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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.) | + | 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.) |
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| − | {{:Team:Mingdao/test9}}
| + | [[File:T--Mingdao--samzzz2.png |300 px]] |
| − | <html lang="en">
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| − | <head>
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| − | <meta charset="UTF-8">
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| − | <link href='https://fonts.googleapis.com/css?family=Arizonia' rel='stylesheet'>
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| − | <link href='https://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet'>
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| − | <title>Description</title>
| + | |
| − | </head>
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| − | <style type="text/css"> | + | <br /><br /> |
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| − | * {
| + | === Mosquito Toll-AMP Signaling === |
| − | margin: 0;
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| − | padding: 0;
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| − | box-sizing: border-box; }
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| − | body {
| + | Mosquito GAM1 promoter is one of the AMP promoters driven by Toll and IMD signaling and activated by mosquito-borne pathogens |
| − | width: 100%;
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| − | margin: 0;
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| − | padding: 0;
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| − | font-family: 'Ubuntu' !important; }
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| | + | [[File:T--Mingdao--samnew11111111111111.png |600 px]] |
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| − | .bg-container {
| + | <br /><br /> |
| − | background-attachment: fixed;
| + | == GENE CLONING == |
| − | overflow: hidden;
| + | === GAM1 Promoter Cloned from the Mosquito Genomic DNA === |
| − | position: relative;
| + | 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 /> |
| − | background-image: url(https://static.igem.org/mediawiki/2018/b/b7/T--Mingdao--ModelingBackground.jpg);
| + | [[File:T--Mingdao--samzzz3.png|500 px]] |
| − | background-repeat: no-repeat;
| + | <br /><br /><br /> |
| − | background-size: 100%;
| + | |
| − | background-position: center;
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| − | }
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| − | .my-main-container {
| + | == FUNCTIONAL ASSAY == |
| − | width: 103.4%;
| + | 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) |
| − | padding: 50px 7%;
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| − | }
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| − | .main-content {
| + | [[File:T--Mingdao--samzzz4.png | 400 px]] |
| − | background-color: rgba(255,255,255, .7);
| + | |
| − | width: 75%;
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| − | margin-left: 11%;
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| − | padding: 50px;
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| − | min-height: 180vh;
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| − | z-index: 10; }
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| − | .text-area {
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| − | margin-bottom: 80px; }
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| − | .text-area h1 {
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| − | font-size: 130px;
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| − | font-weight: 700;
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| − | font-family: 'Arizonia' !important;
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| − | text-align: center;
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| − | margin-bottom: 1rem; }
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| − | .text-area p {
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| − | font-size: 22px;
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| − | font-weight: 500; }
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| − | .m-block img { | + | === Green Fluorescence Observed by E. coli Challenge === |
| − | width: 100%; }
| + | 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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| − | .path-btns {
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| − | width: 25px;
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| − | border-radius: 50%;
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| − | position: absolute;
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| − | background-color: #fff;
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| − | top: 5px;
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| − | left: 27px;
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| − | border: 5px solid #385e66;
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| − | transition: all .3s;
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| − | transform: scale(1.2); }
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| | + | ====GFP positive cell with GAM1-GFP-polyA / pSB1C3 challenged with E. coli==== |
| | + | [[File:T--Mingdao--samzzz7.png | 500 px]] |
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| − | .path {
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| − | height: 100px;
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| − | display: flex; }
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| − | .path .pathSvg {
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| − | display: block; }
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| − | .path .pathWord {
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| − | padding-right: 10%; }
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| − | cursor: pointer;
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| − | fill: #fff; }
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| − | .path .path-btn.path-active {
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| − | fill: #385e66; }
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| − | .path .path-word-sm {
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| − | font-size: 12px; }
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| − | #HQ_page .path .pathWord p {
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| − | font-weight: 700;
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| − | text-align: left !important;
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| − | font-size: 16px;
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| − | }
