Difference between revisions of "Team:NTU-LIHPAO-Taiwan/Parts"

 
(143 intermediate revisions by 2 users not shown)
Line 67: Line 67:
  
 
body {
 
body {
−
background-color: #F0F0F0;
+
background-color: #FFF;
 
}
 
}
 
body,td,th {
 
body,td,th {
Line 73: Line 73:
 
font-family: Calibri, "Arial Narrow", "Agency FB", "Raavi";
 
font-family: Calibri, "Arial Narrow", "Agency FB", "Raavi";
 
}
 
}
 +
 
#Wrapper {
 
#Wrapper {
 
margin-right: auto;
 
margin-right: auto;
Line 80: Line 81:
 
margin-bottom: 0px;
 
margin-bottom: 0px;
 
width: 90%;
 
width: 90%;
−
height: 10000px;
+
height: auto;
 
top: 0px;
 
top: 0px;
 
left: 5%;
 
left: 5%;
 
right: 5%;
 
right: 5%;
 
position: absolute;
 
position: absolute;
 +
bottom: 0px;
 +
padding-top: 0px;
 +
padding-bottom: 0px;
 +
z-index: 0;
 +
}
 +
 +
#Wrapper_Left {
 +
margin-right: auto;
 +
margin-left: auto;
 +
background-color: #F0F0F0;
 +
margin-top: 0px;
 +
margin-bottom: 0px;
 +
width: 5%;
 +
height: 100%;
 +
top: 0px;
 +
left: 0%;
 +
right: auto;
 +
position: fixed;
 +
bottom: 0px;
 +
padding-top: 0px;
 +
padding-bottom: 0px;
 +
z-index: 0;
 +
}
 +
 +
#Wrapper_Right {
 +
margin-right: auto;
 +
margin-left: auto;
 +
background-color: #F0F0F0;
 +
margin-top: 0px;
 +
margin-bottom: 0px;
 +
width: 5%;
 +
height: 100%;
 +
top: 0px;
 +
left: auto;
 +
right: 0%;
 +
position: fixed;
 
bottom: 0px;
 
bottom: 0px;
 
padding-top: 0px;
 
padding-top: 0px;
Line 100: Line 137:
 
height: 58px;
 
height: 58px;
 
top: 0px;
 
top: 0px;
−
left:0px;
+
left: 0px;
−
right:0px;
+
right: 0px;
 
position: fixed;
 
position: fixed;
 
bottom: 0px;
 
bottom: 0px;
Line 113: Line 150:
 
background-repeat: no-repeat;
 
background-repeat: no-repeat;
 
}
 
}
 +
 +
#Wrapper_Bottom {
 +
width: 100%;
 +
height: 100px;
 +
bottom: 0px;
 +
top: auto;
 +
background-color: #FFF;
 +
border-top-style: solid;
 +
border-top-color: #079235;
 +
border-top-width: 3px;
 +
margin-right: auto;
 +
margin-left: auto;
 +
margin-top: 0px;
 +
margin-bottom: 0px;
 +
z-index: 9998;
 +
position: absolute;
 +
}
 +
 +
  
 
/*-------------------------------------------*/
 
/*-------------------------------------------*/
Line 168: Line 224:
 
.Slidemenu {
 
.Slidemenu {
 
z-index: 9999;
 
z-index: 9999;
−
position: fixed;
+
position: absolute;
 
top: 32px;
 
top: 32px;
−
left: auto;
+
bottom: 0px;
 +
left: 51%;
 
right: 15%;
 
right: 15%;
 
list-style:none;
 
list-style:none;
 
margin:0;
 
margin:0;
 
padding:0;
 
padding:0;
 +
position: absolute;
 +
margin-left: -350px;
 
/*background:rgba(100%,100%,100%,0); 主選單色系選擇*/
 
/*background:rgba(100%,100%,100%,0); 主選單色系選擇*/
 
}
 
}
Line 206: Line 265:
 
width: auto;
 
width: auto;
 
font-family: "Agency FB";
 
font-family: "Agency FB";
−
font-size: 18px;
+
font-size: 1em;
 
}
 
}
  
Line 225: Line 284:
 
.Slidemenu li ul li {
 
.Slidemenu li ul li {
 
overflow:hidden;
 
overflow:hidden;
−
height:0;
+
height: 0px;
 +
margin-bottom: 0px;
 
}
 
}
  
Line 234: Line 294:
 
-o-transition:background 300ms ease-in-out;
 
-o-transition:background 300ms ease-in-out;
 
transition:background 300ms ease-in-out;
 
transition:background 300ms ease-in-out;
 +
line-height:1.1em;
 
}
 
}
  
Line 242: Line 303:
 
.Slidemenu ul > li {
 
.Slidemenu ul > li {
 
text-decoration:none;
 
text-decoration:none;
−
float:left;
+
float: left;
−
position:relative;
+
position: relative;
 
height: 27px; /*--改主要選單高度--*/
 
height: 27px; /*--改主要選單高度--*/
−
width: 110px; /*--改主要選單寬度--*/
+
}
 +
 
 +
.width_small {
 +
width: 100px; /*--改主要選單寬度--*/
 
}
 
}
  
Line 259: Line 323:
 
.Slidemenu ul > li:hover ul li {
 
.Slidemenu ul > li:hover ul li {
 
text-decoration:none;
 
text-decoration:none;
−
height: 24px; /*--改拉出的選單高度--*/
+
height: 26px; /*--改拉出的選單高度--*/
 
width: 150px; /*--改拉出的選單寬度--*/
 
width: 150px; /*--改拉出的選單寬度--*/
 
text-align: center;
 
text-align: center;
Line 282: Line 346:
 
}
 
}
  
−
#Content {
+
#Content_Container {
 
position: fixed;
 
position: fixed;
−
top: 0;
 
