Difference between revisions of "Team:Bielefeld-CeBiTec/siRNA"

Revision as of 23:11, 6 September 2018

/* HQ CSS*/ .firstHeading { display: none; }

.mw-content-text { display: none; }

.logo_2018 { display: none; }

globalWrapper {

padding: 0px; margin: 0px; padding-bottom: 0px; }

content {

margin-left:0px; padding:0px; width:100%; margin-top: -7px; }

.mw-content-ltr ul, .mw-content-rtl .mw-content-ltr ul{ margin:0px; }

top_title {

margin-bottom: 0px; }

top_menu_14{

border-bottom: 0px solid black; }

HQ_page p {

margin: 0; }

/*NAVBAR*/

@charset "UTF-8";

body{ margin-top:0px; }

.navigation { height: 70px; background: #002a5c; position: fixed; width:100%; text-decoration: none; }

.brand { position: absolute; padding-left: 20px; float: left; line-height: 70px; text-transform: uppercase; font-size: 1.4em; width: 300px; }

.logo-img{ width: 70%; margin-top: 1%; }

@media only screen and (max-width: 1350px) { .logo-img{ width: 70%; margin-top: 2%; }

.navigation{ position: absolute; }

}

@media only screen and (max-width: 790px) { .logo-img{ width: 70%; margin-top: 3%; }

.navigation{ position: absolute; }

}

.brand a, .brand a:visited { color: #ffffff; text-decoration: none; }

.nav-container { max-width: auto; margin: 0 auto; text-decoration: none; }

nav { float: right; text-decoration: none; } nav ul { list-style: none; margin: 0; padding: 0; text-decoration: none; } nav ul li { float: left; position: relative; text-decoration: none; } nav ul li a, nav ul li a:visited, nav ul li a:focus{ display: block; padding: 0 20px; line-height: 70px; background: #002a5c; color: #ffffff; text-decoration: none; margin-bottom:-4px; border-color:#002a5c; } nav ul li a:hover, nav ul li a:visited:hover { background: #4d9dff; color: #ffffff; text-decoration: none; } nav ul li a:not(:only-child):after, nav ul li a:visited:not(:only-child):after { padding-left: 4px; content: " ▾"; text-decoration: none; } nav ul li ul li { min-width: 190px; text-decoration: none; } nav ul li ul li a { padding: 15px; line-height: 20px; text-decoration: none; text-decoration: none; }

.nav-dropdown { position: absolute; display: none; z-index: 1; box-shadow: 0 3px 12px rgba(0, 0, 0, 0.15); text-decoration: none; }

/* Mobile navigation */ .nav-mobile { display: none; position: absolute; top: 0; right: 0; background: #002a5c; height: 70px; width: 70px; }

@media only screen and (max-width: 1350px) {

.nav-mobile { display: block; }

nav { width: 100%; padding: 70px 0 15px; } nav ul { display: none; max-height: 280px; overflow-y: auto; } nav ul li { float: none; } nav ul li a { padding: 15px; line-height: 20px; text-decoration: none; } nav ul li ul li a { padding-left: 30px; text-decoration: none; }

.nav-dropdown { position: static; } } @media screen and (min-width: 1351px) { .nav-list { display: block !important; } }

nav-toggle {

position: absolute; left: 18px; top: 22px; cursor: pointer; padding: 10px 35px 16px 0px; max-height: 280px; overflow-y: auto; }

nav-toggle span,
nav-toggle span:before,
nav-toggle span:after {

cursor: pointer; border-radius: 1px; height: 5px; width: 35px; background: #ffffff; position: absolute; display: block; content: ""; transition: all 300ms ease-in-out; }

nav-toggle span:before {

top: -10px; }

nav-toggle span:after {

bottom: -10px; }

nav-toggle.active span {

background-color: transparent; }

nav-toggle.active span:before, #nav-toggle.active span:after {

top: 0; }

nav-toggle.active span:before {

transform: rotate(45deg); }

nav-toggle.active span:after {

transform: rotate(-45deg); }

article { max-width: 1000px; margin: 0 auto; padding: 10px; line-height: 1.5; }

.article { max-width: 1000px; margin: 0 auto; padding: 10px; line-height: 1.5; padding-right: 5%; padding-left : 5%;

