Team:Mingdao/Demonstrate

Experiment

Experimental Materials & Flow Chart

Aedes aegypti is a strain of mosquitoes with available full genome sequence data and widely used to be genetically modified. We obtained the lab-reared mosquitoes from Dr. Wu-Chun Tu of National Chung Hsing University, Taiwan. And gDNAs of the mosquitos were freshly prepared for the following experiments.

C6/36 cells from Aedes albopictus has been extensively studied for mosquito gene regulation with high DNA transfection efficiency. The cell line was got from Bioresource Collection and Research Center (BCRC) in Taiwan. The cell culture and maintenance followed the instructions. The Toll signaling and AMP gene expression were tested in this cell line.

All of the experiments were instructed by Dr. Pei-Hong Chen and conducted in our biolab in Mingdao High School with the lab facility including molecular biology equipment, a fluorescence microscope (Leica Microsystems DFC7000 T) and a microplate reader (BioTeK Synergy H1).

C6/36 cells were cultured in T25 flask with MEM media supplemented with 10% FBS, P/S, L-glutamine, sodium pyruvate and maintained in the incubator at 28°C.

C6/36 cells were transfected with DNA in a 96-well plate using liposome-mediated method. The GFP intensity and GFP positive cells were analyzed and observed by the microplate reader and the fluorescence microscope, respectively.

Pest Control

To develop a pest control system, we constructed a GFP reporter expression vector for mosquito cells. We amplified the DNA fragments of a constitutive promoter from Drosophila actin 5c gene and an eukaryotic poly A signal (SV40 polyA) by PCR from pAc5.1 vector. The resulting DNA fragments were assembled with a BioBrick existing part of GFP to generate the reporter vector of Ac5-GFP-polyA / pSB1C3 (K2543004).

To test the reporter system, 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 promoters 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.

Blood Collector

To make a mosquito a natural syringe of blood collector, we want to realize a pathogen digested in midgut of mosquito. We fed Aedes aegypti with glucose water containing heat-killed E. coli. gDNAs were extracted for analysis 6 or 18 hr after feeding.

EXPERIMENT

Aedes aegypti
10% glucose water with or without heat-killed E. coli (109 cells/ml)
gDNA extracted at 0, 6, 18 hours after feeding
2 repeats for every group & one control of gDNA of E. coli
PCR with specific primer for A. aegypti or E. coli

PRIMERS

PRIMERS Aedes: (730 bp)
Act-2F (5′-ATGGTCGGYATGGGNCAGAAGGACTC-3′),
Act-2R (5′-TCGCACTTCATGATSGAGTTGTA-3′)
E. coil: (970 bp)
K12IS-L (5’-CGCGATGGAAGATGCTCTGTA-3’)
K12-R (5’-ATCCTGCGCACCAATCAACAA-3’)

RESULT

Genomic DNAs of Aedes aegypti were confirmed by PCR with Aedes primers in all groups, indicating the integrity of the extracted DNA. Feeding heat-killed bacteria to the mosquitoes, genomic DNAs of E. coli can be detected in the group at 6hr post-feeding and slightly decreased at 18hr post-feeding, demonstrating the existence of non-dividing bacteria at least within overnight in the gut of a mosquito.

Pathogen Surveillance

To engineer mosquitoes as a pathogen surveillance tool, we created a GFP reporter system in the mosquito immune system through Toll & AMP signaling. /p>

The three AMP promoters (GAM1, CecN, DefA) were successfully cloned from gDNA of Aedes aegypti and assembled in front of GFP-polyA in the expression vector.

AMP promoters amplified by PCR

Because CecN and DefA promoter sequences have either EcoRI, XbaI, SpeI or PstI sites, GAM1 promoters were used as a major device for the following studies.

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 and a microplate reader at Ex/Em = 480/520 nm, respectively

RESULT

We successfully assembled three AMP promoters with GFP and poly A to pSB1C3 vector. The function of the devices were tested by challenging with E. coli. The intensities were 5.31-fold, 3.02-fold and 2.29-fold increase for E. coli-induced GAM1, CecN and DefA promoter activities, respectively. The GFP positive cells after induction were clearly observed under fluorescence microscope.

To test the AMP promoter in response to Gram-negative and Gram-positive bacteria, we challenged GAM1 promoter with E. coli and Bacillus subtilis, respectively.

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 or B. subtilis 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. The result further indicated the AMP promoter may be activated in a cross-talk way between Toll and Imd signaling pathways.

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.

Human HIV Testing

Mosquito-borne diseases are those that could be transmitted to people via the bite of infected mosquitoes. Many mosquito-borne pathogens including bacteria and viruses such as Escherichia coli, Staphylococcus aureus, Dengue virus, etc., are able to induce strong immune signaling through the Toll-AMP pathway in a mosquito. However, a lot of human viruses like HIV, HBV, etc. belongs to non-mosquito-borne viruses which are unable to drive the mosquito immune signaling.

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 fusion protein of CD4-Toll was assembled with polyA and driven by Ac5 promoter.

To test feasibility of fusion CD4-Toll chimera, we challenged the mosquito cells co-transfected with GAM1-GFP-polyA and Ac5-CD4-Toll-polyA. Unfortunately, there’s no induction in the expression of CD4-Toll chimera.

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

As our previous data and the research paper (Front Cell Infect Microbiol., 2017) showed, GAM1 promoter can be induced by both Gram-negative and Gram-positive bacteria in a synergetic cross-reactive way involving Toll and Imd signaling. In searching for more specific promoter activity in Toll signaling, we found a Drosophila Toll specific promoter of Drosomycin widely used in Toll signaling studies. In addition, the Drosomycin promoter is also activate in Aedes albopictus cells and reprssed by mosquito virus, Semliki Forest virus (SFV4) (Insect Mol Biol., 2008).

To test feasibility again, 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 demonstrated 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.

This year, we assembled the team, did brainstorming, searched papers and asked a huge question from the daily life experienced by team members when they volunteered in hospital and in Africa. We are proud of ourselves to make a big achievement. We proved the concept of using GE mosquitoes to be a biodegradable syringe, a point-of-care blood testing device and used for blood surveillance. We’ve designed a prototype and did many human practices. We hope our product can come to the world to reach people in need.

Reference

1. Appl Environ Microbiol. (1995) Rapid and accurate identification of Escherichia coli K-12 strains.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC167724/

2. Science (2007) Genome sequence of Aedes aegypti, a major arbovirus vector
https://www.ncbi.nlm.nih.gov/pubmed/17510324

3. Insect Mol Biol. (2008) Semliki Forest virus strongly reduces mosquito host defence signaling.
https://www.ncbi.nlm.nih.gov/pubmed/18811601

4. J Am Mosq Control Assoc. (2010) Universal primers for the amplification and sequence analysis of actin-1 from diverse mosquito species
https://www.ncbi.nlm.nih.gov/pubmed/20649132

5. PLoS One. (2010) Dengue virus inhibits immune responses in Aedes aegypti cells.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0010678

6. Trends Parasitol. (2016) Mosquito Defense Strategies against Viral Infection.
https://www.ncbi.nlm.nih.gov/pubmed/26626596

7.Front Cell Infect Microbiol. (2017) Regulation of Antimicrobial Peptides in Aedes aegypti Aag2 Cells
https://www.ncbi.nlm.nih.gov/pubmed/28217557

8. Crit Rev Eukaryot Gene Expr. (2017) Genetically Modified Aedes aegypti to Control Dengue: A Review.
https://www.ncbi.nlm.nih.gov/pubmed/29283327

Syringe

GFP System

Mosquito Immune Signaling

AMP System