Signalling

Part:BBa_K2543010

Designed by: CHUNG-HSUAN HSIAO   Group: iGEM18_Mingdao   (2018-10-08)


Ac5-hCD4-dToll-polyA / pSB1C3

T--Mingdao--qam1.png

- Ac5 is a strong and constitutive promoter from Drosophila actin 5c gene and commonly used in insect expression system. Human CD4 (hCD4) is a cell marker expressed on the subtype of T helper cell. CD4 acts as a coreceptor to help T cell development and cell function.

- CD4 plays an important role in T cell activation and immune signaling. The extracellular domain of hCD4 (1-396 aa) can form dimer and regulate the function of T cell activation.

- Toll is a transmembrane protein involved in insect immune defense system to recognize pathogens like bacteria, viruses and fungi. Toll activated by pathogens transmits the signaling to express anti-microbial peptide (AMP) to kill the pathogens. - - Drosophila transmembrane domain (808-828 aa) and intracellular domain (829-1097 aa) of Toll (dToll) play an important roll in regulating the immune signaling.

- Polyadenylation (polyA) is one of the process of eukaryotic mRNA translation. It adds a poly(A) tail to protect mRNA from enzymatic degradation and aid in transcription termination. Polyadenylation signal of simian virus 40 (SV40 poly A) has been used widely in mammalian and many eukaryotic gene expression system.

- This construct creates a synthetic human CD4-drosophila Toll chimera receptor system. The system functions not only in response to human HIV virus and also transmit Toll signaling to activate the expression of anti-microbial peptide (AMP)

INTRODUCTION

HIV is a huge epidemic around the world which can cause AIDS in infected people. To identify HIV is very difficult for people in limited-resource countries and individuals who wants privacy. An easy-to-use, cheap and portable testing device is urgently need around the world.

To further engineer the mosquito to recognize HIV, we designed and created a synthetic HIV-specific receptor composed of human CD4 extracellular domain (1-396 aa) and drosophila transmembrane and intracellular domains (808-828 aa and 829-1097 aa, respectively) based on UniProt protein database.

The DNA sequences of human CD4 and Drosophila Toll domains were synthesized by Integrated DNA Technologies, Inc. (IDT). The DNAs were cloned onto pSB1C3 and confirmed by sequencing. The fusion protein of CD4-Toll was further assembled with polyA and driven by Ac5 promoter.


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FUNCTINOAL ASSAY

Ac5 Promoter with Strong Activity

To test the expression vector driven by Ac5 promoter, we cultured a mosquito Aedes albopictus C6/36 cell line and transfected cells with the plasmid of Ac5-GFP-polyA. GFP positive cells and intensity were analyzed 2 days after transfection.

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Signal Driven by CD4-Toll Chimera and Blocked by HIV

To test feasibility of fusion CD4-Toll chimera, we acquired the plasmid of Drosomycin promoter-luciferase from world-renowned insect geneticist, Dr. Jean-Luc Imler and conducted the luc reporter assay with Ac5-CD4-Toll-polyA in the mosquito cells.

T--Mingdao--qam5.png

EXPERIMENTAL DESIGN PRINCIPLE

DEMO VIDEO

See more on our wiki
[http://2018.igem.org/Team:Mingdao/Composite_Part Wiki of MINGDAO Composite Part]

CONCLUSION

This year we developed a human blood virus testing system using GE mosquitoes. The synthetic CD4-Toll chimera can trigger mosquito Toll-AMP signaling with Drosomycin protomer that is able to be repressed by the existence of gp120 of HIV particle. We will replace the luciferase gene with GFP in the Drosomycin reporter vector for the future application.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 104
    Illegal XhoI site found at 3691
    Illegal XhoI site found at 4153
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3760
    Illegal NgoMIV site found at 5079
    Illegal AgeI site found at 3742
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2923
    Illegal BsaI site found at 4549
    Illegal BsaI.rc site found at 2533

References

CD4

1. UniProtKB - P01730 (CD4_HUMAN)
2. Retrovirology. (2006) Association between disruption of CD4 receptor dimerization and increased human immunodeficiency virus type 1 entry
3. J Immunol. (2006) Evidence for a domain-swapped CD4 dimer as the coreceptor for binding to class II MHC.
4. J Immunol. (2006) Triggering of T cell activation via CD4 dimers.
5. J Biol Chem. (2014) Disulfide reduction in CD4 domain 1 or 2 is essential for interaction with HIV glycoprotein 120 (gp120), which impairs thioredoxin-driven CD4 dimerization.

Toll

1. UniProtKB - P08953 (TOLL_DROME)
2. Cell. (1988) The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein.
3. Nat Rev Immunol. (2006) Toll-like receptors as molecular switches.
4. J Immunol. (2011) The Drosophila Toll signaling pathway.



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