteins from a single open reading frame (ORF) effectively. To minimize the risk of homologous recombination, it is important to use different 2A peptide sequences if more than two genes are being linked. The 2A peptide system has thus far worked successfully in all eukaryotic systems tested, from mammalian cells, yeast, and plants.(A El et al，2004)[[File:abc.jpg]]
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== '''GSG linker''' ==
 
== '''GSG linker''' ==
  
 
GSG linker is an oligopeptide of “Gly-Ser-Gly” between your protein and 2A peptide to enhance cleavage.( Andrea L et al，2012)
 
GSG linker is an oligopeptide of “Gly-Ser-Gly” between your protein and 2A peptide to enhance cleavage.( Andrea L et al，2012)
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== '''Improvements by AFCM-EGYPT 2020''' ==
 +
Team:<html><a href="https://2020.igem.org/Team:AFCM-Egypt">AFCM-Egypt 2020</a></html>has improved this part by inserting V5 epitope tag (GKPUPNPLLGLDST) and 3xFlag epitope tag immediately preceding 2A which has been shown to improve 2A cleavage efficiency. and provided characterization from literature that P2A has superior cleavage ability over other 2A peptides all of which is presented in the following part <html><a href="https://parts.igem.org/Part:BBa_K3504018">BBa_K3504018</a></html>
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[[Image:P2A_Sbol.png|thumb|Right|Figure 2. P2A+GSG enhancement using V5 epitope tag (GKPUPNPLLGLDST) and 3xFlag epitope tag.]]
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[[Image:P2A_Characterization.jpg|thumb|left|Figure 1. Comparison of different 2A peptides according to trastuzumab expression.]]<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />
 
=='''Characterization done by SUSTech_Shenzhen 2019'''==
 
=='''Characterization done by SUSTech_Shenzhen 2019'''==
  
 
SUSTech_Shenzhen 2019 iGEM team improved the characterization of this part by linked the 2A sequence with two target protein (cytokine IL-10 and eGFP, enhanced green fluorescent protein) and measured the expression level of the desired protein. By the measurement data, we conformed the feasibility of 2A peptide in a new chassis (HeLa cell). We also provided quantitative data to analyze the influence of 2A's cleavage function on the expression level of downstream protein.
 
SUSTech_Shenzhen 2019 iGEM team improved the characterization of this part by linked the 2A sequence with two target protein (cytokine IL-10 and eGFP, enhanced green fluorescent protein) and measured the expression level of the desired protein. By the measurement data, we conformed the feasibility of 2A peptide in a new chassis (HeLa cell). We also provided quantitative data to analyze the influence of 2A's cleavage function on the expression level of downstream protein.
  
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First, we obtained the DNA sequence of 2A peptide from 2019 iGEM distribution and linked our target protein sequence to construct two plasmid: 5xUAS-EGFP and 5xUAS-IL10-2A-EGFP, 5xUAS is the binding site of <a href="https://parts.igem.org/Part:BBa_K2986003">part BBa_K2986003</a>(GAVPO, a light-switchable trans activator).
+
First, we obtained the DNA sequence of 2A peptide from 2019 iGEM distribution and linked our target protein sequence to construct two plasmid: 5xUAS-EGFP and 5xUAS-IL10-2A-EGFP, 5xUAS is the binding site of <html><a href="https://parts.igem.org/Part:BBa_K2986003">part BBa_K2986003</a></html>(GAVPO, a light-switchable trans activator).
 
(figure1-2: plasmid construction)
 
(figure1-2: plasmid construction)
  
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<td>
 
<td>
 
<figure>
 
<figure>
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     <img src="https://2019.igem.org/wiki/images/2/29/T--SUSTech_Shenzhen--p2Aplasmid-1.png" alt="eGFP plasmid construction" style="width:75%;" />
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     <img src="https://2019.igem.org/wiki/images/2/29/T--SUSTech_Shenzhen--p2Aplasmid-1.png" alt="Figure 1: eGFP plasmid construction" style="width:100%;" />
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     <figcaption>eGFP plasmid construction</figcaption>
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     <figcaption>Figure1: eGFP plasmid construction</figcaption>
 