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| − | #HQ_page .text-area p {
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| − | font-size: 22px;
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| − | font-family: 'Ubuntu';
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| − | font-weight: 500; }
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| − | img.center {
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| − | display: block;
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| − | margin: 0 auto; }
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| − | .text-area h2{
| + | ====GFP with GAM1-GFP-polyA/pSB1C3 induced by both Gram (-) E. coli and Gram (+) B. subtilis bacteria==== |
| − | text-align: center;
| + | |
| − | margin-bottom: 2rem !important;
| + | |
| − | margin-top: 2rem;
| + | |
| − | color: black !important;
| + | |
| − | font-size: 40px;
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| − | font-weight:bold;
| + | |
| − | font-family:serif;
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| − | }
| + | |
| − | .text-area h3{
| + | |
| − | text-align: left;
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| − | margin-bottom: 2rem !important;
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| − | margin-top: 2rem;
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| − | color: #4682B4 !important;
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| − | font-size: 28px;
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| − | font-weight:bold;
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| − | }
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| − | .text-area h4{
| + | [[File:T--Mingdao--samzzz8.png | 500 px]] |
| − | text-align: left;
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| − | margin-bottom: 2rem !important;
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| − | margin-top: 2rem;
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| − | color: #f9000c !important;
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| − | font-size: 30px;
| + | |
| − | font-weight:bold;
| + | |
| − | }
| + | |
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| − | .top {
| + | ====GFP signal dose-dependently increased with bacteria concentrations==== |
| − | position: fixed;
| + | |
| − | right: 50px;
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| − | bottom: 50px;
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| − | height: 50px;
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| − | width: 50px;
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| − | cursor: pointer; }
| + | |
| − | .top img {
| + | |
| − | width: 100%; }
| + | |
| − | img.pic {
| + | |
| − | display: block !important;
| + | |
| − | margin: 40px auto !important;
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| − | }
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| − | | + | |
| − | video {
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| − | position:relative;
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| − | width: 100%;
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| − | height:auto;
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| − | margin: 20px auto !important;
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| − | }
| + | |
| − | </style>
| + | |
| − | | + | |
| − | <body>
| + | |
| − | <div class="bg-container" style="max-height:none;">
| + | |
| − | <img class="top-picture" src="https://static.igem.org/mediawiki/2018/c/cd/T--Mingdao--Phil13-3.png" style="width:100%">
| + | |
| − | <div class="my-main-container">
| + | |
| − | <div class="main-content">
| + | |
| − | <div class="text-area">
| + | |
| − | <h1 id = "d-introduction"></h1>
| + | |
| − | | + | |
| − | <img class="center" src="https://static.igem.org/mediawiki/2018/a/a0/T--Mingdao--phil13M1.png" alt="" style="width: 80%; margin-bottom: 20px;">
| + | |
| − | <br /><br />
| + | |
| − | <h4> GAM1 promoter cloned from the mosquito genomic DNA </h4>
| + | |
| − | <p style="text-indent:2em">
| + | |
| − | 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 />
| + | |
| − | </p>
| + | |
| − | <img class="center" src="https://static.igem.org/mediawiki/2018/e/e8/T--Mingdao--phil13M3.png" alt="" style="width: 80%; margin-bottom: 20px;">
| + | |
| − |
| + | |
| − | <br /><br /><br /><br />
| + | |
| − | <p style="text-indent:2em">
| + | |
| − | To test the function of GAM1 promoter, the part was assembled with GFP and polyA (Part: BBa_K2543005, GAM1-GFP-polyA/pSB1C3)
| + | |
| − | <br /><br />
| + | |
| − | </p>
| + | |
| − | <img class="center" src="https://static.igem.org/mediawiki/2018/f/fb/T--Mingdao--phil13M4.png" alt="" style="width: 80%; margin-bottom: 20px;">
| + | |
| − | <br /><br /><br /><br />
| + | |
| − | <p style="text-indent:2em">
| + | |
| − | Mosquito GAM1 promoter is one of the AMP promoters driven by Toll signaling and activated by mosquito-borne pathogens
| + | |
| − | <br /><br />
| + | |
| − | </p>
| + | |
| − | <img class="center" src="https://static.igem.org/mediawiki/2018/b/b4/T--Mingdao--phil21.png" alt="" style="width: 60%; margin-bottom: 20px;">
| + | |
| − | <br />
| + | |
| − | <h4> Green fluorescence observed by E. coli challenge </h4>
| + | |
| − | <p style="text-indent:2em">
| + | |
| − | 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.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <h3>EXPERIMENT</h3>
| + | |
| − | <p>
| + | |
| − | ↓ C6/36 cells were seeded at the density of 1.8 x 10<sup>5</sup> cell/well in a 96-well plate<br />
| + | |
| − | ↓ Cells were transfected with the AMP-GFP-polyA vectors<br />
| + | |
| − | ↓ E. coli was added on 2 days post-transfection at MOI=10<br />
| + | |
| − | ↓ GFP positive cells and intensity were analyzed by a fluorescence microscope
| + | |
| − | <br /><br />
| + | |
| − | <img class="center" src="https://static.igem.org/mediawiki/2018/f/f4/T--Mingdao--phil13M6.png" alt="" style="width: 80%; margin-bottom: 20px;">
| + | |
| − | | + | |
| − | | + | |
| − | <h3>RESULT</h3>
| + | |
| − | <p style="text-indent:2em">
| + | |
| − | The figure showed ~50% GFP positive cells were present in the existence of E. coli under fluorescence microscope.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <br /><br />
| + | |
| − | <h4> GFP induced by both Gram (+) and Gram (-) bacteria </h4>
| + | |
| − | <h3>EXPERIMENT</h3>
| + | |
| − | <p>
| + | |
| − | ↓ C6/36 cells were seeded at the density of 1.8 x 10<sup>5</sup> cell/well in a 96-well plate<br />
| + | |
| − | ↓ Cells were transfected with the AMP-GFP-polyA vectors<br />
| + | |
| − | ↓ E. coli was added on 2 days post-transfection at MOI=10<br />
| + | |
| − | ↓ GFP intensity was measured by a microplate reader at Ex/Em = 480/520 nm.
