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| <!-- 极速模式 --> | | <!-- 极速模式 --> |
− | <title>Application</title> | + | <title>Proof</title> |
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| padding-left: 20%; | | padding-left: 20%; |
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| + | .text-content article div.carousel-inner img { |
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| + | .text-content article a.btn { |
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| <body> | | <body> |
| <header class="header"> | | <header class="header"> |
| + | <div class="navbar-logo"> |
| + | <a class="navbar-brand" href="https://igem.org/Main_Page"> |
| + | <img alt="Brand" src="https://2016.igem.org/wiki/skins/Igem/images/IGEM_white_letters.png"> |
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| </ul> | | </ul> |
| </li> | | </li> |
− | <li class="dropdown1"><a class="down-scroll" href="https://2016.igem.org/Team:HUST-China/Model">MODELING</a> | + | <li class="dropdown1"><a class="down-scroll" href="#">MODELING</a> |
| <ul class="dropdown2"> | | <ul class="dropdown2"> |
| <li><a href="https://2016.igem.org/Team:HUST-China/Model">Overview</a></li> | | <li><a href="https://2016.igem.org/Team:HUST-China/Model">Overview</a></li> |
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| </ul> | | </ul> |
| </li> | | </li> |
− | <li class="dropdown1"><a class="down-scroll" href="">PARTS</a> | + | <li class="dropdown1"><a class="down-scroll" href="#">PARTS</a> |
| <ul class="dropdown2"> | | <ul class="dropdown2"> |
| <li><a href="https://2016.igem.org/Team:HUST-China/Parts">Summary</a></li> | | <li><a href="https://2016.igem.org/Team:HUST-China/Parts">Summary</a></li> |
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| </ul> | | </ul> |
| </li> | | </li> |
− | <li class="dropdown1"><a class="down-scroll" href="">TEAM</a> | + | <li class="dropdown1"><a class="down-scroll" href="#">TEAM</a> |
| <ul class="dropdown2"> | | <ul class="dropdown2"> |
| <li><a href="https://2016.igem.org/Team:HUST-China/Team">Team Roster</a></li> | | <li><a href="https://2016.igem.org/Team:HUST-China/Team">Team Roster</a></li> |
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| <div class="container-fluid member-head"> | | <div class="container-fluid member-head"> |
| <div class="container"> | | <div class="container"> |
− | <h2 class="text-center">Proof Of Concept</h2> | + | <h2 class="text-center">Proof of Concept</h2> |
| </div> | | </div> |
| </div> | | </div> |
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| <h3>Hello world</h3> | | <h3>Hello world</h3> |
| <p>fdwfewf</p> --> | | <p>fdwfewf</p> --> |
− | <h2 id="location_Pro_ver">Prokaryotic version:</h2> | + | <h2 id="location_Pro_ver">Prokaryotic version</h2> |
| <p> | | <p> |
| It is a tri-stable system adapted from bacteriophage λ operon. In the operon, promoter RE is activated by CII trascriptional activator. Ftsh is an ATP-dependent host metalloprotease which will normally degrade CII, while CIII serves as an inhibitor of Ftsh to free CII. CI can function as an inhibitor to block pR, while Cro can bind pRM to stop the expression of downstream gene. The positive feedback control under pRE is used to enhance pulse signal 1 and convert it into robust stable signal. | | It is a tri-stable system adapted from bacteriophage λ operon. In the operon, promoter RE is activated by CII trascriptional activator. Ftsh is an ATP-dependent host metalloprotease which will normally degrade CII, while CIII serves as an inhibitor of Ftsh to free CII. CI can function as an inhibitor to block pR, while Cro can bind pRM to stop the expression of downstream gene. The positive feedback control under pRE is used to enhance pulse signal 1 and convert it into robust stable signal. |
