Team:Mingdao/Parts

Description

GAM1 promoter cloned from the mosquito genomic DNA

The DNA fragment of GAM1 promoter was amplified from gDNA of Aedes aegypti by PCR. The PCR products were cloned onto pSB1C3 vector and the sequence was confirmed by sequencing.

To test the function of GAM1 promoter, the part was assembled with GFP and polyA (Part: BBa_K2543005, GAM1-GFP-polyA/pSB1C3)

Mosquito GAM1 promoter is one of the AMP promoters driven by Toll and IMD signaling and activated by mosquito-borne pathogens

Green fluorescence observed by E. coli challenge

To test the function of the devices, C6/36 cells were transfected with the vectors. And the mosquito cells were challenged with bacteria on 2 days after transfection.

EXPERIMENT

↓ C6/36 cells were seeded at the density of 1.8 x 10⁵ cell/well in a 96-well plate
↓ Cells were transfected with the GAM1-GFP-polyA vector
↓ E. coli was added on 2 days post-transfection at MOI=10
↓ GFP positive cells and intensity were analyzed by a fluorescence microscope

RESULT

The figure showed ~50% GFP positive cells were present in the existence of E. coli under fluorescence microscope.

GFP induced by both Gram (+) and Gram (-) bacteria

EXPERIMENT

↓ C6/36 cells were seeded at the density of 1.8 x 10⁵ cell/well in a 96-well plate
↓ Cells were transfected with the AMP-GFP-polyA vectors
↓ E. coli was added on 2 days post-transfection at MOI=10
↓ GFP intensity was measured by a microplate reader at Ex/Em = 480/520 nm.

RESULT

The data represented in C6/36 cells showed that GAM1 promoter was not only activated by Gram-negative E. coli but also induced by Gram-positive B. subtilis.

GFP signal increased with bacteria concentrations

To verify the application of GAM1 promoter as a biosensor to measure the amounts of pathogens, E. coli at various concentrations were added onto the mosquito cells transfected with the GAM1-GFP-polyA / pSB1C3

EXPERIMENT

↓ C6/36 cells were seeded at the density of 1.8 x 10⁵ cell/well in a 96-well plate
↓ Cells were transfected with GAM1-GFP-polyA or Ac5-GFP-polyA vectors
↓ E. coli at MOI=2, 4, 8, 16, 32 were added on 2 days post-transfection
↓ GFP intensity was measured by a microplate reader at Ex/Em = 480/520 nm.

RESULT

As figures shown above, the green fluorescence intensities driven by GAM1 promoter were increased dose-dependently in the presence of E. coli at MOIs from 2 to 32. The fluorescence expressed by Ac5 promoter was not influenced at the same condition. These results demonstrated GAM1-GFP reporter system can used in the mosquito cells as a biosensor in response of different concentrations of bacteria.

CONCLUSION

Taken together, we created a GFP reporter system driven under AMP promoter by Toll signaling. The expression of GFP can be induced by bacteria in a dose-dependent manner. The green fluorescence observed under microscope further proved the concept of GE mosquito cells as a pathogen surveillance tool.

Glowing mosquitoes with GAM1-GFP reporter and bacteria

To demonstrate in adult mosquitoes, we collaborated with iGEM Team NCHU_Taichung to microinject DNA into Aedes aegypti. We prepared the plasmid of GAM1-GFP-polyA / pSB1C3 and heat-killed E. coli. A member who works in Entomology Department of National Chung Hsing University take us to the mosquito lab and helped us inject the materials to the midgut of Aedes aegypti.

The mosquitoes injected with DNA plus E. coli showed fluorescence signal in Gel Imaging System and Blue LED Box.

Reference

1. PNAS (2001) Gambicin: a novel immune responsive antimicrobial peptide from the malaria vector Anopheles gambiae.

2. Insect Mol Biol. (2007) Regulated expression of microinjected DNA in adult Aedes aegypti mosquitoes.

3. PLoS Pathog. (2008) The Aedes aegypti toll pathway controls dengue virus infection.

4. Front Cell Infect Microbiol. (2017) Regulation of Antimicrobial Peptides in Aedes aegypti Aag2 Cells

5. PNAS (2017) Insect pathogenic fungus interacts with the gut microbiota to accelerate mosquito mortality.

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.

Mosquito Toll-Amp signaling plays an important roll in response to and regulating blood infectious pathogens.

To genetically engineer mosquitoes as blood testing tools, we’ve collected DNA materials as BioBrick parts for future assembly.

First, we designed a universal pathogen blood testing kit consisting of BioBrick parts of GAM1 promoter, GFP and polyA, as well as Ac5 promoter as a positive expression control.

Second, we designed a HIV viral blood testing kit consisting of BioBrick parts of Ac5 promoter, CD4 extracellular domain, Drosophila transmembrane and intracellular domains, and poly A, as well as Ac5-GFP-polyA as a positive expression control.