| + | |
| − | <br /><br />
| + | |
| − | <img class="center" src="https://static.igem.org/mediawiki/2018/d/d1/T--Mingdao--phil13M7.png" alt="" style="width: 60%; margin-bottom: 20px;">
| + | |
| − | <h3>RESULT</h3>
| + | |
| − | <p style="text-indent:2em">
| + | |
| − | 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.
| + | |
| − | </p>
| + | |
| − | | + | |
| − | <br /><br /><br />
| + | |
| − | <h4> GFP signal increased with bacteria concentrations </h4>
| + | |
| − | <p style="text-indent:2em">
| + | |
| | 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 | | 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 |
| − | </p>
| |
| | | | |
| − | <h3>EXPERIMENT</h3>
| + | [[File:T--Mingdao--samzzz9.png | 500 px]] |
| − | <p>
| + | |
| − | ↓ C6/36 cells were seeded at the density of 1.8 x 10<sup>5</sup> cell/well in a 96-well plate<br />
| + | |
| − | ↓ Cells were transfected with GAM1-GFP-polyA or Ac5-GFP-polyA vectors<br />
| + | |
| − | ↓ E. coli at MOI=2, 4, 8, 16, 32 were added on 2 days post-transfection<br />
| + | |
| − | ↓ GFP intensity was measured by a microplate reader at Ex/Em = 480/520 nm.<br />
| + | |
| − | </p>
| + | |
| − | <br />
| + | |
| − | <img class="center" src="https://static.igem.org/mediawiki/2018/4/4a/T--Mingdao--phil28.png" alt="" style="width: 60%; margin-bottom: 20px;"><br />
| + | |
| − | <img class="center" src="https://static.igem.org/mediawiki/2018/d/d9/T--Mingdao--phil29.png" alt="" style="width: 60%; margin-bottom: 20px;">
| + | |
| | | | |
| − | <h3>RESULT</h3>
| |
| − | <p style="text-indent:2em">
| |
| − | 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.
| |
| − | </p>
| |
| − |
| |
| − | <h3>CONCLUSION</h3>
| |
| − | <p style="text-indent:2em">
| |
| | 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. | | 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. |
| − | </p>
| |
| − | <br /><br /><br /><br />
| |
| − | <h4>Glowing mosquito with GAM1-GFP reporter and bacteria</h4>
| |
| − | <p style="text-indent:2em">
| |
| − | 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.
| |
| − | </p>
| |
| − | <br />
| |
| − | <p style="text-indent:2em">
| |
| − | The mosquito injected with DNA plus E. coli showed fluorescence signal in Gel Imaging System and Blue LED Box.
| |
| − | </p>
| |
| − | <br />
| |
| − | <img class="center" src="https://static.igem.org/mediawiki/2018/7/70/T--Mingdao--phil13M9.png" alt="" style="width: 50%; margin-bottom: 20px;">
| |
| − |
| |
| − | <br /><br />
| |
| − | <h2>Reference</h2>
| |
| − | <p>
| |
| − | 1. <a href=https://www.ncbi.nlm.nih.gov/pubmed/11606751>PNAS (2001) Gambicin: a novel immune responsive antimicrobial peptide from the malaria vector Anopheles gambiae.</a> <br />
| |
| − | <p>
| |
| − | 2. <a href=https://www.ncbi.nlm.nih.gov/pubmed/17257211> Insect Mol Biol. (2007) Regulated expression of microinjected DNA in adult Aedes aegypti mosquitoes.</a> <br />
| |
| − | <p>
| |
| − | 3. <a href=https://www.ncbi.nlm.nih.gov/pubmed/18604274> PLoS Pathog. (2008) The Aedes aegypti toll pathway controls dengue virus infection.