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| <!-- 图片 --> | | <!-- 图片 --> |
| <img src="https://static.igem.org/mediawiki/2016/8/88/T--HUST-China--Description-Fig-Eukaryote.png" alt="" class="img-responsive"> | | <img src="https://static.igem.org/mediawiki/2016/8/88/T--HUST-China--Description-Fig-Eukaryote.png" alt="" class="img-responsive"> |
− | <p class="text-center">Fig*: circuit of prokaryote version serving as tri-stable signal filter</p> | + | <p style="text-align:center">Fig 1: circuit of prokaryote version serving as tri-stable signal filter</p> |
| <!-- <h2>Creation based on Signal Filter</h2> --> | | <!-- <h2>Creation based on Signal Filter</h2> --> |
| <!-- 文字 --> | | <!-- 文字 --> |
| <p> | | <p> |
− | When signal 1 is sensed, a double promoter structure will help induce cro and CII downstream pRE. At the same time constitutively produced CIII will guarantee enough CII to enhance the downstream transcription of the pRE. Thus, there forms a positive feedback loop to direct a fast and strong expression of cro. A quantity of Cro represses transcription under pRM by binding to cro binding site and blocks gene of interest 2’s expression, so it turns to a stable state of expressing gene of interest 1. | + | When pulse 1 is sensed, a double promoter structure will help induce cro and cII downstream pRE. At the same time constitutively produced CIII will guarantee enough CII to enhance the downstream transcription of the pRE. Thus, there forms a positive feedback loop to direct a fast and strong expression of cro. A quantity of Cro represses transcription under pRM by binding to cro binding site and blocks gene of interest 2’s expression, so it turns to a stable state of expressing gene of interest 1. |
| </p> | | </p> |
| <!-- <img src="" alt=""> --> | | <!-- <img src="" alt=""> --> |
− | <p>When signal 2 comes, under a certain inducible promoter, CI will be expressed and then binds to CI binding site within pR promoter, blocking gene of interest 1 and CIII’s expressions. With CIII’s reduction, FtsH gradually degrades CII to interrupt the stable state. Therefore, expression of cro immediately drops down, allowing transcription from pRM. The system turns to another stable state of gene of interest 2’s expression. | + | <p>When pulse 2 comes, under a certain inducible promoter, CI will be expressed and then binds to CI binding site within pR promoter, blocking gene of interest 1 and CIII’s expressions. With CIII’s reduction, FtsH gradually degrades CII to interrupt the stable state. Therefore, expression of cro immediately drops down, allowing transcription from pRM. The system turns to another stable state of gene of interest 2’s expression. |
| </p> | | </p> |
| <p> | | <p> |
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| <div id="myCarousel" class="carousel slide"> | | <div id="myCarousel" class="carousel slide"> |
| <!-- 轮播(Carousel)指标 --> | | <!-- 轮播(Carousel)指标 --> |
− | <ol class="carousel-indicators">
| + | <ol class="carousel-indicators"> |
− | <li data-target="#myCarousel" data-slide-to="0" class="active"></li>
| + | <li data-target="#myCarousel" data-slide-to="0" class="active"></li> |
− | <li data-target="#myCarousel" data-slide-to="1"></li>
| + | <li data-target="#myCarousel" data-slide-to="1"></li> |
− | <li data-target="#myCarousel" data-slide-to="2"></li>
| + | <li data-target="#myCarousel" data-slide-to="2"></li> |
− | <!-- <li data-target="#myCarousel" data-slide-to="3"></li> -->
| + | <li data-target="#myCarousel" data-slide-to="3"></li> |
− | </ol>
| + | <li data-target="#myCarousel" data-slide-to="4"></li> |
