Difference between revisions of "Team:Mingdao/Composite Part"

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 Toll transmembrane and intracellular domains (808-828 aa and 829-1097 aa, respectively) based on UniProt protein database.

The DNA fragments of human CD4 and Drosophila Toll domains were synthesized by Integrated DNA Technologies, Inc. (IDT). The DNAs were cloned onto pSB1C3 as BioBrick basic parts and confirmed by sequencing. The CD4 and Toll was further assembled with Ac5 promoter and polyA to become BioBrcik composite parts. Finally, the fusion protein was made as Ac5-hCD4-dToll-polyA / pSB1C3 (BBa_K2543010)

Ac5 promoter with strong activity

To test the gene expression driven by Ac5 promoter, we cultured a mosquito Aedes albopictus C6/36 cell line and transfected cells with the plasmid of Ac5-GFP-polyA (K2543004). GFP positive cells and intensity were analyzed 2 days after transfection.

EXPERIMENT

↓ C6/36 cells (1.8 x 10⁵ cells/well in a 96-well plate)
↓ Liposome-mediated transfection and culture for 2 more days
↓ Read fluorescence intensity at Ex/Em = 480/520 nm with a microplate reader
↓ Observe GFP+ cells under a fluorescence microscope

RESULT

As data shown here, Ac5 is a strong and constitutive promoter which can drive GFP to high expression level in mosquito cells. And we can transfect more than 50% of GFP positive cell with liposome-mediated DNA delivery.

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.

EXPERIMENT

↓ C6/36 cells (1.8 x 10⁵ cells/well in a 96-well plate)
↓ Liposome-mediated transfection and culture for 2 more days
↓ Add gp120 of HIV (1 μg/ml*) or not and incubate for 24 hours
↓ Cell lysis and luciferase assay
*The concentration of gp120 in the serum of HIV-infected people is between 0.12~1 μg/ml.

RESULT

The result indicated that luciferase activity driven by Drosomycin promoter can be triggered by CD4-Toll chimera. The activity was decreased in the presence of gp120 of HIV. The finding demonstrates the possibility that GE mosquito created by our project could be applied to detect HIV virus in infected human blood.

Design principle of GE mosquito

Reference

1. UniProtKB - P01730 (CD4_HUMAN)

2. UniProtKB - P08953 (TOLL_DROME)

3. Cell. (1988) The Toll gene of Drosophila, required for dorsal-ventral embryonic polarity, appears to encode a transmembrane protein.

4. J Immunol. (2006) Evidence for a domain-swapped CD4 dimer as the coreceptor for binding to class II MHC.

5. J Immunol. (2006) Triggering of T cell activation via CD4 dimers.

6. Nat Rev Immunol. (2006) Toll-like receptors as molecular switches.

7. Retrovirology. (2006) Association between disruption of CD4 receptor dimerization and increased human immunodeficiency virus type 1 entry

8. J Immunol. (2011) The Drosophila Toll signaling pathway.

9. 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.