| |
| − | </a> <br />
| |
| − | <p>
| |
| − | 4. <a href=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291090/>Front Cell Infect Microbiol. (2017) Regulation of Antimicrobial Peptides in Aedes aegypti Aag2 Cells</a> <br />
| |
| − | <p>
| |
| − | 5. <a href=https://www.ncbi.nlm.nih.gov/pubmed/28533370>PNAS (2017) Insect pathogenic fungus interacts with the gut microbiota to accelerate mosquito mortality.</a> <br />
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| − | <p>
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| − |
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| − | </p>
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| − |
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| − | </div>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="path-btns" style="left:0;">
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| − | <div class="path">
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| − | <div class="pathSvg">
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| − | <svg width="80" height = "100">
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| − | <rect x ="36" y="20" width="6" height="80" style="fill:#385e66"/>
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| − | </svg>
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| − | </div>
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| − | <div id="d-introduction-btn" class="path-dot"></div>
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| − | <div class="pathWord path-word-sm">
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| − | <p>Introduction</p>
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| − | </div>
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| − | </div>
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| − | <div class="path">
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| − | <div class="pathSvg">
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| − | <svg width="80" height = "100">
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| − | <rect x ="36" y="20" width="6" height="80" style="fill:#385e66"/>
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| − | </svg>
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| − | </div>
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| − | <div id="d-model1-btn" class="path-dot" style="top: 100px"></div>
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| − | <div class="pathWord path-word-sm">
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| − | <p>Model 1</p>
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| − | </div>
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| − | </div>
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| − | <div class="path">
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| − | <div class="pathSvg">
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| − | <svg width="80" height = "100">
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| − | <rect x ="36" y="20" width="6" height="80" style="fill:#385e66"/>
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| − | </svg>
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| − | </div>
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| − | <div id="d-model2-btn" class="path-dot" style="top: 200px"></div>
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| − | <div class="pathWord path-word-sm">
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| − | <p>Model 2</p>
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| − | </div>
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| − | </div>
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| − | <div class="path">
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| − | <div class="pathSvg">
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| − | <svg width="80" height = "100">
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| − | <rect x ="36" y="20" width="0" height="80" style="fill:#385e66"/>
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| − | </svg>
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| − | </div>
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| − | <div id="d-conclusion-btn" class="path-dot" style="top: 300px"></div>
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| − | <div class="pathWord path-word-sm">
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| − | <p>Conclusion</p>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="top">
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| − | <img class="center" src="https://static.igem.org/mediawiki/2017/5/52/T--CSMU_NCHU_Taiwan--top.png" alt="">
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| − | </body>
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| − | $(document).ready(function(){
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| − | $('.top').on('click', function(){
| |
| − | $('html, body').animate({scrollTop: '0px'}, 500);
| |
| − | });
| |
| − | $("#d-introduction-btn").css('background-color', '#385e66');
| |
| − | var scroll_pos = 0;
| |
| − | $(document).scroll(function() {
| |
| − | scroll_pos = $(this).scrollTop();
| |
| − |
| |
| − | d_introduction_pos = $("#d-introduction").offset().top -100
| |
| − | d_model1 = $("#d-model1").offset().top -100
| |
| − | d_model2= $("#d-model2").offset().top -100
| |
| − | d_conclusion_pos = $("#d-conclusion").offset().top -100
| |
| − |
| |
| − | // introduction
| |
| − | if(scroll_pos < d_model1_pos) {
| |
| − | $(".path-dot").css('background-color', '#fff')
| |
| − | $("#d-introduction-btn").css('background-color', '#385e66');
| |
| − | // model1
| |
| − | } else if(scroll_pos < d_model2_pos){
| |
| − | if(scroll_pos >= d_model1_pos){
| |
| − | $(".path-dot").css('background-color', '#fff')
| |
| − | $("#d-model1-btn").css('background-color', '#385e66');}
| |
| − |
| |
| − | // model2
| |
| − | } else if(scroll_pos < d_conclusion_pos){
| |
| − | if(scroll_pos >= d_model2_pos){
| |
| − | $(".path-dot").css('background-color', '#fff')
| |
| − | $("#d-model2-btn").css('background-color', '#385e66');}
| |
| − | }
| |
| − |
| |
| − | // conclusion
| |
| − | else if( scroll_pos >= d_conclusion_pos) {
| |
| − | $(".path-dot").css('background-color', '#fff')
| |
| − | $("#d-conclusion-btn").css('background-color', '#385e66');
| |
| − | }
| |
| − | });
| |
| − | });
| |
| | | | |
| − | </script>
| |
| | | | |
| − | </html>
| + | === Glowing mosquito with GAM1-GFP reporter and bacteria=== |
| − | {{:Team:Mingdao/test6}}
| + | 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. |
| | | | |
| | | | |
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.)
Mosquito GAM1 promoter is one of the AMP promoters driven by Toll and IMD signaling and activated by mosquito-borne pathogens
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.
To test the function of GAM1 promoter, the part was assembled with GFP and polyA (Part: BBa_K2543005, GAM1-GFP-polyA/pSB1C3)
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 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
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 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.
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.