−
left: 5.2%;
 
 
width: 17.8%;
 
width: 17.8%;
 
height: 100%;
 
height: 100%;
 +
left: 5.2%;
 +
right: auto;
 +
background: #f7fbff;
 +
z-index: 5;
 +
}
 +
 +
#Content {
 +
position: absolute;
 +
top: 60px;
 +
width: 100%;
 +
height: auto;
 
padding: 0;
 
padding: 0;
 
margin: 0;
 
margin: 0;
−
background: #f7fbff;
 
 
transition: 0.5s;
 
transition: 0.5s;
 
font-size: 0.8rem;
 
font-size: 0.8rem;
−
z-index: 5;
 
 
font-family: Calibri;
 
font-family: Calibri;
 
}
 
}
Line 312: Line 383:
 
position: absolute;
 
position: absolute;
 
top: 5rem;
 
top: 5rem;
−
left: 2rem;
+
left: 10%;
 +
width: 90%;
 
}
 
}
  
Line 325: Line 397:
 
padding-top: 5px;
 
padding-top: 5px;
 
padding-bottom: 5px;
 
padding-bottom: 5px;
 +
line-height: 30px;
 
}
 
}
  
Line 331: Line 404:
 
overflow-y: auto;
 
overflow-y: auto;
 
transition: 0.5s;
 
transition: 0.5s;
−
padding-left: 2rem;
+
width: 85%;
 +
left: 15%;
 +
position: relative;
 
}
 
}
  
 
.sub-Content > li {
 
.sub-Content > li {
−
padding-top: 8px;
+
padding-top: 10px;
−
padding-bottom: 8px;
+
padding-bottom: 10px;
 
}
 
}
  
Line 349: Line 424:
 
opacity: 0.5;
 
opacity: 0.5;
 
}
 
}
 +
 +
#Healthin_Logo_Content {
 +
width: 100%;
 +
height: auto;
 +
}
 +
 +
#Healthin_Logo {
 +
top: 55px;
 +
padding-left: 3%;
 +
padding-right: 3%;
 +
position: absolute;
 +
width: 94%;
 +
height: 94%;
 +
}
 +
 +
 +
 +
/*-------------------------------------------*/
 +
/*---------------Introduction----------------*/
 +
/*-------------------------------------------*/
 +
 +
.Container_Bottom1 {
 +
height: 70px;
 +
width: 100%;
 +
}
 +
 +
.Container_Bottom2 {
 +
height: 70px;
 +
width: 100%;
 +
padding-top: 10px;
 +
border-top-style: solid;
 +
border-top-color: #079235;
 +
border-top-width: 3px;
 +
}
 +
 +
.Intro_Picture {
 +
width: 100%;
 +
height: auto;
 +
background-color: #FFF;
 +
position: absolute;
 +
}
 +
 +
#NTUschool_logo {
 +
background-color: #FFF;
 +
padding-left: 9.6%;
 +
padding-right: 60.4%;
 +
position: absolute;
 +
width: 30%;
 +
height: 100%;
 +
}
 +
 +
#LIHPAO_logo {
 +
background-color: #FFF;
 +
padding-left: 66.4%;
 +
padding-right: 9.6%;
 +
position: absolute;
 +
width: 24%;
 +
height: 100%;
 +
}
 +
 +
#NTUBST_logo {
 +
background-color: #FFF;
 +
padding-left: 49.2%;
 +
padding-right: 43.2%;
 +
position: absolute;
 +
width: 7.6%;
 +
height: 100%;
 +
}
 +
 +
.Text_Sponsor {
 +
color: #767676;
 +
font-size: 15px;
 +
font-family: Calibri;
 +
text-align: center;
 +
line-height: 20px;
 +
}
 +
 +
 +
/*-------------------------------------------*/
 +
/*---------------Article Picture-------------*/
 +
/*-------------------------------------------*/
 +
 +
.Container_Article_Picture {
 +
height: 280px;
 +
width: 100%;
 +
padding-top: 25px;
 +
padding-bottom: 25px;
 +
}
 +
 +
.Article_Picture {
 +
width: 480px;
 +
height: auto;
 +
left: 50%;
 +
right: auto;
 +
background-color: #FFF;
 +
margin: 0 0 0 -240px;
 +
position: absolute;
 +
}
 +
 +
.Article_PictureText {
 +
width: 430px;
 +
height: auto;
 +
padding-left: 25px;
 +
padding-right: 25px;
 +
border-top-width: 2px;
 +
border-top-style: dashed;
 +
border-top-color: #fe5838;
 +
}
 +
 +
.Container_Article_Picture1 {
 +
height: 400px;
 +
width: 100%;
 +
padding-top: 25px;
 +
padding-bottom: 25px;
 +
}
 +
 +
.Article_Picture1 {
 +
width: 368px;
 +
height: auto;
 +
left: 50%;
 +
right: auto;
 +
background-color: #FFF;
 +
margin: 0 0 0 -184px;
 +
position: absolute;
 +
}
 +
 +
.Article_PictureText1 {
 +
width: 318px;
 +
height: auto;
 +
padding-left: 25px;
 +
padding-right: 25px;
 +
border-top-width: 2px;
 +
border-top-style: dashed;
 +
border-top-color: #fe5838;
 +
}
 +
 +
.Text_Picture {
 +
font-size: 18px;
 +
font-family: Calibri;
 +
text-align: justify;
 +
line-height: 26px;
 +
color: #08923a;
 +
}
 +
  
 
/*-------------------------------------------*/
 
/*-------------------------------------------*/
Line 355: Line 574:
  
 
#Articles {
 
#Articles {
−
width: 76%;
+
width: 100%;
−
height: 10000px;
+
height: auto;
−
left: 22%;
+
−
right: 2%;
+
 
top: -10px;
 
top: -10px;
 
position: absolute;
 
position: absolute;
 
background-color: #FFF;
 
background-color: #FFF;
−
z-index: 0;
+
z-index: 1;
 
color: #000;
 
color: #000;
 