</figure>
 
</figure>
 
</td>
 
</td>
 
<td>
 
<td>
 
<figure>
 
<figure>
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     <img src="https://2019.igem.org/wiki/images/4/4f/T--SUSTech_Shenzhen--p2Aplasmid-2.png" alt="IL10-2A-eGFP plasmid construction" style="width:75%;" />
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     <img src="https://2019.igem.org/wiki/images/4/4f/T--SUSTech_Shenzhen--p2Aplasmid-2.png" alt="Figure2: IL10-2A-eGFP plasmid construction" style="width:100%;" />
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     <figcaption>IL10-2A-eGFP plasmid construction</figcaption>
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     <figcaption>Figure2: IL10-2A-eGFP plasmid construction</figcaption>
 
</figure>
 
</figure>
 
</td>
 
</td>
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</html>
 
</html>
 
Then, we transfect the plasmid sequence and the BBa_K2986003 into HeLa cell line, and measure the induced protein expression level. We used flow cytometry to measure the eGFP expression level, and ELISA (enzyme linked immunosorbent assay) method to measure the IL-10 expression level.
 
Then, we transfect the plasmid sequence and the BBa_K2986003 into HeLa cell line, and measure the induced protein expression level. We used flow cytometry to measure the eGFP expression level, and ELISA (enzyme linked immunosorbent assay) method to measure the IL-10 expression level.
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(figure3-4：flow cytometry of eGFP expression level) (figure4-5: ELISA test of IL-10)
 
(figure3-4：flow cytometry of eGFP expression level) (figure4-5: ELISA test of IL-10)
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3: https://2019.igem.org/File:T--SUSTech_Shenzhen--p2Aflow.png
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4: https://2019.igem.org/File:T--SUSTech_Shenzhen--p2Afigure.png
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<html>
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5: https://2019.igem.org/File:T--SUSTech_Shenzhen--p2AELISA-1.jpeg
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<table>
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6: https://2019.igem.org/File:T--SUSTech_Shenzhen--p2AELISA-2.jpg
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<tr>
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<td>
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<figure>
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    <img src="https://2019.igem.org/wiki/images/6/6b/T--SUSTech_Shenzhen--p2Aflow.png" alt="Figure 3: Flow cytometry of eGFP expression level of two cell lines" style="width:100%;" />
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    <figcaption>Figure 3: Flow cytometry of eGFP expression level of two cell lines</figcaption>
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</figure>
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</td>
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<td>
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<figure>
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    <img src="https://2019.igem.org/wiki/images/c/c4/T--SUSTech_Shenzhen--p2Afigure.png" alt="Figure 4: Comparison of eGFP expression level of two cell lines (with our without 2A sequence)"style="width:100%;" />
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    <figcaption>Figure 4: Comparison of eGFP expression level of two cell lines (with our without 2A sequence)</figcaption>
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</figure>
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</td>
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</tr>
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</table>
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<table>
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<tr>
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<td>
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<figure>
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    <img src="https://2019.igem.org/wiki/images/8/87/T--SUSTech_Shenzhen--p2AELISA-1.jpeg" alt="Figure 5: IL10 ELISA test" style="width:72%;" />
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    <figcaption>Figure 5: IL10 ELISA test</figcaption>
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</figure>
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</td>
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<td>
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<figure>
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    <img src="https://2019.igem.org/wiki/images/c/c4/T--SUSTech_Shenzhen--p2AELISA-2.jpg" alt="Figure 6: Result of IL10 ELISA test" style="width:100%;" />
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    <figcaption>Figure 6: Result of IL10 ELISA test</figcaption>
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</figure>
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</td>
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</tr>
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</table>
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</html>
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From figure 3-6, we conformed that this part is functional in HeLa cell line: the IL-10 and eGFP is cleaved by 2A peptide and successfully expressed.
 
From figure 3-6, we conformed that this part is functional in HeLa cell line: the IL-10 and eGFP is cleaved by 2A peptide and successfully expressed.

