Difference between revisions of "Part:BBa K2543005"
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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.). GAM1-GFP-polyA can act as a reporter system which is activated by mosquito-borne pathogens.
 
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-GFP-polyA can act as a reporter system which is activated by mosquito-borne pathogens.
 
    
 
    
 +
 +
=== Mosquito Toll-AMP Signaling ===
 +
 +
Mosquito GAM1 promoter is one of the AMP promoters driven by Toll signaling and activated by mosquito-borne pathogens
 +
 +
[[File:T--Mingdao--samzzz5.png |300 px]]
 +
 +
<br /><br />
 +
 +
 +
=== 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====
 +
[[File:T--Mingdao--samzzz7.png | 500 px]]
 +
 +
 +
====GFP with GAM1-GFP-polyA/pSB1C3 induced by both Gram (-) E. coli and Gram (+) B. subtilis bacteria====
 +
 +
[[File:T--Mingdao--samzzz8.png | 500 px]]
 +
 +
 +
====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
 +
 +
[[File:T--Mingdao--samzzz9.png | 500 px]]
 +
 +
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.
 +
       
 +
 +
[[File:T--Mingdao--samzzz10.png | 500 px]]
 +
 +
 +
 +
== 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.
 +
  
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2543005 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2543005 SequenceAndFeatures</partinfo>
 +
 +
 +
===References===
 +
 +
1. [https://www.ncbi.nlm.nih.gov/pubmed/11606751 PNAS (2001) Gambicin: a novel immune responsive antimicrobial peptide from the malaria vector Anopheles gambiae.]<br />
 +
2. [https://www.ncbi.nlm.nih.gov/pubmed/17257211 Insect Mol Biol. (2007) Regulated expression of microinjected DNA in adult Aedes aegypti mosquitoes]<br />
 +
3. [https://www.ncbi.nlm.nih.gov/pubmed/18604274 PLoS Pathog. (2008) The Aedes aegypti toll pathway controls dengue virus infection.]<br />
 +
4. [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291090/ Front Cell Infect Microbiol. (2017) Regulation of Antimicrobial Peptides in Aedes aegypti Aag2 Cells]<br />
 +
5. [https://www.ncbi.nlm.nih.gov/pubmed/28533370 PNAS (2017) Insect pathogenic fungus interacts with the gut microbiota to accelerate mosquito mortality.]<br />
 +
  
  

Revision as of 02:28, 14 October 2018


GAM1-GFP-polyA / pSB1C3

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-GFP-polyA can act as a reporter system which is activated by mosquito-borne pathogens.


Mosquito Toll-AMP Signaling

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

T--Mingdao--samzzz5.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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2641
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1505


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.