}

/* W3 Schools templates*/

.w3-top,.w3-bottom{position:fixed;width:100%;z-index:1;} .w3-top{top:0;} .w3-bottom{bottom:0;}

footer{display:block;width:98%}

.w3-container:after,.w3-container:before{content:"";display:table;clear:both;width:98%;}

.w3-container{}

.w3-padding-32{padding-top:32px!important;padding-bottom:32px!important;}

.w3-center .w3-bar{display:inline-block;width:auto;}

.w3-center{text-align:center!important;}

.w3-black,.w3-hover-black:hover{color:#fff!important;background-color:#000!important;}

.w3-xlarge{font-size:24px!important;}

/*OWN STYLES*/

body { font-family: "Lato", sans-serif; }

@media only screen and (min-width: 1010px){ .main_content {

background-color: white; border: 1px solid #000000; border-radius: 0px; box-shadow: 1px 1px 3px rgba(0, 0, 0, 0.35); min-height: 100px; border-top: none; border-bottom: none; margin-left:25%; margin-right:25%; padding-bottom: 4%; min-height: 420px; position: sticky; margin-top: -100px; } }

@media only screen and (max-width: 1009px){ .main_content {

background-color: white; border: 1px solid #000000; border-radius: 0px; box-shadow: 1px 1px 3px rgba(0, 0, 0, 0.35); min-height: 100px; border-top: none; border-bottom: none; margin-left:0; margin-right:0; padding-bottom: 4%; position: sticky; margin-top: -100px; } }

container{

position: relative; min-height: 100%; height: 100%; }

.container{ padding-top:0px; background-color: #001633; }

.section{ margin: 1%; }

.half{ max-width: 48%; }

.half.left { float: left; margin-right: 1%; }

.half.right { float: right; margin-left: 1%;

}

article, .article { text-align: justify; display: block; margin: auto; font-size: 16px; padding-bottom: 20px; }

article, .article, .contentline { width: 90%; font-size: 16px; padding-bottom: 30px; }

.contentline article, .contentline .article { width: 100%; }

h1, h2, h3, h4, h5, h6 { width: 92%; margin: auto; margin-top: 0px; padding-top: 20px; padding-bottom: 15px; }

.p { text-align: justify; }

.title { font-size: 40px; padding-top: 5%; padding-left: 3%; padding-bottom: 40px; font-weight: bold; background-color: rgb(255,255,255); margin-top: -70px; position:sticky; line-height:1.5em; text-align:left; }

h2 { padding-left: 3%; font-size:14; }

br { display: block; /* makes it have a width */ content: ""; /* clears default height */ margin-top: 20px; /* change this to whatever height you want it */ }

p { font-size:14px; }

body { padding-top: 0px; }

article{ padding-right: 5%; padding-left : 5%; }

page-arrow-top {

display: none; cursor: pointer; padding: 0; margin: 0; position: fixed; bottom: 20px; right: 50px; }

.half { width: 48%; }

.half.left { float: left; margin-right: 1%; }

.half.right { float: right; margin-left: 1%; }

.sidenavi{

margin-top: 50px; margin-left: 4%; display: block; position: -webkit-sticky; position: sticky; top: 150px; float: left; background-color: #001633; border-radius: 30px; border-color: white; border-style: solid; }

.side_list{ display: block; margin-right: 10px; margin-bottom: 15px; margin-top: 10px; margin-left: 10px; color: white; }

.side_link{ color: white; font-size: 20px; text-decoration:none; padding: 10px; }