| + | <li data-target="#myCarousel" data-slide-to="5"></li> |
| + | </ol> |
| <!-- 轮播(Carousel)项目 --> | | <!-- 轮播(Carousel)项目 --> |
| <div class="carousel-inner"> | | <div class="carousel-inner"> |
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| + | <div class="item active"> |
| <div class="container"> | | <div class="container"> |
| <div class="row"> | | <div class="row"> |
| <div class="col-md-6" style="margin:100px 0"> | | <div class="col-md-6" style="margin:100px 0"> |
| <div class="caption"> | | <div class="caption"> |
− | <p> | + | <p style="margin-left:100px"> |
− | A prokaryote version of our Signal Fliter. Users can define two input signals and three combinations of output. For more details, see to our description page | + | According to the Flourescence measurement curve, we can see clearly that GFP level increased over time and it showed significant difference from CK. See more details in our Results page. |
| </p> | | </p> |
− | <p><a href="#" class="btn btn-primary" role="button">More details</a></p> | + | <p><a href="https://2016.igem.org/Team:HUST-China/Results#location_pro&pro" class="btn btn-primary" role="button">More details</a></p> |
| </div> | | </div> |
| </div> | | </div> |
| <div class="col-md-6" style="margin:100px 0"> | | <div class="col-md-6" style="margin:100px 0"> |
− | <img src="https://static.igem.org/mediawiki/2016/8/88/T--HUST-China--Description-Fig-Eukaryote.png" alt="" class="img-responsive"> | + | <img src="https://static.igem.org/mediawiki/2016/e/ef/T--HUST-China--CII-pRE_plate.png" alt="" class="img-responsive"> |
| </div> | | </div> |
| </div> | | </div> |
| </div> | | </div> |
| </div> | | </div> |
− | <div class="item">
| + | |
| + | <div class="item"> |
| <div class="container"> | | <div class="container"> |
| <div class="row"> | | <div class="row"> |
− | <div class="col-md-6 " style="margin:100px 0"> | + | <div class="col-md-6" style="margin:100px 0"> |
| <div class="caption"> | | <div class="caption"> |
− | <p> | + | <p style="margin-left:70px"> |
− | Small in scale, big in thoughts! THis summer, we held a fabulous HUST-Cheering! conferrence. And anlong with our integrated Practices,see to HP page. | + | We did Fluorescence microscope detection after 30, 120 and 240 minutes induction. According to the figture below, we can tell qualitively that pRE leakage are at relative low level and CII can efficiently activate the promoter. See more details in our Results page. |
| </p> | | </p> |
− | <p><a href="#" class="btn btn-primary" role="button">More details</a></p> | + | <p><a href="https://2016.igem.org/Team:HUST-China/Results#location_pro&pro" class="btn btn-primary" role="button">More details</a></p> |
| </div> | | </div> |
| </div> | | </div> |
| <div class="col-md-6" style="margin:100px 0"> | | <div class="col-md-6" style="margin:100px 0"> |
− | <img src="https://static.igem.org/mediawiki/2016/6/64/T--HUST-China--Integrated_Practices-Fig2-4.jpeg" alt="" class="img-responsive"> | + | <img src="https://static.igem.org/mediawiki/2016/4/4b/T--HUST-China--Experiments-CII-pRE_Flou-detec.png" alt="" class="img-responsive"> |
| </div> | | </div> |
| </div> | | </div> |
| </div> | | </div> |
| </div> | | </div> |
− | <div class="item">
| + | <div class="item"> |
| <div class="container"> | | <div class="container"> |
| <div class="row"> | | <div class="row"> |
| <div class="col-md-6" style="margin:100px 0"> | | <div class="col-md-6" style="margin:100px 0"> |
| <div class="caption"> | | <div class="caption"> |
− | <p> | + | <p style="margin-left:100px"> |
− | Fluorescence measurement is the main characterization method this year. We employed GFP and RFP as reporters to build valid data of our circuits. For more results see to Wetlab Results!
