Team:Mingdao/Basic Part

Basic Part

GAM1 promoter / pSB1C3

Part: BBa_K2543001

GAM1 is an inducible promoter from mosquitoes and regulated by Toll signaling in the mosquito defense system. The promoter drives the immune responsive antimicrobial peptide (AMP), Gambicin, to kill both Gram-positive and Gram-negative bacteria. And it can control Dengue virus infection and malaria parasite through Toll pathway. It works both in mosquitoes (e.g, Anopheles gambiae, Aedes aegypti, Aedes albopictus, etc.) and insect cell lines (e.g, Drosophila S2 cells, Aag2 cells, C6/36 cells, etc.)

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.

AMP promoters amplified by PCR

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

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.

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.

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.

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.

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

Introduction

Model 1

Model 2

Conclusion