.side_list > a:hover{ text-decoration:none; color: white; }

.side_list > a{ text-decoration:none; font-size: 18px; padding: 10px; width:100%; cursor: pointer; color: white; }

@media only screen and (max-width: 1150px){ .sidenavi{

margin-top: 50px; margin-left: 1%; display: block; position: -webkit-sticky; position: sticky; top: 150px; float: left; background-color: #001633; border-radius: 30px; border-color: white; border-style: solid; }

.side_list{ display: block; margin-right: 10px; margin-bottom: 15px; margin-top: 10px; margin-left: 10px; color: white; }

.side_link{ color: white; font-size: 10px; text-decoration:none; padding: 10px; }

.side_list > a:hover{ text-decoration:none; color: white; }

.side_list > a{ text-decoration:none; font-size: 16px; padding: 12px; width:100%; cursor: pointer; color: white; }

}

@media only screen and (max-width: 1685px){ .sidenavi{

margin-top: 50px; margin-left: 1%; display: block; position: -webkit-sticky; position: sticky; top: 150px; float: left; background-color: #001633; border-radius: 30px; border-color: white; border-style: solid; }

.side_list{ display: block; margin-right: 10px; margin-bottom: 15px; margin-top: 10px; margin-left: 10px; color: white; }

.side_link{ color: white; font-size: 10px; text-decoration:none; padding: 10px; }

.side_list > a:hover{ text-decoration:none; color: white; }

.side_list > a{ text-decoration:none; font-size: 16px; padding: 8px; width:100%; cursor: pointer; color: white; }

}

@media only screen and (max-width: 1300px){ .sidenavi{ display: none;

} }

figure { text-align: center; margin-bottom: 30px;

}

figcaption{ text-align: justify; font-size: 13px; margin-top:10px; }

.figure.eighty { width:80%; }

.figure.ten { width:10%; }

.figure.twenty { width:20%; }

.figure.thirty { width:30%; }

.figure.fourty { width:40%; }

.figure.fifty { width:50%; }

.figure.sixty { width:60%; }

.figure.seventy { width:70%; }

.figure.ninety { width:90%; }

.figure.hundred { width:100%; }

table, th, td { border: 1px solid black; border-collapse: collapse; font-size:17px; } th, td { padding: 15px; text-align: left; } table#t01 tr:first-child td{ background-color: #eee; }

table#t01 tr td:first-child { background-color: #eee; color: black; }

table.centern{ margin-left:15%; margin-right:15%; margin-top: 100px; margin-top: 5%; }

HQ_page table {

border: 1px solid #ccc; border-collapse: collapse; width: 70%; margin: auto; margin-bottom: 0px; margin-top: 100px; margin-right: auto; margin-left: auto; }

hr { margin-top: 10%; margin-bottom: 20px; border: 0; text-align: center; border-top: 2px solid #000000; margin-left: auto; margin-right: auto;

}

.reftext { display: block; text-align: justify; margin-left: 5%; margin-right: 5%; font-size: 14px; padding-bottom: 20px; line-height: 20px; }

/*Footer*/ .fa-facebook-official:before { content: "\f230"; color: white; }

.fa-instagram:before { content: "\f230"; color: white; }

.fa-twitter:before { content: "\f230"; color: white; }

myDIV {

margin-top: 10px; display:none; }

.refbtn{ background-color: white; border: none; font-size: 150%; font-weight: bold; color: black; margin-left: 5%; margin-bottom: 26px; margin-top: 30px;

}

a.shifted-anchor { display: block; position:relative; top: -100px; }

active{

text-decoration: none; }

html,body { background-color: #001633; }

myVideo {

position: static; top:0; left:0 right: 0; bottom: 0; height: auto; margin-top: 70px; z-index:1; }

.title_picture{ width:100%; padding-top:40px; }

@-webkit-keyframes animateBubble { 0% { margin-top: 310%; } 100% { margin-top: 5%; } }