Latest revision as of 14:21, 20 October 2020

GSG linker+P2A


P2A

2A peptide sequences were found in Picornaviruses to mediate "cleavage" between two proteins. We use 2A peptide-linked multicistronic vectors to express multiple proteins from a single open reading frame (ORF) effectively. To minimize the risk of homologous recombination, it is important to use different 2A peptide sequences if more than two genes are being linked. The 2A peptide system has thus far worked successfully in all eukaryotic systems tested, from mammalian cells, yeast, and plants.(A El et al，2004)Abc.jpg

GSG linker

GSG linker is an oligopeptide of “Gly-Ser-Gly” between your protein and 2A peptide to enhance cleavage.( Andrea L et al，2012)

Improvements by AFCM-EGYPT 2020

Team:AFCM-Egypt 2020has improved this part by inserting V5 epitope tag (GKPUPNPLLGLDST) and 3xFlag epitope tag immediately preceding 2A which has been shown to improve 2A cleavage efficiency. and provided characterization from literature that P2A has superior cleavage ability over other 2A peptides all of which is presented in the following part BBa_K3504018

Figure 2. P2A+GSG enhancement using V5 epitope tag (GKPUPNPLLGLDST) and 3xFlag epitope tag.
Figure 1. Comparison of different 2A peptides according to trastuzumab expression.














Characterization done by SUSTech_Shenzhen 2019

SUSTech_Shenzhen 2019 iGEM team improved the characterization of this part by linked the 2A sequence with two target protein (cytokine IL-10 and eGFP, enhanced green fluorescent protein) and measured the expression level of the desired protein. By the measurement data, we conformed the feasibility of 2A peptide in a new chassis (HeLa cell). We also provided quantitative data to analyze the influence of 2A's cleavage function on the expression level of downstream protein.

First, we obtained the DNA sequence of 2A peptide from 2019 iGEM distribution and linked our target protein sequence to construct two plasmid: 5xUAS-EGFP and 5xUAS-IL10-2A-EGFP, 5xUAS is the binding site of part BBa_K2986003(GAVPO, a light-switchable trans activator). (figure1-2: plasmid construction)

Figure 1: eGFP plasmid construction
Figure1: eGFP plasmid construction
Figure2: IL10-2A-eGFP plasmid construction
Figure2: IL10-2A-eGFP plasmid construction
Then, we transfect the plasmid sequence and the BBa_K2986003 into HeLa cell line, and measure the induced protein expression level. We used flow cytometry to measure the eGFP expression level, and ELISA (enzyme linked immunosorbent assay) method to measure the IL-10 expression level. (figure3-4：flow cytometry of eGFP expression level) (figure4-5: ELISA test of IL-10)

Figure 3: Flow cytometry of eGFP expression level of two cell lines
Figure 3: Flow cytometry of eGFP expression level of two cell lines
Figure 4: Comparison of eGFP expression level of two cell lines (with our without 2A sequence)
Figure 4: Comparison of eGFP expression level of two cell lines (with our without 2A sequence)
Figure 5: IL10 ELISA test
Figure 5: IL10 ELISA test
Figure 6: Result of IL10 ELISA test
Figure 6: Result of IL10 ELISA test


From figure 3-6, we conformed that this part is functional in HeLa cell line: the IL-10 and eGFP is cleaved by 2A peptide and successfully expressed. Our quantitative data from figure 3-4 also provided another characterization feature of 2A peptide: expression level of the latter protein linked after 2A (eGFP level in the IL10-2A-eGFP cell line) is lower than the control group without 2A (eGFP cell line). The eGFP expression level after 2A is decreased 29.64%.


References

Abdelhak El Amrani,Abdellah Barakate,Barak M. Askari, Xuejun Li, Alison G. Roberts,Martin D. Ryan, and Claire Halpin.(2004)Coordinate Expression and Independent Subcellular Targeting of Multiple Proteins from a Single Transgene.Plant Physiology.Vol. 135, pp. 16–24

Andrea L. Szymczak-Workman, Kate M. Vignali, and Dario A.A. Vignali.(2012)Design and construction of 2A vectors.Cold Spring Harb Protoc :199-204.


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
    COMPATIBLE WITH RFC[1000]