}
 
}
  
 
.ContentBox {
 
.ContentBox {
−
width: 100%;
+
width: 76%;
−
height: 675px;
+
height: auto;
 +
left: 22%;
 +
right: 2%;
 +
position: absolute;
 +
background-color: #FFF;
 
}
 
}
  
 
.Text1 {
 
.Text1 {
−
font-size: 36px;
+
font-size: 28px;
 
font-family: Calibri;
 
font-family: Calibri;
 
left: 20px;
 
left: 20px;
Line 380: Line 601:
 
padding-bottom: 10px;
 
padding-bottom: 10px;
 
padding-left: 20px;
 
padding-left: 20px;
−
border-bottom-width: 1px;
+
border-bottom-width: 2px;
−
border-bottom-style: solid;
+
border-bottom-style: dashed;
−
border-bottom-color: #000;
+
border-bottom-color: #fe5838;
 +
color: #c00000;
 
}
 
}
  
 
.Text2 {
 
.Text2 {
−
font-size: 28px;
+
font-size: 24px;
 
font-family: Calibri;
 
font-family: Calibri;
 
padding-top: 30px;
 
padding-top: 30px;
Line 392: Line 614:
 
padding-bottom: 10px;
 
padding-bottom: 10px;
 
padding-left: 50px;
 
padding-left: 50px;
 +
color: #00b050;
 
}
 
}
  
Line 405: Line 628:
 
}
 
}
  
−
.Text_underline {
+
.Text_TitleUnderline {
−
text-decoration: underline;
+
font-size: 22px;
 +
font-family: Calibri;
 +
text-align: justify;
 +
padding-top: 10px;
 +
padding-bottom: 0px;
 +
line-height: 26px;
 +
color: #0070c0;
 +
text-decoration: underline;
 
}
 
}
  
Line 412: Line 642:
 
font-style: italic;
 
font-style: italic;
 
}
 
}
 +
 +
ol.part1 {
 +
list-style-type: none;
 +
margin-left: 0px;
 +
}
 +
 +
ol.part2 {
 +
list-style-type: decimal;
 +
margin-left: 0px;
 +
}
 +
 +
ol.part2 li {
 +
padding-top: 10px;
 +
padding-bottom: 10px;
 +
}
 +
 +
ol.part3 {
 +
list-style-type: lower-alpha;
 +
}
 +
 +
 +
/*-------------------------------------------*/
 +
/*-------------------Media-------------------*/
 +
/*-------------------------------------------*/
 +
 +
@media screen and (min-width: 0px) and (max-width: 1200px) {
 +
 +
#NTUXLIHPAO_icon {
 +
height: 40px;
 +
width: 200px;
 +
position: absolute;
 +
left: 1%;
 +
top: 18px;
 +
right: auto;
 +
bottom: auto;
 +
z-index: 9999;
 +
}
 +
 +
 +
#width_small {
 +
width: 95px; /*--改主要選單寬度--*/
 +
}
 +
 +
.Slidemenu {
 +
z-index: 9999;
 +
position: absolute;
 +
top: 32px;
 +
bottom: 0px;
 +
left: 49%;
 +
right: auto;
 +
list-style:none;
 +
margin:0;
 +
padding:0;
 +
position: absolute;
 +
margin-left: -320px;
 +
/*background:rgba(100%,100%,100%,0); 主選單色系選擇*/
 +
}
 +
}
 +
 +
@media screen and (min-width: 901px) and (max-width: 1050px) {
 +
 +
.Container_Bottom2 {
 +
height: 60px;
 +
width: 100%;
 +
padding-top: 10px;
 +
border-top-style: solid;
 +
border-top-color: #079235;
 +
border-top-width: 3px;
 +
}
 +
}
 +
 +
@media screen and (min-width: 0px) and (max-width: 900px) {
 +
 +
.Container_Bottom2 {
 +
height: 50px;
 +
width: 100%;
 +
padding-top: 10px;
 +
border-top-style: solid;
 +
border-top-color: #079235;
 +
border-top-width: 3px;
 +
}
 +
}
 +
  
 
</style>
 
</style>
Line 424: Line 737:
  
 
<body>
 
<body>
 +
 +
<div id="Wrapper_Left"></div>
 +
<div id="Wrapper_Right"></div>
  
 
<div id="Wrapper">
 
<div id="Wrapper">
Line 437: Line 753:
 
<div class="Slidemenu">
 
<div class="Slidemenu">
 
<ul>
 
<ul>
−
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan">Home</a>
+
<li><div class=width_small><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan">Home</a></div>
−
        </li>
+
−
       
+
−
<li><a href="#">Team</a>
+
−
<ul class="subs">
+
−
<li><a href="#">Sub Item</a></li>
+
−
<li><a href="#">Sub Item</a></li>
+
−
<li><a href="#">Sub Item</a></li>
+
−
</ul>
+
−
        </li>
+
−
       
+
−
<li id=Position_Now><a>Project</a>
+
−
<ul class="subs">
+
−
<li><a href="#">Abstract</a></li>
+
−
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Overview">Overview</a></li>
+
−
<li><a href="#">Parts</a></li>
+
−
</ul>
+
 
</li>
 
</li>
−
       
+
 
−
<li><a href="#">Results</a>
+
<li><div class=width_small span style="cursor:default"><a>Team</a></div>
 
<ul class="subs">
 
<ul class="subs">
−
<li><a href="#">Sub Item</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Team">Team</a></li>
−
<li><a href="#">Sub Item</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Attributions">Attributions</a></li>
−
<li><a href="#">Sub Item</a></li>
+
 
</ul>
 
</ul>
 
</li>
 
</li>
−
               
+
−
<li><a href="#">Modeling</a>
+
<li><div class=width_small span style="cursor:default"><a>Project</a></div>
 