@-moz-keyframes animateBubble { 0% { margin-top: 310%; } 100% { margin-top: 5%; } }

@keyframes animateBubble { 0% { margin-top: 310%; } 100% { margin-top: 5%; } }

@-webkit-keyframes animateBubbleSeite { 0% { margin-top: 40px; } 100% { margin-top: -200%; } }

@-moz-keyframes animateBubbleSeite { 0% { margin-top: 40px; } 100% { margin-top: -200%; } }

@keyframes animateBubbleSeite { 0% { margin-top: 40px; } 100% { margin-top: -200%; } }

@-webkit-keyframes sideWays { 0% { margin-left:0px; } 100% { margin-left:50px; } }

@-moz-keyframes sideWays { 0% { margin-left:0px; } 100% { margin-left:50px; } }

@keyframes sideWays { 0% { margin-left:0px; } 100% { margin-left:50px; } }

.s1 { -webkit-animation: animateBubbleSeite 120s linear infinite, sideWays 2s ease-in-out infinite alternate; -moz-animation: animateBubbleSeite 120s linear infinite, sideWays 2s ease-in-out infinite alternate; animation: animateBubbleSeite 120s linear infinite, sideWays 2s ease-in-out infinite alternate;

left: 5%; top: 95%; z-index:1; overflow: hidden;

-webkit-transform: scale(0.1); -moz-transform: scale(0.1); transform: scale(0.1); }

.x1 { -webkit-animation: animateBubble 100s linear infinite, sideWays 2s ease-in-out infinite alternate; -moz-animation: animateBubble 100s linear infinite, sideWays 2s ease-in-out infinite alternate; animation: animateBubble 100s linear infinite, sideWays 2s ease-in-out infinite alternate;

left: -5%; top: 20%; z-index:1; overflow: hidden;

-webkit-transform: scale(0.1); -moz-transform: scale(0.1); transform: scale(0.1); }

.x2 { -webkit-animation: animateBubble 80s linear infinite, sideWays 4s ease-in-out infinite alternate; -moz-animation: animateBubble 80s linear infinite, sideWays 4s ease-in-out infinite alternate; animation: animateBubble 80s linear infinite, sideWays 4s ease-in-out infinite alternate;

left: 5%; top: 20%; z-index:1; overflow: hidden; -webkit-transform: scale(0.2); -moz-transform: scale(0.2); transform: scale(0.2); }

.x3 { -webkit-animation: animateBubble 120s linear infinite, sideWays 2s ease-in-out infinite alternate; -moz-animation: animateBubble 120s linear infinite, sideWays 2s ease-in-out infinite alternate; animation: animateBubble 120s linear infinite, sideWays 2s ease-in-out infinite alternate;

left: 10%; top: 20%; overflow: hidden; z-index:1; -webkit-transform: scale(0.2); -moz-transform: scale(0.2); transform: scale(0.2); }

.x4 { -webkit-animation: animateBubble 90s linear infinite, sideWays 3s ease-in-out infinite alternate; -moz-animation: animateBubble 90s linear infinite, sideWays 3s ease-in-out infinite alternate; animation: animateBubble 90s linear infinite, sideWays 3s ease-in-out infinite alternate;

left: 20%; top: 20%; overflow: hidden; z-index:1; -webkit-transform: scale(0.2); -moz-transform: scale(0.2); transform: scale(0.2); }

.x5 { -webkit-animation: animateBubble 70s linear infinite, sideWays 4s ease-in-out infinite alternate; -moz-animation: animateBubble 70s linear infinite, sideWays 4s ease-in-out infinite alternate; animation: animateBubble 70s linear infinite, sideWays 4s ease-in-out infinite alternate;

left: 30%; top: 20%; overflow: hidden; z-index:1; -webkit-transform: scale(0.1); -moz-transform: scale(0.1); transform: scale(0.1); }

