Difference between revisions of "Team:FAFU-CHINA"

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        font-family: Georgia, Times, "Times New Roman", serif;
 +
padding: 10px 15px 5px 10px;
 +
border-bottom: 1px solid #ccc;
 +
margin-bottom: 0px;
 +
}
 +
 
 +
.newsItem {
 +
border-bottom: 1px solid #ccc;
 +
display: block;
 +
padding: 5px 15px 0px 10px;
 +
margin-bottom: 0px;
 +
}
 +
 
 +
.newsItem h3 {
 +
width: auto;
 +
display: inline;
 +
font-size: 14px;
 +
font-family: Arial, Helvetica, sans-serif;
 +
padding: 0px;
 +
margin: 0px;
 +
}
 +
 
 +
.newsItem img {
 +
float: right;
 +
clear: right;
 +
width: 80px;
 +
padding-left: 15px;
 +
display: inline-block;
 +
}
 +
 
 +
.newsItem .newsDate {
 +
font-style: italic;
 +
font-size: 14px;
 +
display: inline-block;
 +
color: #999;
 +
float: right;
 +
padding: 0px 0px 10px 15px;
 +
}
 +
 
 +
.newsItem p, .newsItem ul, .newsItem li {
 +
padding: 0px;
 +
margin-left: 20px;
 +
font-size: 12px;
 +
line-height: 1.2;
 +
}
 +
 
 +
.newsItem p {margin-top: 10px;}
 +
 
 +
.annItem {
 +
border: 2px solid #414141;
 +
display: block;
 +
padding: 0px 15px 10px 10px;
 +
margin-bottom: 18px;
 +
position: relative;
 +
height: 160px;
 +
vertical-align: middle;
 +
}
 +
 
 +
.annItem .annCentered {
 +
display: table-cell;
 +
vertical-align: middle;
 +
height: 160px;
 +
}
 +
 
 +
.annCentered h3 {
 +
padding: 0px;
 +
display: block;
 +
margin-left: auto;
 +
margin-right: auto;
 +
text-align: center;
 +
}
 +
 
 +
.annItem ul, .annItem li {padding: 0px; margin: 0px;}
 +
.annItem li {list-style: none; margin: 5px}
 +
 
 +
 
 +
 
 +
</style>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<style>
 +
 
 +
*, *:after, *:before { -webkit-box-sizing: border-box; box-sizing: border-box; }
 +
.clearfix:before, .clearfix:after { content: ''; display: table; }
 +
.clearfix:after { clear: both; }
 +
 
 +
.container {
 +
background: white;
 +
width: 974px;
 +
margin: 0 auto;
 +
margin-bottom: 100px;
 +
}
 +
 
 +
body {
 +
min-width: 960px;
 +
/*background-image: url("https://static.igem.org/mediawiki/2014/7/74/Rutgers_stripes.png");*/
 +
/*background-image: url("https://static.igem.org/mediawiki/2014/6/69/Rutgers_bg_hoffman.png");*/
 +
background-image: url("https://static.igem.org/mediawiki/2014/5/5d/Rutgers_bg_hoffman2.png");
 +
font-weight: 400 !important;
 +
font-family: Arial, Helvetica, sans-serif;
 +
}
 +
 
 +
a {
 +
color: black;
 +
text-decoration: none !important;
 +
outline: none;
 +
}
 +
 
 +
a:hover, a:focus {
 +
color: #74777b;
 +
}
 +
 
 +
/*section {
 +
font-family: Arial, Helvetica, sans-serif;
 +
}*/
 +
 
 +
h2 {
 +
display:inline-block;
 +
padding-left: 20px;
 +
padding-right: 20px;
 +
border-bottom: 1px dotted black;
 +
font-family: Arial, Helvetica, sans-serif !important;
 +
}
 +
 
 +
iframe {
 +
border: 1px dotted black;
 +
margin: 0px;
 +
}
 +
 
 +
section ul.reglist {
 +
list-style-type: circle !important;
 +
margin-left: 25px;
 +
}
 +
 
 +
section ul.reglist li {
 +
list-style-type: circle !important;
 +
text-align: justify !important;
 +
text-justify: inter-word !important;
 +
font-size: 16px !important;
 +
 
 +
padding-right: 25px;
 +
line-height: 110%;
 +
}
 +
 
 +
table.dotted p {
 +
text-align: justify !important;
 +
text-justify: inter-word !important;
 +
font-size: 16px !important;
 +
line-height: 110%;
 +
}
 +
 
