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 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 AMP-GFP-polyA vectors
↓ 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 mosquito 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 mosquito 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.