<ul class="subs">
 
<ul class="subs">
−
<li><a href="#">Sub Item</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Description">Description</a></li>
−
<li><a href="#">Sub Item</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Design">Design</a></li>
−
<li><a href="#">Sub Item</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Results">Results</a></li>
 +
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Modeling">Modeling</a></li>
 +
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Experiments">Protocols</a></li>
 
</ul>
 
</ul>
 
</li>
 
</li>
−
       
+
−
<li><a href="#">Notebook</a>
+
<li><div class=width_small span style="cursor:default"><div id=Position_Now><a>Parts</a></div></div>
 
<ul class="subs">
 
<ul class="subs">
−
<li><a href="#">Sub Item</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Parts">Team Parts</a></li>
−
<li><a href="#">Sub Item</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Basic_Part">Basic Parts</a></li>
−
<li><a href="#">Sub Item</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Composite_Part">Composite Parts</a></li>
 
</ul>
 
</ul>
 +
</li>
 +
 +
<li><div class=width_small><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Notebook">Notebook</a></div>
 
</li>
 
</li>
 
          
 
          
−
<li><a href="#">Safety</a>
+
<li><div class=width_small><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Safety">Safety</a></div>
−
<ul class="subs">
+
−
<li><a href="#">Sub Item</a></li>
+
−
<li><a href="#">Sub Item</a></li>
+
−
<li><a href="#">Sub Item</a></li>
+
−
</ul>
+
 
</li>
 
</li>
−
 
+
−
<li><a href="#">Human Practice</a>
+
<li><div class=width_small span style="cursor:default"><a>Society</a></div>
 
<ul class="subs">
 
<ul class="subs">
−
<li><a href="#">Sub Item</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Practices">Human Practices</a></li>
−
<li><a href="#">Sub Item</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Collaborations">Collaborations</a></li>
−
<li><a href="#">Sub Item</a></li>
+
<li><a href="https://2015.igem.org/Team:NTU-LIHPAO-Taiwan/Entrepreneurship">Entrepreneurship</a></li>
 
</ul>
 
</ul>
 
</li>
 
</li>
Line 498: Line 797:
 
</div>
 
</div>
 
</div>
 
</div>
−
   
+
 
 +
<div id="Content_Container">
 +
<div id="Healthin_Logo_Content"><div id="Healthin_Logo"><img src="https://static.igem.org/mediawiki/2015/6/69/Healthin_White.png" style="max-width: 100%; height: auto"/></div></div>
 
<div id="Content">
 
<div id="Content">
 
<ul class="main-Content">
 
<ul class="main-Content">
 
<li>
 
<li>
−
<span class="title">Overview</span>
+
<span class="title">Prototype</span>
 
<ul class="sub-Content">
 
<ul class="sub-Content">
−
<li><a href="#First1">Pigout / Stay in Shape</a></li>
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<li><a href="#First">Prototype</a></li>
 
</ul>
 
</ul>
 
</li>
 
</li>
 
<li>
 
<li>
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<span class="title">Background</span>
+
<span class="title">Main Part</span>
 
<ul class="sub-Content">
 
<ul class="sub-Content">
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<li><a href="#Second1">CPP-PYY</a></li>
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<li><a href="#Second1">Part:BBa_K1841001</a></li>
−
<li><a href="#Second2">Nisin Selection</a></li>
+
<li><a href="#Second2">Part:BBa_K1841004</a></li>
−
<li><a href="#Second3">Suicide</a></li>
+
<li><a href="#Second3">Part:BBa_K1841007</a></li>
 
</ul>
 
</ul>
 
</li>
 
</li>
 
<li>
 
<li>
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<span class="title">Design</span>
+
<span class="title">Test Part</span>
 
<ul class="sub-Content">
 
<ul class="sub-Content">
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<li><a href="#Third1">CPP-PYY</a></li>
+
<li><a href="#Third1">RFP</a></li>
−
<li><a href="#Third2">Nisin Selection</a></li>
+
<li><a href="#Third2">His-PYY</a></li>
−
<li><a href="#Third3">Suicide</a></li>
+
<li><a href="#Third3">Signal peptide-PYY-His</a></li>
 +
<li><a href="#Third4">Y2R</a></li>
 
</ul>
 
</ul>
 
</li>
 
</li>
 
<li>
 
<li>
−
<span class="title">Result</span>
+
<span class="title">References</span>
 
<ul class="sub-Content">
 
<ul class="sub-Content">
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<li><a href="#Fourth1">CPP-PYY</a></li>
+
<li><a href="#Fourth">References</a></li>
−
<li><a href="#Fourth2">Nisin Selection</a></li>
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−
<li><a href="#Fourth3">Suicide</a></li>
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</ul>
 
</ul>
 
</li>
 
</li>
 
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<div class="Text_italic"><i>Lactobacillus casei</i></div>
 
 
<div id="Articles">
 
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<div class="ContentBox">
 
<div class="ContentBox">
 
<div class="ContentHolder">
 
<div class="ContentHolder">
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<div class="Text1">Overview</div>
+
<div class="Text1">Prototype</div>
−
<div class="Text2" id="First1">Pigout Versus Stay in Shape</div>
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<div class="Text2" id="First">Prototype</div>
 
<div class="Text3">
 
<div class="Text3">
−
This year, iGEM team of NTU-LIHPAO-Taiwan take note of the problem that the obesity condition in Taiwan has deteriorated. Moreover, the market is flooded with those unverified slimming drugs that harm the public health simultaneously. Therefore, we hope to initiate our project from the key peptide “Peptide YY (PYY)” which can control appetite, and take advantage of the probiotic characteristic of <i>Lactobacillus casei</i>, with cell penetrating peptides which contain large developmental potential in oral peptide drugs. We hope that we can make <i>Lactobacillus casei</i> secret CPP-PYY complex which can be another newly created oral peptide drugs.
+
The iGEM Team NTU-LIHPAO-Taiwan 2015 had built a new biological system for the iGEM community. The original design of the system making up the (Product) contains the three elements listed below, in a probiotic – <i>Lactobacillus casei</i> ATCC 393.
 