 +
section .fp {
 +
width: 750px;
 +
margin: 0 auto;
 +
}
 +
section .fp p {
 +
text-align: justify !important;
 +
text-justify: inter-word !important;
 +
font-size: 16px !important;
 +
line-height: 110%;
 +
padding-bottom: 10px;
 +
}
 +
 
 +
table {
 +
margin: 0 auto !important;
 +
border-collapse: collapse;
 +
}
 +
 
 +
table.dotted img {
 +
display: block;
 +
margin: 0 auto; /* !important;*/
 +
}
 +
 
 +
table.dotted th {
 +
background: gray;
 +
border: 1px solid black !important;
 +
}
 +
 
 +
table.dotted td {
 +
border: 1px dotted black !important;
 +
vertical-align: top;
 +
}
 +
 
 +
table.abstract td {
 +
width: 700px;
 +
padding: 0 0px 25px 0px !important;
 +
}
 +
 
 +
table.parts td {
 +
vertical-align: middle;
 +
}
 +
 
 +
table.accomp td {
 +
width: 430px;
 +
padding: 0 0px 25px 0px !important;
 +
}
 +
table.accomp td img {
 +
width: 250px;
 +
}
 +
 
 +
table.attrib td {
 +
width: 700px;
 +
padding: 10px !important;
 +
text-align: left;
 +
}
 +
table.attrib td img {
 +
float: left;
 +
margin: 10px;
 +
}
 +
table.attrib td p {
 +
padding: 10px;
 +
}
 +
 
 +
/*.h2wide h2 {
 +
display: inline-block;
 +
font-family: Arial, Helvetica, sans-serif;
 +
height: 34px;
 +
padding: 0;
 +
margin: 25px auto;
 +
}
 +
.h2wide {
 +
}
 +
.h2left {
 +
display: inline-block;
 +
width: 0;
 +
height: 0;
 +
border-top: 3px solid transparent;
 +
border-right: 150px solid red;
 +
-webkit-filter: drop-shadow(0 0 1px red);
 +
filter: drop-shadow(0 0 1px red);
 +
}
 +
.h2right {
 +
display: inline-block;
 +
width: 0;
 +
height: 0;
 +
border-top: 3px solid transparent;
 +
border-left: 150px solid red;
 +
-webkit-filter: drop-shadow(0 0 1px red);
 +
filter: drop-shadow(0 0 1px red);
 +
}*/
 +
 
 +
.no-flexbox .support {
 +
display: block;
 +
}
 +
 
 +
.container > section {
 +
/*padding: 5em 0;*/
 +
font-size: 16px; /* (was 1.25em) */
 +
min-height: 100%;
 +
}
 +
 
 +
img.RU_logo {
 +
width: 600px;
 +
display: block;
 +
margin: 10px auto;
 +
margin-bottom: 0;
 +
}
 +
 
 +
img.iGEM_logo {
 +
margin: 5px 0 0 40px;
 +
}
 +
 
 +
img.down_arrow {
 +
display: block;
 +
margin: 40px auto;
 +
}
 +
 
 +
/* Nav */
 +
 
 +
.tabs {
 +
position: relative;
 +
/*overflow: hidden;*/
 +
margin: 0 auto;
 +
width: 100%;
 +
font-weight: 300;
 +
font-size: 1.25em;
 +
}
 +
 
 +
.tabs nav {
 +
text-align: center;
 +
/*margin-bottom: 30px;*/
 +
}
 +
 
 +
.tabs nav ul {
 +
position: relative;
 +
display: -ms-flexbox;
 +
display: -webkit-flex;
 +
display: -moz-flex;
 +
display: -ms-flex;
 +
display: flex;
 +
margin: 0 auto;
 +
padding: 0;
 +
max-width: 1200px;
 +
list-style: none;
 +
-ms-box-orient: horizontal;
 +
-ms-box-pack: center;
 +
-webkit-flex-flow: row wrap;
 +
-moz-flex-flow: row wrap;
 +
-ms-flex-flow: row wrap;
 +
flex-flow: row wrap;
 +
-webkit-justify-content: center;
 +
-moz-justify-content: center;
 +
-ms-justify-content: center;
 +
justify-content: center;
 +
}
 +
 
 +
.tabs nav ul li {
 +
position: relative;
 +
z-index: 1;
 +
display: block;
 +
margin: 0;
 +
text-align: center;
 +
-webkit-flex: 1;
 +
-moz-flex: 1;
 +
-ms-flex: 1;
 +
flex: 1;
 +
}
 +
 