| + | As the Relative Fluorescent intensity measurement data shows, CI can inhibit pR in minor degree but the leakage expression under pR can’t be ignored, so we should consider to increase the binding sites within pR or the amount of CI coding sequence in the circuit. See more details in our Results page. |
| </p> | | </p> |
− | <p><a href="#" class="btn btn-primary" role="button">More details</a></p> | + | <p><a href="https://2016.igem.org/Team:HUST-China/Results#location_pro&pro" class="btn btn-primary" role="button">More details</a></p> |
| </div> | | </div> |
| </div> | | </div> |
| <div class="col-md-6" style="margin:100px 0"> | | <div class="col-md-6" style="margin:100px 0"> |
− | <img src="https://static.igem.org/mediawiki/2016/f/fb/T--HUST-China--home-flou.png" alt="" class="img-responsive"> | + | <img src="https://static.igem.org/mediawiki/2016/d/d1/T--HUST-China--Experiments-CI-pR_plate.png" alt="" class="img-responsive"> |
| </div> | | </div> |
| </div> | | </div> |
| </div> | | </div> |
| </div> | | </div> |
− | </div>
| + | <div class="item"> |
− | <!-- 轮播(Carousel)导航 -->
| + | |
− | <a class="carousel-control left" href="#myCarousel" data-slide="prev" style="font-size:100px;">‹</a>
| + | |
− | <a class="carousel-control right" href="#myCarousel" data-slide="next" style="vertical-align:middle;font-size:100px">›</a>
| + | |
− | </div>
| + | |
− | </div>
| + | |
− | <h2 id="location_Euk_ver">Eukaryotic version:</h2>
| + | |
− | <p>It is a bi-stable system derived from Arabidopsis thaliana stress response system. Enzyme catalysis is the core of this design to increase the efficiency of state transition.
| + | |
− | </p>
| + | |
− | <img src="https://static.igem.org/mediawiki/2016/1/1e/T--HUST-China--Description-Fig-prokaryote.png" alt="" class="img-responsive">
| + | |
− | <p class="text-center">Fig*: circuit of Eukaryote version serving as bii-stable signal filter</p>
| + | |
− | <p>Clade A protein phosphatases type 2C(PP2CA) and Sucrose Nonfermenting1 Related Subfamily 2 (SnRK2s) protein kinase are both components of Abscisic acid signaling network in Arabidopsis thaliana. ABF2 is a leucine zipper transcription factor which basically binds ABA-response element (ABRE). ABF2 can be phosphorylated by SnRK2s and be efficiently dephosphorylated by PP2CA. Also, in the absence of Abscisic acid, SnRK2 kinases can be inactivated by PP2Cs thus shut down the gene expression efficiently.
| + | |
− | </p>
| + | |
− | <p>When signal 1(ON) comes, promoter RD29A drives expression of SnRK2.2, which can phosphorylate ABF2 (constitutively expressed). Compared with protein co-facter association, enzyme catalysis can produce a large quantity of phosphorylated ABF2 in a short time, then enhance pRD29A to turn on the gene of interest’s expression. When signal 2(OFF) comes, gene PP2CA expresses, dephosphorylates ABF2 and inactivates SnRK2.2, thus turning the system into OFF state.
| + | |
− | </p>
| + | |
− | <div class="container-fluid index-details1 index-details">
| + | |
− | <div id="myCarousel2" class="carousel slide">
| + | |
− | <!-- 轮播(Carousel)指标 -->
| + | |
− | <ol class="carousel-indicators">
| + | |
− | <li data-target="#myCarousel2" data-slide-to="0" class="active"></li>
| + | |
− | <li data-target="#myCarousel2" data-slide-to="1"></li>
| + | |
− | <li data-target="#myCarousel2" data-slide-to="2"></li>
| + | |
− | <!-- <li data-target="#myCarousel2" data-slide-to="3"></li> -->