</div>
 
</div>
−
<div class="Text1">Background</div>
+
<div class="Container_Article_Picture1">
−
<div class="Text2" id="Second1">CPP-PYY</div>
+
<div class="Article_Picture1">
−
<div class="Text3"><div class="Text_underline">Cell Penetrating Peptide (CPP)</div></div>
+
<img src="https://static.igem.org/mediawiki/2015/a/a8/NTU_LASMID.jpg" width="368px"/>
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<div class="Text3">
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<div class="Article_PictureText1"><div class="Text_Picture">[Fig.1-1] Designed Plasmid</div></div>
−
CPP is a kind of short segment peptide that can spontaneously carry macromolecules such as DNA, proteins, and peptides to penetrate cell membrane. Generally speaking, they often contain less than 40 amino acids and also called protein transduction domain (PTDs) or membrane transduction domain (MTDs). CPP which is first discovered and extensively studied is TAT protein derived from human immunodeficiency virus-1 (HIV-1) and antennapedia homeodomain (Antp) transcription factor comes from Drosophila melanogaster. The shortest segments diagnosed from the sequences are TAT and penetratin, and more and more CPPs come into existence from the following studies. There are also synthetic amino acid sequences such as R9.
+
</div>
 
</div>
 
</div>
 +
 +
<div class="Text1">Main Part</div>
 +
<div class="Text2" id="Second1">Part: BBa_K1841001</div>
 +
<div class="Text3"><div class="Text_TitleUnderline">nisI</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
However, the mechanism of CPP penetrating cell membrane is still vague, and different CPPs, CPP-cargo complexes, secondary structures have significant effect on distinct cells’ penetrating mechanism. Currently, there are majorly two pathways: energy-independent direct penetration and energy-consuming endocytosis, and two pathways both have three same steps: membrane interaction, membrane permeation, secreting CPPs to cytoplasm.
+
Research from Torsten <i>et al.</i> revealed that the highest level of acquired nisin tolerance was achieved after coordinated expression of all four nisin immunity genes, containing <i>nisI</i>, <i>nisF</i>, <i>nisE</i>, and <i>nisG</i>.<a href="#Reference1">[1]</a>  Functional analyses provided evidence that NisI acts as a nisin-sequestering protein and that NisFEG acts as a nisin exporter that expels nisin molecules from the cytoplasmic membrane into the environment.
−
Direct penetration, first of all, positively charged CPPs draw negatively charged molecules on membrane such as HS, phospholipid dilayer, with integral proteins folded causing membrane temporarily collapse making CPP penetrate cell membrane, and it may form an inverted micelle or a pore. It increases partial hydrogen concentration that CPPs enter the cytoplasm, forming the concentration gradient, it makes CPPs move from one side to the other, and whether TAT penetrate the cell depends on the concentration and cargo’s properties.  
+
 
</div>
 
</div>
−
<div class="Text3">
+
<div class="Container_Article_Picture">
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Endocytosis uses pinocytosis, macropinocytosis, receptor-mediated endocytosis to form vesicles, transporting CPPs into cytoplasm. Because in the early phase scientists recognized that CPP can penetrate cell membrane in 4oC so that they deducted CPP majorly penetrate cell membrane with direct penetration. Nonetheless, current studies show that endocytosis more or less involves in the penetrating process of different CPPs in various conditions.  
+
<div class="Article_Picture">
 +
<img src="https://static.igem.org/mediawiki/2015/3/34/NTU-Team-nisI.jpg" width="480px"/>
 +
<div class="Article_PictureText"><div class="Text_Picture">[Fig.2-1] Nisin Selection</div></div>
 +
</div>
 
</div>
 
</div>
−
<div class="Text3"><div class="Text_underline">Peptide YY (PYY)</div></div>
+
<div class="Text2" id="Second2">Part: BBa_K1841004</div>
 +
<div class="Text3"><div class="Text_TitleUnderline">plac promoter</div></div>
 
<div class="Text3">
 
<div class="Text3">
−
Peptide YY is a short peptide that can restrain our appetite. Because the peptide’s head and tail are both amino acid, tyrosine (Y), it is named peptide YY (PYY). PYY has two forms: PYY 1-36 is the unmodified form, and PYY 3-36 is the kind of PYY cut off two amino acids in N-terminal side by dipeptidyl peptidase-IV. Each contains 60% and 40% of all PYY.
+
Lactose operon of <i>Lactobacillus casei</i> In <i>Lactobacillus casei</i> ATCC393 [pLZ15–], the lactose genes are grouped in a cluster transcribed as single operon. The cluster lacTEGF encodes an antiterminator protein (LacT), lactose-specific elements (LacE and LacF) of the phosphotransferase system (PTS) and a phospho-β-galactosidase (LacG).
 +
The promoter region contains a cre element (catabolite responsive element) overlapping the –35 region, which is followed by a highly conserved sequence, the ribonucleic antiterminator (RAT) sequence, and a terminator structure.  
 +
The antiterminator activity of LacT is also negatively controlled by glucose, possibly by PTS-mediated phosphorylation as explained below.<a href="#Reference2">[2]</a>
 +
<div class="Container_Article_Picture">
 +
<div class="Article_Picture">
 +
<img src="https://static.igem.org/mediawiki/2015/a/a4/NTU-Team-plac-cl.jpg" width="480px"/>
 +
<div class="Article_PictureText"><div class="Text_Picture">[Fig.2-2-1] Suicide Sense Part</div></div>
 +
</div>
 