.x6 { -webkit-animation: animateBubble 115s linear infinite, sideWays 2s ease-in-out infinite alternate; -moz-animation: animateBubble 115s linear infinite, sideWays 2s ease-in-out infinite alternate; animation: animateBubble 115s linear infinite, sideWays 2s ease-in-out infinite alternate;

left: 50%; top: 20%; overflow: hidden; z-index:1; -webkit-transform: scale(0.1); -moz-transform: scale(0.1); transform: scale(0.1); }

.x7 { -webkit-animation: animateBubble 120s linear infinite, sideWays 2s ease-in-out infinite alternate; -moz-animation: animateBubble 120s linear infinite, sideWays 2s ease-in-out infinite alternate; animation: animateBubble 120s linear infinite, sideWays 2s ease-in-out infinite alternate;

left: 65%; top: 20%; overflow: hidden; z-index:1; -webkit-transform: scale(0.1); -moz-transform: scale(0.1); transform: scale(0.1); }

.x8 { -webkit-animation: animateBubble 100s linear infinite, sideWays 3s ease-in-out infinite alternate; -moz-animation: animateBubble 100s linear infinite, sideWays 3s ease-in-out infinite alternate; animation: animateBubble 100s linear infinite, sideWays 3s ease-in-out infinite alternate;

left: 80%; top: 20%; overflow: hidden; z-index:1; -webkit-transform: scale(0.2); -moz-transform: scale(0.2); transform: scale(0.2); }

.x9 { -webkit-animation: animateBubble 80s linear infinite, sideWays 4s ease-in-out infinite alternate; -moz-animation: animateBubble 80s linear infinite, sideWays 4s ease-in-out infinite alternate; animation: animateBubble 80s linear infinite, sideWays 4s ease-in-out infinite alternate;

left: 90%; top: 20%; overflow: hidden; z-index:1; -webkit-transform: scale(0.1); -moz-transform: scale(0.1); transform: scale(0.1); }

.x10 { -webkit-animation: animateBubble 120s linear infinite, sideWays 2s ease-in-out infinite alternate; -moz-animation: animateBubble 120s linear infinite, sideWays 2s ease-in-out infinite alternate; animation: animateBubble 120s linear infinite, sideWays 2s ease-in-out infinite alternate;

left: 80%; top: 20%; overflow: hidden; z-index:1; -webkit-transform: scale(0.1); -moz-transform: scale(0.1); transform: scale(0.1); }

.bubble { -webkit-border-radius: 50%; -moz-border-radius: 50%; border-radius: 50%;

-webkit-box-shadow: 0 20px 30px rgba(0, 0, 0, 0.2), inset 0px 10px 30px 5px rgba(255, 255, 255, 1); -moz-box-shadow: 0 20px 30px rgba(0, 0, 0, 0.2), inset 0px 10px 30px 5px rgba(255, 255, 255, 1); box-shadow: 0 20px 30px rgba(0, 0, 0, 0.2), inset 0px 10px 30px 5px rgba(255, 255, 255, 1);

height: 200px; position: absolute; width: 200px; }

.bubble:after { background: -moz-radial-gradient(center, ellipse cover, rgba(255,255,255,0.5) 0%, rgba(255,255,255,0) 0%); /* FF3.6+ */ background: -webkit-gradient(radial, center center, 0px, center center, 100%, color-stop(0%,rgba(255,255,255,0.5)), color-stop(0%,rgba(255,255,255,0))); /* Chrome,Safari4+ */ background: -webkit-radial-gradient(center, ellipse cover, rgba(255,255,255,0.5) 0%,rgba(255,255,255,0) 0%); /* Chrome10+,Safari5.1+ */ background: -o-radial-gradient(center, ellipse cover, rgba(255,255,255,0.5) 0%,rgba(255,255,255,0) 0%); /* Opera 12+ */ background: -ms-radial-gradient(center, ellipse cover, rgba(255,255,255,0.5) 0%,rgba(255,255,255,0) 0%); /* IE10+ */ background: radial-gradient(ellipse at center, rgba(255,255,255,0.5) 0%,rgba(255,255,255,0) 0%); /* W3C */ filter: progid:DXImageTransform.Microsoft.gradient( startColorstr='#80ffffff', endColorstr='#00ffffff',GradientType=1 ); /* IE6-9 fallback on horizontal gradient */

