Difference between revisions of "Part:BBa K2543001"
 
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<partinfo>BBa_K2543001 short</partinfo>
 
<partinfo>BBa_K2543001 short</partinfo>
  
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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.)  
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GAM1 is an inducible promoter from mosquitoes and regulated by Toll and IMD 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.)  
  
 
[[File:T--Mingdao--samzzz2.png |300 px]]
 
[[File:T--Mingdao--samzzz2.png |300 px]]
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=== Mosquito Toll-AMP Signaling ===
 
=== Mosquito Toll-AMP Signaling ===
  
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Mosquito GAM1 promoter is one of the AMP promoters driven by Toll signaling and activated by mosquito-borne pathogens
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Mosquito GAM1 promoter is one of the AMP promoters driven by Toll and IMD signaling and activated by mosquito-borne pathogens
  
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[[File:T--Mingdao--samzzz5.png |300 px]]
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[[File:T--Mingdao--samnew11111111111111.png |600 px]]
  
 
<br /><br />
 
<br /><br />
 
== GENE CLONING ==
 
== GENE CLONING ==
 
=== GAM1 Promoter Cloned from the Mosquito Genomic DNA ===
 
=== GAM1 Promoter Cloned from the Mosquito Genomic DNA ===
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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.
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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.<br /><br />
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[[File:T--Mingdao--samzzz3.png|400 px]]     
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[[File:T--Mingdao--samzzz3.png|500 px]]     
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<br /><br /><br />
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== FUNCTIONAL ASSAY ==  
 
== FUNCTIONAL ASSAY ==  
 
To test the function of GAM1 promoter, the part was assembled with GFP and polyA (Part: [https://parts.igem.org/Part:BBa_K2543005 BBa_K2543005], GAM1-GFP-polyA/pSB1C3)
 
To test the function of GAM1 promoter, the part was assembled with GFP and polyA (Part: [https://parts.igem.org/Part:BBa_K2543005 BBa_K2543005], GAM1-GFP-polyA/pSB1C3)
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To test the function of the devices, C6/36 cells were transfected with the plasmid vectors. And the mosquito cells were challenged with bacteria on 2 days after transfection.
 
To test the function of the devices, C6/36 cells were transfected with the plasmid vectors. And the mosquito cells were challenged with bacteria on 2 days after transfection.
  
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[[File:T--Mingdao--samzzz6.png | 400 px]]
 
  
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====GFP positive cell with GAM1-GFP-polyA / pSB1C3 challenged with E. coli====
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[[File:T--Mingdao--samzzz7.png | 500 px]]
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====GFP with GAM1-GFP-polyA/pSB1C3 induced by both Gram (-) E. coli and Gram (+) B. subtilis bacteria====
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[[File:T--Mingdao--samzzz8.png | 500 px]]
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====GFP signal dose-dependently increased with bacteria concentrations====
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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
 +
 +
[[File:T--Mingdao--samzzz9.png | 500 px]]
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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.
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=== Glowing mosquito with GAM1-GFP reporter and bacteria===
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To demonstrate in adult mosquitoes, we microinjected GAM1-GFP expression vector with heat-killed E. coli into the midgut of Aedes aegypti with the help of the expert in the Mosquito Lab at National Chung Hsing University.
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[[File:T--Mingdao--samzzz10.png | 500 px]]
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== CONCLUSION ==
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This year, we developed a mosquito blood surveillance system with GFP reporter and GAM1 promoter that can be induced by pathogens like E. coli, B. subtilit, etc. We demonstrated in mosquito cells by observing green fluorescence under microscope and GFP intensity by a microplate reader. Further, the adult female mosquito with our reporter plasmid DNA can detect the existence of E. coli.
  
  

Latest revision as of 03:38, 18 October 2018


GAM1 promoter / pSB1C3

GAM1 is an inducible promoter from mosquitoes and regulated by Toll and IMD 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.)

T--Mingdao--samzzz2.png



Mosquito Toll-AMP Signaling

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

T--Mingdao--samnew11111111111111.png



GENE CLONING

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.

T--Mingdao--samzzz3.png



FUNCTIONAL ASSAY

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

T--Mingdao--samzzz4.png


Green Fluorescence Observed by E. coli Challenge

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


GFP positive cell with GAM1-GFP-polyA / pSB1C3 challenged with E. coli

T--Mingdao--samzzz7.png


GFP with GAM1-GFP-polyA/pSB1C3 induced by both Gram (-) E. coli and Gram (+) B. subtilis bacteria

T--Mingdao--samzzz8.png


GFP signal dose-dependently 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

T--Mingdao--samzzz9.png

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 microinjected GAM1-GFP expression vector with heat-killed E. coli into the midgut of Aedes aegypti with the help of the expert in the Mosquito Lab at National Chung Hsing University.


T--Mingdao--samzzz10.png


CONCLUSION

This year, we developed a mosquito blood surveillance system with GFP reporter and GAM1 promoter that can be induced by pathogens like E. coli, B. subtilit, etc. We demonstrated in mosquito cells by observing green fluorescence under microscope and GFP intensity by a microplate reader. Further, the adult female mosquito with our reporter plasmid DNA can detect the existence of E. coli.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

References

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.