</div>
 
</div>
−
<div class="Text3">
+
<div class="Container_Article_Picture">
−
In the situation of PYY binding to the receptors, PYY 1-36’s affinity to Y1, Y2, Y4,and Y5 are all high. However, because PYY 3-36 is cut off two amino acids in N-terminal side causing conformational change, its affinity to Y2 is higher than others. Since both two types of PYY don’t require disulfide bond to stable its structure, it can spontaneously become a stable and activated form in the solution.  
+
<div class="Article_Picture">
−
PYY is classified as gastrointestinal(GI) hormone. After intestine absorbs micromolecule nutrients, ileum and colon epithelial cells will secret PYY to blood. As PYY contact hypothalamus by blood circulation.
+
<img src="https://static.igem.org/mediawiki/2015/9/9a/NTU-Team-nuca.jpg" width="480px"/>
 +
<div class="Article_PictureText"><div class="Text_Picture">[Fig.2-2-2] Suicide Kill Part</div></div>
 +
</div>
 
</div>
 
</div>
−
 
+
<div class="Text2" id="Second3">Part: BBa_K1841007</div>
−
<div class="Text2" id="Second2">Nisin Selection</div>
+
<div class="Text3"><div class="Text_TitleUnderline">TAT-PYY</div></div>
−
<div class="Text3"><div class="Text_underline">Lactic Acid Bacteria (LAB)</div></div>
+
 
<div class="Text3">
 
<div class="Text3">
−
In our project, we choose <i>Lactobacillus casei</i> ATCC393 as the study material which belongs to the diverse family of lactic acid bacteria. Lactic acid bacteria are not a formal term in taxonomy; as a matter of fact, the lactic acid bacteria are referred to as a group of microorganism that are able to metabolite carbohydrates to produce lactic acid with the yield over 50%. [1] One notable fact is that lactic acid bacteria are long be used in the manufacture of dairy products, and therefore they are generally regarded as safe (GRAS). Moreover, they are the most representative probiotics in the intestines.
+
TAT can bring the big molecule substance like DNA, protein, peptide to penetrate the cell. Usually, named as protein transduction domain or membrane transduction domain, it is less than 40 amino acid.<a href="#Reference3">[3]</a> PYY belongs to the gastrointestinal hormones. Once the intestine detects the nutrition, the epidermal cell, L cell of ileum and colon will secrete the PYY. While the blood pass the hypothalamus, PYY will bind to the neuropeptide Y receptor in ventromedial nuclei causing the sense of satiation.<a href="#Reference4">[4]</a>
 +
</div>
 +
<div class="Container_Article_Picture">
 +
<div class="Article_Picture">
 +
<img src="https://static.igem.org/mediawiki/2015/6/6a/NTU-Team-tat-pyy-his.jpg" width="480px"/>
 +
<div class="Article_PictureText"><div class="Text_Picture">[Fig.2-3] Cell penetrating peptide + PYY</div></div>
 +
</div>
 
</div>
 
</div>
−
<div class="Text3"><div class="Text_underline">Nisin</div></div>
 
 
<div class="Text3">
 
<div class="Text3">
−
Since we are aim to produce PYY for the further use in human beings, a food-grade experimental procedure must be conducted. Here we choose nisin, a kind of bacteriocins excreted by <i>Lactococcus lactis</i>, as our selection marker. Bacteriocins are antimicrobial peptides produced by bacteria to kill or inhibit the growth of similar or closely related bacterial strain(s). [2] For nisin, however, it can form pores in the bacterial cytoplasmic membrane and decrease the membrane potential; thus, it has a broader range of target cells. [3] Nisin-producing organisms have their specific way to protect their cell membrane from the nisin pore-forming activity. We use this characteristic of nisin immunity to select cells containing out target plasmids. The detailed self-protection mechanism is discussed in the Design section.
+
In this part, we use the constitutive promoter BBa_J23100 to produce signal peptide-PYY-histidine fusion protein. With the His-tag, we can easily purify the signal peptide-PYY from other non-His-tag protein. This part is constructed to test the amount of the PYY that can secret out of the <i>L. casei</i>.
 +
</div>
 +
<div class="Text1">Test Part</div>
 +
<div class="Text2" id="Third1">RFP</div>
 +
<div class="Container_Article_Picture">
 +
<div class="Article_Picture">
 +
<img src="https://static.igem.org/mediawiki/2015/4/4d/NTU-Team-plac-gfp.jpg" width="480px"/>
 +
<div class="Article_PictureText"><div class="Text_Picture">[Fig.3-1] Suicide sense test</div></div>
 +
</div>
 
</div>
 
</div>
−
               
 
−
<div class="Text2" id="Second3">Suicide</div>
 
−
<div class="Text3"><div class="Text_underline">Programmed Cell Death</div></div>
 
 
<div class="Text3">
 
<div class="Text3">
−
The prototype of our product is an oral capsule which can function well while it reaches the human intestines. Although <i>Lactobacillus casei</i> ATCC393 as a probiotic would not do harm to the consumers’ healthy, for safety concern, cell apoptosis should be introduced due to several reasons. To begin with, we want to control the quantity of PYY within a proper range so as not to cause side-effects. What we value the most is the yield of PYY produced by the time the cell died; therefore, the concentration of bacteria being put into the capsule can be determined with its penetration rate taken into consideration. Another critical reason is that the genes transfer among bacteria and with the environment must be diminished. Otherwise, it may not only interfere the gut flora, but also contaminate the surroundings. With those concerns, we found a desirable part in the iGEM biobricks, namely NcuA. [4] It is a thermonuclease that degrades both plasmid and chromosomal DNA. For more information, please go to the Design section.
+
In this part, we use the RFP protein to testify the function of the lactose operon.
 +
</div>
 +
<div class="Text2" id="Third2">His-PYY</div>
 +
<div class="Container_Article_Picture">
 +
<div class="Article_Picture">
 +
<img src="https://static.igem.org/mediawiki/2015/1/12/NTU-Team-his-pyy.jpg" width="480px"/>
 +
<div class="Article_PictureText"><div class="Text_Picture">[Fig.3-2] PYY</div></div>
 +
</div>
 