| + | |
− | </ol>
| + | |
− | <!-- 轮播(Carousel)项目 -->
| + | |
− | <div class="carousel-inner">
| + | |
− | <div class="item active">
| + | |
| <div class="container"> | | <div class="container"> |
| <div class="row"> | | <div class="row"> |
| <div class="col-md-6" style="margin:100px 0"> | | <div class="col-md-6" style="margin:100px 0"> |
| <div class="caption"> | | <div class="caption"> |
− | <p> | + | <p style="margin-left:100px"> |
− | A prokaryote version of our Signal Fliter. Users can define two input signals and three combinations of output. For more details, see to our description page
| + | We detected GFP reporter in E.coli after induction of 20minute, 120minutes and 240minutes through 20 times of amplification. See more details in our Results page. |
| </p> | | </p> |
− | <p><a href="#" class="btn btn-primary" role="button">More details</a></p> | + | <p><a href="https://2016.igem.org/Team:HUST-China/Results#location_pro&pro" class="btn btn-primary" role="button">More details</a></p> |
| </div> | | </div> |
| </div> | | </div> |
| <div class="col-md-6" style="margin:100px 0"> | | <div class="col-md-6" style="margin:100px 0"> |
− | <img src="https://static.igem.org/mediawiki/2016/8/88/T--HUST-China--Description-Fig-Eukaryote.png" alt="" class="img-responsive"> | + | <img src="https://static.igem.org/mediawiki/2016/6/63/T--HUST-China--Experiments-CI-pR_Flou-detec.png" alt="" class="img-responsive"> |
| </div> | | </div> |
| </div> | | </div> |
| </div> | | </div> |
| </div> | | </div> |
− | <div class="item">
| + | <div class="item"> |
| <div class="container"> | | <div class="container"> |
| <div class="row"> | | <div class="row"> |
− | <div class="col-md-6 " style="margin:100px 0"> | + | <div class="col-md-6" style="margin:100px 0"> |
| <div class="caption"> | | <div class="caption"> |
− | <p> | + | <p style="margin-left:100px"> |
− | Small in scale, big in thoughts! THis summer, we held a fabulous HUST-Cheering! conferrence. And anlong with our integrated Practices,see to HP page.
| + | We characterized cro and pRM inhibition by the same method as CI and pR’s. See more details in our Results page. |
| </p> | | </p> |
− | <p><a href="#" class="btn btn-primary" role="button">More details</a></p> | + | <p><a href="https://2016.igem.org/Team:HUST-China/Results#location_pro&pro" class="btn btn-primary" role="button">More details</a></p> |
| </div> | | </div> |
| </div> | | </div> |
| <div class="col-md-6" style="margin:100px 0"> | | <div class="col-md-6" style="margin:100px 0"> |
− | <img src="https://static.igem.org/mediawiki/2016/6/64/T--HUST-China--Integrated_Practices-Fig2-4.jpeg" alt="" class="img-responsive"> | + | <img src="https://static.igem.org/mediawiki/2016/1/15/T--HUST-China--CI-pR_inhibition.png" alt="" class="img-responsive"> |
| </div> | | </div> |
| </div> | | </div> |
| </div> | | </div> |
| </div> | | </div> |
− | <div class="item">
| + | <div class="item"> |
| <div class="container"> | | <div class="container"> |
| <div class="row"> | | <div class="row"> |
| <div class="col-md-6" style="margin:100px 0"> | | <div class="col-md-6" style="margin:100px 0"> |
| <div class="caption"> | | <div class="caption"> |
− | <p> | + | <p style="margin-left:100px"> |
− | Fluorescence measurement is the main characterization method this year. We employed GFP and RFP as reporters to build valid data of our circuits. For more results see to Wetlab Results!
