Difference between revisions of "Part:BBa K4586019"

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==Usage==
 
==Usage==
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We used this basic part in our design to mediate the transfer of our transcription module VP64 into the nucleus to carry out its action on the target promoter that regulates the expression of our therapeutic agent (Cass12k).
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This part implemented in our design is a linker peptide to link between the elements of our internal domain the syn notch receptor which are the zinc finger peptide (ZF21.16) and, SV40 NLS and VP64 that represent our potent transcription module.
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==Literature Characterization==
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The study used G4S Linker (E7O2V) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody. The Engineered Cell Line was provided by the Lohmueller Lab, University of Pittsburgh. To express flow cytometry for testing of the G4S Linker.
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<html><div align="center"style="border:solid #17252A; width:50%;float:center;"><img style="                              max-width:850px;
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width:75%;
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height:auto;
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position: relative;
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top: 50%;
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left: 35%;
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transform: translate( -50%);
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padding-bottom:25px;
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padding-top:25px;
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"src="https://static.igem.wiki/teams/4586/wiki/literature-characterisation-parts/capture.png">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>The study used express flow cytometry to assess live Jurkat cells, which appear blue (negative), and engineered Jurkat cells, which appear green (positive). To express an scFv-based anti-CD19 CAR containing a G4S linker.
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  </span></p></div></html>
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<br><br><br><br>
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<html><div align="center"style="border:solid #17252A; width:50%;float:center;"><img style="                              max-width:850px;
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width:75%;
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height:auto;
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position: relative;
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top: 50%;
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left: 35%;
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transform: translate( -50%);
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padding-bottom:25px;
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padding-top:25px;
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"src="https://static.igem.wiki/teams/4586/wiki/literature-characterisation-parts/g4s-linker.png">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>Flow cytometry was used to watch the cell binding of GLPB30 antibodies to CAR T-cell constructs containing G4S-linked ScFvs. (A) By using an anti-human PE secondary antibody after an anti-G4S GLPB30 antibody, they could find 3–23/B3 CAR-transgene expression. Two days after CD3+ pan-T cell mRNA transduction, staining was carried out. (B) N-terminal MYC tags could be found on anti-CD19 CAR with an (G4S)3
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linker, and they could be observed on lentiviral-transfected pan-T cells. (c) In contrast to MYC-negative cells, MYC-positive CAR T cells showed higher GLPB30 staining.
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</span></p></div></html>
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==References==
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Köseer, Ayse Sedef, et al. "Immunotargeting of cancer stem cells." Cancers 15.5 (2023): 1608
  
  

Latest revision as of 15:24, 24 September 2023


G4S linker

Part Description

This part codes for the poly-Glycine-Serine (G4S) linker peptide that is a type of flexible,unstructured synthetic peptide linker sequence. G4S linker is composed of a core pentapeptide Gly-Gly-Gly-Gly-Ser that is repeated and usually found within scFV-based synthetic receptors as CARs and syn notch receptors.

Usage

This part implemented in our design is a linker peptide to link between the elements of our internal domain the syn notch receptor which are the zinc finger peptide (ZF21.16) and, SV40 NLS and VP64 that represent our potent transcription module.

Literature Characterization

The study used G4S Linker (E7O2V) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody. The Engineered Cell Line was provided by the Lohmueller Lab, University of Pittsburgh. To express flow cytometry for testing of the G4S Linker.

The study used express flow cytometry to assess live Jurkat cells, which appear blue (negative), and engineered Jurkat cells, which appear green (positive). To express an scFv-based anti-CD19 CAR containing a G4S linker.





Flow cytometry was used to watch the cell binding of GLPB30 antibodies to CAR T-cell constructs containing G4S-linked ScFvs. (A) By using an anti-human PE secondary antibody after an anti-G4S GLPB30 antibody, they could find 3–23/B3 CAR-transgene expression. Two days after CD3+ pan-T cell mRNA transduction, staining was carried out. (B) N-terminal MYC tags could be found on anti-CD19 CAR with an (G4S)3 linker, and they could be observed on lentiviral-transfected pan-T cells. (c) In contrast to MYC-negative cells, MYC-positive CAR T cells showed higher GLPB30 staining.

References

Köseer, Ayse Sedef, et al. "Immunotargeting of cancer stem cells." Cancers 15.5 (2023): 1608




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]