</div>
 
</div>
−
 
−
<div class="Text1">Design</div>
 
−
<div class="Text2" id="Third1">CPP-PYY</div>
 
−
<div class="Text2" id="Third2">Nisin Selection</div>
 
−
<div class="Text3">(Fig. Promoter-RBS-nisI-Ter)</div>
 
 
<div class="Text3">
 
<div class="Text3">
−
Studies have showed that for nisin resistance, the immunity lipoprotein NisI as well as the ABC transporter-homologous system NisF/E/G is involved. Functional analysis suggests that NisI acts as nisin-intercepting protein, while NisF/E/G complex acts as exporter that expels the unwanted nisin molecules from cytoplasm to the outer environment. [4] Researchers find that NisI seems to play a more crucial role in nisin immunity than the NisF/E/G complex. [5] Through experiments, either of each expressing in the heterologous bacteria is able to protect the host cells.
+
In this part, we use the constitutive promoter BBa_J23100 to produce histidine-PYY fusion protein. With the His-tag, we can easily purify the PYY from other non-His-tag protein. This part is constructed to test the amount of the PYY under the constitutive promoter in the <i>L. casei</i>.
 +
</div>
 +
<div class="Text2" id="Third3">Signal peptide-PYY-His</div>
 +
<div class="Container_Article_Picture">
 +
<div class="Article_Picture">
 +
<img src="https://static.igem.org/mediawiki/2015/3/39/NTU-Team-sp-pyy-his.jpg" width="480px"/>
 +
<div class="Article_PictureText"><div class="Text_Picture">[Fig.3-3] Signal peptide + PYY</div></div>
 +
</div>
 
</div>
 
</div>
−
<div class="Text2" id="Third3">Suicide</div>
 
−
<div class="Text1">Result</div>
 
−
<div class="Text2" id="Fourth1">CPP-PYY</div>
 
−
<div class="Text2" id="Fourth2">Nisin Selection</div>
 
−
<div class="Text3">(Fig. Promoter-RBS-nisI-Ter)</div>
 
−
<div class="Text3">(Fig. plasmid)</div>
 
 
<div class="Text3">
 
<div class="Text3">
−
Studies have showed that for nisin resistance, the immunity lipoprotein NisI as well as the ABC transporter-homologous system NisF/E/G is involved. Functional analysis suggests that NisI acts as nisin-intercepting protein, while NisF/E/G complex acts as exporter that expels the unwanted nisin molecules from cytoplasm to the outer environment. [5] Researchers find that NisI seems to play a more crucial role in nisin immunity than the NisF/E/G complex. [6] Through experiments, either of each expressing in the heterologous bacteria is able to protect the host cells. [5] Moreover, the expression of nisI in <i>Lactobacillus plantarum</i> was assessed to be at the same level as in <i>Lactococcus lactis</i>. [6]
+
In this part, we use the constitutive promoter BBa_J23100 to produce signal peptide-PYY-histidine fusion protein. With the His-tag, we can easily purify the signal peptide-PYY from other non-His-tag protein. This part is constructed to test the amount of the PYY that can secret out of the <i>L. casei</i>.
 +
</div>
 +
<div class="Text2" id="Third4">Y2R</div>
 +
<div class="Container_Article_Picture">
 +
<div class="Article_Picture">
 +
<img src="https://static.igem.org/mediawiki/2015/e/ea/NTU-Team-y2r.jpg" width="480px"/>
 +
<div class="Article_PictureText"><div class="Text_Picture">[Fig.3-4] Y2R</div></div>
 +
</div>
 
</div>
 
</div>
 
<div class="Text3">
 
<div class="Text3">
−
The figure above shows our gene circuit for nisin selection. The promoter we chose was pUO19 from <i>Escherichia coli</i> which is also functional in <i>Lactobacillus casei</i> and the gene <i>nisI</i> helps <i>Lactobacillus casei</i> transform from nisin-sensitive into nisin-resistant. The fraction enlarged was latter proceeded ligation with CPP-PYY circuit, enabling the following selection.
+
In this part, we use the constitutive promoter BBa_J23100 to produce Y2R. This part is constructed to verify the function of PYY.
 +
</div>
 +
<div class="Text1">References</div>
 +
<div class="Text2" id="Fourth">References</div>
 +
<div class="Text3" id="Reference1">
 +
[1] Torsten, S., Stefan, H., Irina, S., and K. D. Entian. Function of Lactococcus lactis Nisin Immunity Genes nisI and nisFEG after Coordinated Expression in the Surrogate Host Bacillus subtilis. The Jurnal of Biological Chemistry, USA. , Vol. 278, pp. 89 -94 (2003)
 +
</div>
 +
<div class="Text3" id="Reference2">
 +
[2] Yu-Kuo Tsai, Hung- Wen Chen, Ta-Chun Lo and Thy-Hou Lin. Specific point mutation in <i>Lactobacillus casei</i> ATCC 27139 cause the phenotype switch from Lac- to Lac+. Microbiology, pp. 751-760(2009)
 +
</div>
 +
<div class="Text3" id="Reference3">
 +
[3] Ju-Chen Cheng, 2009, Investigation on the Characteristic of Heparin-Binding Haemagglutinin 3 Adhesin (HBHA)-Related Peptides and their Transcytosis
 +
</div>
 +
<div class="Text3" id="Reference4">
 +
[4] C. W. le Roux and S. R. Bloom. Peptide YY, appetite and food intake.(2005)
 
