Team:Mingdao/Part Collection

Part Collection

Part Collection

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.

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.

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

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.

UNIVERSAL PATHOGEN BLOOD TESTING KIT I

HUMAN HIV VIRUS BLOOD TESTING KIT II

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.)

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.

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

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.

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

Composite Part

Ac5-hCD4-dToll-polyA / pSB1C3

Part: BBa_K2543010

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).

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.

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.

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

Introduction

Model 1

Model 2

Conclusion