| + | We use IPTG with final concentration of 1mM to induce the GFP-LVAssrAtag and measure the relative fluorescence through plate reader with Excitation light 495nm. See more details in our Results page. |
| </p> | | </p> |
− | <p><a href="#" class="btn btn-primary" role="button">More details</a></p> | + | <p><a href="https://2016.igem.org/Team:HUST-China/Results#location_tri_fun" class="btn btn-primary" role="button">More details</a></p> |
| </div> | | </div> |
| </div> | | </div> |
| <div class="col-md-6" style="margin:100px 0"> | | <div class="col-md-6" style="margin:100px 0"> |
− | <img src="https://static.igem.org/mediawiki/2016/f/fb/T--HUST-China--home-flou.png" alt="" class="img-responsive"> | + | <img src="https://static.igem.org/mediawiki/2016/f/fa/T--HUST-China--Experiments-LVAssrA.png" alt="" class="img-responsive"> |
| </div> | | </div> |
| </div> | | </div> |
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| </div> | | </div> |
| </div> | | </div> |
| + | <!-- 轮播(Carousel)导航 --> |
| + | <a class="carousel-control left" href="#myCarousel" data-slide="prev" style="font-size:100px;">‹</a> |
| + | <a class="carousel-control right" href="#myCarousel" data-slide="next" style="vertical-align:middle;font-size:100px">›</a> |
| + | </div> |
| + | </div> |
| + | <h2 id="location_Euk_ver">Eukaryotic version</h2> |
| + | <p>It is a bi-stable system derived from Arabidopsis thaliana stress response system. Enzyme catalysis is the core of this design to increase the efficiency of state transition. |
| + | </p> |
| + | <img src="https://static.igem.org/mediawiki/2016/1/1e/T--HUST-China--Description-Fig-prokaryote.png" alt="" class="img-responsive"> |
| + | <p style="text-align:center">Fig 2: circuit of Eukaryote version serving as bii-stable signal filter</p> |
| + | <p>Clade A protein phosphatases type 2C (PP2CA) and Sucrose Nonfermenting1 Related Subfamily 2 (SnRK2s) protein kinase are both components of Abscisic acid signaling network in Arabidopsis thaliana. ABF2 is a leucine zipper transcription factor which basically binds ABA-response element (ABRE). ABF2 can be phosphorylated by SnRK2s and be efficiently dephosphorylated by PP2CA. Also, in the absence of Abscisic acid, SnRK2 kinases can be inactivated by PP2Cs thus shut down the gene expression efficiently. |
| + | </p> |
| + | <p>When signal 1 (ON) comes, promoter RD29A drives expression of SnRK2.2, which can phosphorylate ABF2 (constitutively expressed). Compared with protein co-facter association, enzyme catalysis can produce a large quantity of phosphorylated ABF2 in a short time, then enhance pRD29A to turn on the gene of interest’s expression. When signal 2 (OFF) comes, gene PP2CA expresses, dephosphorylates ABF2 and inactivates SnRK2.2, thus turning the system into OFF state. |
| + | </p> |
| + | <div class="container-fluid index-details1 index-details"> |
| + | <div id="myCarousel2" class="carousel slide"> |
| + | <!-- 轮播(Carousel)指标 --> |
| + | <ol class="carousel-indicators"> |
| + | <li data-target="#myCarousel" data-slide-to="0" class="active"></li> |
| + | <li data-target="#myCarousel" data-slide-to="1"></li> |
| + | </ol> |
| + | <!-- 轮播(Carousel)项目 --> |
| + | <div class="carousel-inner"> |
| + | <div class="item active"> |
| + | <div class="container"> |
| + | <div class="row"> |
| + | <div class="col-md-6" style="margin:100px 0"> |
| + | <div class="caption"> |
| + | <p style="margin-left:70px"> |
| + | We ran a SDS-PAGE of the culture supernatants of induced cells to identify the three recombinant proteins. See more details in our Results page. |
| + | </p> |
| + | <p><a href="https://2016.igem.org/Team:HUST-China/Results#location_pro_exp" class="btn btn-primary" role="button">More details</a></p> |
| + | </div> |
| + | </div> |
| + | <div class="col-md-6" style="margin:100px 0"> |
| + | <img src="https://static.igem.org/mediawiki/2016/b/b1/T--HUST-China--SDS.png" alt="" class="img-responsive"> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | <div class="item"> |
| + | <div class="container"> |
| + | <div class="row"> |
| + | <div class="col-md-6" style="margin:100px 0"> |
| + | <div class="caption"> |
| + | <p> |
| + | We simulated our project in cellular level and Intestinal level to help us figure out the feasibility of it and guide us the direction of further improvement. See more results in our Model page. |
| + | </p> |
| + | <p><a href="https://2016.igem.org/Team:HUST-China/Model" class="btn btn-primary" role="button">More details</a></p> |
| + | </div> |
| + | </div> |
| + | <div class="col-md-6" style="margin:100px 0"> |
| + | <img src="https://static.igem.org/mediawiki/2016/5/59/T--HUST-China--modeling-application-LA_decomposition.png" alt="" class="img-responsive"> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | </div> |
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