 +
.tabs nav a {
 +
position: relative;
 +
display: block;
 +
overflow: hidden;
 +
text-overflow: ellipsis;
 +
white-space: nowrap;
 +
line-height: 2.5;
 +
}
 +
 
 +
.tabs nav a span {
 +
vertical-align: middle;
 +
font-size: 1em;
 +
}
 +
 
 +
.tabs nav li.tab-current a {
 +
color: black;
 +
}
 +
 
 +
.tabs nav a:focus {
 +
outline: none;
 +
}
 +
 
 +
/* nav list elements */
 +
 
 +
.tabs-style-circlefill {
 +
max-width: 960px;
 +
/*border: 0px solid #2CC185;*/
 +
}
 +
 
 +
.tabs-style-circlefill nav {
 +
padding: 0px 50px;
 +
/*border: 1px solid #C5C5C5;*/
 +
/*border: 1px solid red;
 +
border-style: solid none;*/
 +
-webkit-box-shadow: 0px -5px 5px -5px rgba(255,0,0,1), 0px 5px 5px -5px rgba(255,0,0,1);
 +
-moz-box-shadow: 0px -5px 5px -5px rgba(255,0,0,1), 0px 5px 5px -5px rgba(255,0,0,1);
 +
box-shadow: 0px -5px 5px -5px rgba(255,0,0,1), 0px 5px 5px -5px rgba(255,0,0,1);
 +
}
 +
 
 +
.tabs-style-circlefill nav ul li {
 +
overflow: hidden;
 +
margin: 0px;
 +
/*border-right: 1px solid lightgray;*/
 +
}
 +
 
 +
.tabs-style-circlefill nav li a {
 +
/*padding: 1.5em 0;*/
 +
color: black;
 +
font-size: 1em;
 +
}
 +
 
 +
.tabs-style-circlefill nav li:first-child {
 +
border-left: none;
 +
}
 +
 
 +
.tabs-style-circlefill nav li:last-child {
 +
border: none;
 +
}
 +
 
 +
.tabs-style-circlefill nav li::before {
 +
position: absolute;
 +
top: 50%;
 +
left: 50%;
 +
margin: 0;
 +
width: 0;
 +
height: 0;
 +
/*border: 10px solid black;*/
 +
border-radius: 50%;
 +
background: red;
 +
content: '';
 +
-webkit-transition: all 0.3s;
 +
transition: all 0.3s;
 +
}
 +
 
 +
.tabs-style-circlefill nav li.tab-current::before {
 +
margin: -100px 0 0 -100px;
 +
height: 200px;
 +
width: 200px;
 +
}
 +
 
 +
.tabs-style-circlefill nav a {
 +
-webkit-transition: color 0.3s;
 +
transition: color 0.3s;
 +
}
 +
 
 +
.tabs-style-circlefill nav a span {
 +
/*display: none;*/
 +
}
 +
 
 +
.tabs-style-circlefill nav li.tab-current a {
 +
color: #fff;
 +
}
 +
 
 +
.tabs-style-circlefill .icon::before {
 +
display: block;
 +
margin: 0;
 +
pointer-events: none;
 +
}
 +
 
 +
.tabs-style-circlefill .content-wrap {
 +
/*border-top: 1px solid #2CC185;*/
 +
overflow: hidden;
 +
}
 +
 
 +
/* Content */
 +
.content-wrap {
 +
position: relative;
 +
}
 +
 
 +
.content-wrap section {
 +
display: none;
 +
margin: 0 auto;
 +
padding: 50px;
 +
text-align: center;
 +
}
 +
 
 +
.content-wrap section.content-current {
 +
display: block;
 +
}
 +
 
 +
.content-wrap section table.picturetable {
 +
text-align: center;
 +
margin: 0 auto;
 +
}
 +
 
 +
.content-wrap section table.picturetable td {
 +
padding: 0 5px 20px 5px;
 +
}
 +
 
 +
.content-wrap section table.picturetable img {
 +
height: 130px;
 +
}
 +
 
 +
.content-wrap section p {
 +
margin: 0;
 +
font-size: 12px;
 +
text-align: center;
 +
}
 +
 
 +
.content-wrap section .thumbnail_container {
 +
margin: 30px auto;
 +
margin-top: 0;
 +
display: inline-block;
 +
}
 +
 
 +
.content-wrap section .thumbnail_container img {
 +
margin: 5px;
 +
height: 130px;
 +
}
 +
 
 +
.content-wrap section .acknowledgements {
 +
text-align: left;
 +
font-size: 12px;
 +
width: 500px;
 +
margin: 0 auto;
 +
}
 +
 