-webkit-border-radius: 50%; -moz-border-radius: 50%; border-radius: 50%;

-webkit-box-shadow: inset 0 20px 30px rgba(255, 255, 255, 0.3); -moz-box-shadow: inset 0 20px 30px rgba(255, 255, 255, 0.3); box-shadow: inset 0 20px 30px rgba(255, 255, 255, 0.3);

content: ""; height: 180px; left: 10px; position: absolute; width: 180px; }

@media only screen and (max-width: 1700px) {

.bubble.x1, .bubble.x2, .bubble.x8, .bubble.x9, .bubble.x10{ display:none; }

}

@media only screen and (max-width: 1200px) {

.bubble.x1, .bubble.x2, .bubble.x7, .bubble.x8, .bubble.x9, .bubble.x10{ display:none; } }

.timer_box{

   border: white;
   border-style: solid;
   padding-bottom: 125px;
   padding-left: 10px;
   padding_top:10px;
   marging-bottom: 100px;

}

gold {

 float: left;
 font-size: 50px;
 margin-top: 10px;
 width: 50%;
 color:white;

text-align: center;

}

gold_text {

 float: left;
 font-size: 20px;
 margin-top: 0px;
 width: 50%;
 color:white;

text-align: center; }

silver {

 float: left;
 text-align: center;
 font-size: 50px;
 margin-top: 10px;
 width: 50%;
 color:white;

}

silver_text {

 float: left;
 text-align: center;
 font-size: 20px;
 margin-top: 0px;
 width: 50%;
 color:white;

}

.img_text{ text-align:center; position:absolute; }

home_imgs {

   margin-top:100px;
   width:60%;

}

@media only screen and (max-width: 1300px) {

home_imgs{

width:70%;

}

silver{

font-size: 30px;

}

gold{

font-size: 30px;

}

silver_text{

font-size: 20px;

}

gold_text{

font-size: 20px; }

}

@media only screen and (max-width: 900px) {

.timer_box{

   border: white;
   border-style: solid;
   padding-bottom: 100px;
   padding-left: 10px;
   padding_top:10px;
   marging-bottom: 100px;

}

home_imgs{

margin-top:50px; width:90%; }

silver{

font-size: 20px;

}

gold{

font-size: 20px;

}

silver_text{

font-size: 15px;

}

gold_text{

font-size: 15px; }

}

@media only screen and (max-width: 600px) {

.timer_box{

   border: white;
   border-style: solid;
   padding-bottom: 100px;
   padding-left: 10px;
   padding_top:10px;
   marging-bottom: 100px;

}

home_imgs{

margin-top:15px; width:100%; }

silver{

font-size: 15px;

}

gold{

font-size: 15px;

}

silver_text{

font-size: 10px;

}

gold_text{

font-size: 10px; }

}

@media only screen and (max-width: 400px) {

.timer_box{

   border: white;
   border-style: solid;
   padding-bottom: 50px;
   padding-left: 10px;
   padding_top:10px;
   marging-bottom: 100px;

}

home_imgs{

margin-top:5px; width:100%; }

silver{

font-size: 10px;

}

gold{

font-size: 10px;

}

silver_text{

font-size: 5px;