</div>
 
</div>
  
−
<div class="Text2" id="Fourth3">Suicide</div>
+
<div class="Container_Bottom1"></div>
−
<div class="Text3">(Fig. plac-RBS-RFP-Ter)</div>
+
<div class="Container_Bottom2">
−
<div class="Text3">(Fig. plac-RBS-CI-Ter)</div>
+
<div class="Intro_Picture">
−
<div class="Text3">(Fig. pCI-RBS-NcuA-Ter)</div>
+
<div id="NTUschool_logo"><a href="http://www.ntu.edu.tw/english/index.html"><img src="https://static.igem.org/mediawiki/2015/d/de/NTUschool_icon.png"  style="max-width: 100%; max-height: 70px"/></a></div>
−
 
+
<div id="LIHPAO_logo"><a href="http://www.llsc.com.tw/"><img src="https://static.igem.org/mediawiki/2015/d/d4/LIHPAO_logo.png" style="max-width: 100%; max-height: 70px"/></a></div>
 +
<div id="NTUBST_logo"><a href="http://www.bst.ntu.edu.tw/BST-new/NTUBST.html"><img src="https://static.igem.org/mediawiki/2015/6/68/NTUBST_logo.png" style="max-width: 100%; max-height: 70px"/></a></div>
 +
</div>
 +
</div>
 +
<div class="Text_Sponsor">
 +
Maintained by the iGEM team NTU-LIHPAO-Taiwan&nbsp;&nbsp;&nbsp;&nbsp;©2015 NTU-LIHPAO-Taiwan
 +
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Latest revision as of 13:19, 18 September 2015

NTU-LIHPAO-Taiwan

Prototype
Prototype
The iGEM Team NTU-LIHPAO-Taiwan 2015 had built a new biological system for the iGEM community. The original design of the system making up the (Product) contains the three elements listed below, in a probiotic – Lactobacillus casei ATCC 393.
[Fig.1-1] Designed Plasmid
Main Part
Part: BBa_K1841001
nisI
Research from Torsten et al. revealed that the highest level of acquired nisin tolerance was achieved after coordinated expression of all four nisin immunity genes, containing nisI, nisF, nisE, and nisG.[1] Functional analyses provided evidence that NisI acts as a nisin-sequestering protein and that NisFEG acts as a nisin exporter that expels nisin molecules from the cytoplasmic membrane into the environment.
[Fig.2-1] Nisin Selection
Part: BBa_K1841004
plac promoter
Lactose operon of Lactobacillus casei In Lactobacillus casei ATCC393 [pLZ15–], the lactose genes are grouped in a cluster transcribed as single operon. The cluster lacTEGF encodes an antiterminator protein (LacT), lactose-specific elements (LacE and LacF) of the phosphotransferase system (PTS) and a phospho-β-galactosidase (LacG). The promoter region contains a cre element (catabolite responsive element) overlapping the –35 region, which is followed by a highly conserved sequence, the ribonucleic antiterminator (RAT) sequence, and a terminator structure. The antiterminator activity of LacT is also negatively controlled by glucose, possibly by PTS-mediated phosphorylation as explained below.[2]
[Fig.2-2-1] Suicide Sense Part
[Fig.2-2-2] Suicide Kill Part
Part: BBa_K1841007
TAT-PYY
TAT can bring the big molecule substance like DNA, protein, peptide to penetrate the cell. Usually, named as protein transduction domain or membrane transduction domain, it is less than 40 amino acid.[3] PYY belongs to the gastrointestinal hormones. Once the intestine detects the nutrition, the epidermal cell, L cell of ileum and colon will secrete the PYY. While the blood pass the hypothalamus, PYY will bind to the neuropeptide Y receptor in ventromedial nuclei causing the sense of satiation.[4]
[Fig.2-3] Cell penetrating peptide + PYY
In this part, we use the constitutive promoter BBa_J23100 to produce signal peptide-PYY-histidine fusion protein. With the His-tag, we can easily purify the signal peptide-PYY from other non-His-tag protein. This part is constructed to test the amount of the PYY that can secret out of the L. casei.
Test Part
RFP
[Fig.3-1] Suicide sense test
In this part, we use the RFP protein to testify the function of the lactose operon.
His-PYY
[Fig.3-2] PYY
In this part, we use the constitutive promoter BBa_J23100 to produce histidine-PYY fusion protein. With the His-tag, we can easily purify the PYY from other non-His-tag protein. This part is constructed to test the amount of the PYY under the constitutive promoter in the L. casei.
Signal peptide-PYY-His
[Fig.3-3] Signal peptide + PYY
In this part, we use the constitutive promoter BBa_J23100 to produce signal peptide-PYY-histidine fusion protein. With the His-tag, we can easily purify the signal peptide-PYY from other non-His-tag protein. This part is constructed to test the amount of the PYY that can secret out of the L. casei.
Y2R
[Fig.3-4] Y2R
In this part, we use the constitutive promoter BBa_J23100 to produce Y2R. This part is constructed to verify the function of PYY.
References
References
[1] Torsten, S., Stefan, H., Irina, S., and K. D. Entian. Function of Lactococcus lactis Nisin Immunity Genes nisI and nisFEG after Coordinated Expression in the Surrogate Host Bacillus subtilis. The Jurnal of Biological Chemistry, USA. , Vol. 278, pp. 89 -94 (2003)
[2] Yu-Kuo Tsai, Hung- Wen Chen, Ta-Chun Lo and Thy-Hou Lin. Specific point mutation in Lactobacillus casei ATCC 27139 cause the phenotype switch from Lac- to Lac+. Microbiology, pp. 751-760(2009)
[3] Ju-Chen Cheng, 2009, Investigation on the Characteristic of Heparin-Binding Haemagglutinin 3 Adhesin (HBHA)-Related Peptides and their Transcytosis
[4] C. W. le Roux and S. R. Bloom. Peptide YY, appetite and food intake.(2005)
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