}

gold_text{

font-size: 5px; }

}

siRNA/RNAi

In Synthetic Biology it is often required to perform knockouts in the context of metabolic engineering. Currently knockouts are usually carried out by using the CRISPR/Cas9 system. One problem of these knockouts is that they are permanent and that the knockout of housekeeping genes can inhibit growth. In certain cases a knockout can lead to the death of the cell, making it impossible to knock a gene out for metabolic engineering. We want to propose an alternative: siRNAs and RNAi. The RNA interference system (RNAi) can be used to knock down genes by degradation of the corresponding mRNA, making the knockout inducible by using an inducible promoter. This enables us to let production cells grow until a desired cell density or production phase and lets us induce the knockdown when needed.
Protein biosynthesis is strongly regulated by different post- and pretranslational mechanisms. Post-translational regulation is realized by small interfering RNAs (siRNA)which block the ribosome binding site (RBS) and inhibit transcription by the RNA interference (RNAi) mechanism. Both mechanisms use the RNA chaperone Hfq to facilitate the hybridization of siRNA and mRNA (Na et al., 2013).
After transcription, RNAs are protected from degradation by a triphosphate at the 5’ terminus. Monophosphorylated ends are produced through maturation of the RNA by internal cleavage or by cleavage of pyrophosphate at the 5’ terminus (Foley et al., 2015).
Cleavage of the 5’ terminal phosphates is also the first step in RNA degradation. RNase E plays a major role in RNA degradation as well as in RNAi, as it does not only degrade monophosphorylated RNAs but can also accept a monophosphorylated 5’ terminus as an activator that triggers a conformational change by interaction with the 5’ sensing pocket. It has been shown that this allows the RNase E to cleave mRNA. Therefore, a complementary siRNA with unpaired bases at the 5’ terminus bound to an mRNA can induce cleavage of the mRNA. In a test four unpaired bases were sufficient and cleavage occurred 6 bp downstream of the homologue region. The required complementary sequence was rather short being only 9 bp long.
To enhance the degrading capability of siRNAs the 5’ terminus can be tailored to match the substrate specificity of the RNA pyrophosphohydrolase RppH by having a C at Position 2 from the 5’ terminus (Bandyra et al., 2012).
To find and design possible siRNAs for RNAi we designed and tested a program which also predicts secondary structures and the non-complementary 5’ terminus.
*Link here* Additionally we designed a two vector system to scan single siRNAs or siRNA libraries for the most effective RNAi silencing of a given target gene.

Two Vector System

To compare and scan multiple siRNAs or RNA libraries for RNAi we developed a two vector system comprised of an expression Vektor for the siRNAs and a Target Vektor for the production of a target sequence and the marker protein.
There are two versions of the Target Vektor. Version one is comprised of the PTet promoter, a lacZ-alpha cassette, a linker and the reporter protein AmilCP. To enable flourescence measurements a version containing the Blue Flourescent Protein (BFP) has been constructed as well. With higher silencing effectivity the absorption declines which can lead to false positive results in cases where the reporter protein does not work as expected. The second version has the lacZ-alpha cassette linked to the LacI protein. If transcribed correctly this inhibits the Lac promoter which in turn produces the reporter protein AmilCP if not inhibited.

Figure X: Map of Biobrick XXX, The target vektor of our siRNA test system.

This setup prevents false positive results as the absorption increases with higher silencing activity. Prior to use one of both biobricks needs to be cloned into another vector to achieve two different antibiotic resistances. The lacZ-alpha cassette can be replaced using the BbsI restriction enzyme. After the ligation of a target sequence into the Target vector it can be transformed. A library of possible siRNAs can be inserted into the expression vector followed by a transformation into competent cells containing the target vector. The emerging colonies then can be scanned for maximum silencing efficiency. Multiple colonies can be tested simultaneously in 96 well plates.

RNAi versus Knockouts

Using the siRNA Testing System

@@ Line 86: / Line 86: @@
   <figure role="group">
+<img class="figure onehundred" src="https://static.igem.org/mediawiki/2018/9/94/T--Bielefeld-CeBiTec--ALE-siRNA_Testsystem_Übersichtsgrafik.png">
 <!--
 </br>