 +
 
 +
/* Fallback */
 +
.no-js .content-wrap section {
 +
display: block;
 +
padding-bottom: 2em;
 +
border-bottom: 1px solid rgba(255,255,255,0.6);
 +
}
 +
 
 +
.no-flexbox nav ul {
 +
display: block;
 +
}
 +
 
 +
.no-flexbox nav ul li {
 +
min-width: 15%;
 +
display: inline-block;
 +
}
 +
</style>
 +
 
 +
 
 +
 
 +
</p><p>
 +
 
 +
<style>
 +
/* Thanks, Northwestern - https://2014.igem.org/Team:Northwestern/Templates/Reset */
 +
 
 +
/* Redesigning the topmenu */
 +
/***************************/
 +
 
 +
#footer-box, #siteSub, #search-controls, .firstHeading, #contentSub, #catlinks,  #p-logo {
 +
  display:none;
 +
}
 +
#bodyContent a[href ^="mailto:"],
 +
.link-mailto {
 +
    background: none;
 +
    padding-right: 0;
 +
}
 +
#bodyContent a[href ^="https://"], .link-https {
 +
    background: none;
 +
    padding-right: 0;
 +
}
 +
#menubar {
 +
margin-left: 0px;
 +
background: none;
 +
}
 +
 
 +
.right-menu li a, .left-menu li a {
 +
color: white !important;
 +
background-color: transparent;
 +
font-size: 10px;
 +
font-weight: bold;
 +
}
 +
.right-menu li a {
 +
color: black !important;
 +
}
 +
 
 +
.rright-menu li a:hover, .left-menu:hover a {
 +
color: black !important;
 +
}
 +
 
 +
#top-section {
 +
height: 15px;
 +
background: white;
 +
}
 +
 
 +
#content {
 +
width: 100%;
 +
}
 +
h1, h2, h3, h4, h5, h6 {
 +
text-decoration: none;
 +
border: none;
 +
}
 +
 
 +
</style>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<style>
 +
 
 +
*, *:after, *:before { -webkit-box-sizing: border-box; box-sizing: border-box; }
 +
.clearfix:before, .clearfix:after { content: ''; display: table; }
 +
.clearfix:after { clear: both; }
 +
 
 +
.container {
 +
background: white;
 +
width: 974px;
 +
margin: 0 auto;
 +
margin-bottom: 100px;
 +
}
 +
 
 +
body {
 +
min-width: 960px;
 +
/*background-image: url("https://static.igem.org/mediawiki/2014/7/74/Rutgers_stripes.png");*/
 +
/*background-image: url("https://static.igem.org/mediawiki/2014/6/69/Rutgers_bg_hoffman.png");*/
 +
background-image: url("https://static.igem.org/mediawiki/2014/5/5d/Rutgers_bg_hoffman2.png");
 +
font-weight: 400 !important;
 +
font-family: Arial, Helvetica, sans-serif;
 +
}
 +
 
 +
a {
 +
color: black;
 +
text-decoration: none !important;
 +
outline: none;
 +
}
 +
 
 +
a:hover, a:focus {
 +
color: #74777b;
 +
}
 +
 
 +
/*section {
 +
font-family: Arial, Helvetica, sans-serif;
 +
}*/
 +
 
 +
h2 {
 +
display:inline-block;
 +
padding-left: 20px;
 +
padding-right: 20px;
 +
border-bottom: 1px dotted black;
 +
font-family: Arial, Helvetica, sans-serif !important;
 +
}
 +
 
 +
iframe {
 +
border: 1px dotted black;
 +
margin: 0px;
 +
}
 +
 
 +
section ul.reglist {
 +
list-style-type: circle !important;
 +
margin-left: 25px;
 +
}
 +
 
 +
section ul.reglist li {
 +
list-style-type: circle !important;
 +
text-align: justify !important;
 +
text-justify: inter-word !important;
 +
font-size: 16px !important;
 +
 
 +
padding-right: 25px;
 +
line-height: 110%;
 +
}
 +
 
 +
table.dotted p {
 +
text-align: justify !important;
 +
text-justify: inter-word !important;
 +
font-size: 16px !important;
 +
line-height: 110%;
 +
}
 +
 
 +
section .fp {
 +
width: 750px;
 +
margin: 0 auto;
 +
}
 +
section .fp p {
 +
text-align: justify !important;
 +
text-justify: inter-word !important;
 +
font-size: 16px !important;
 +
line-height: 110%;
 +
padding-bottom: 10px;
 +
}
 +
 
 +
table {
 +
margin: 0 auto !important;
 +
border-collapse: collapse;
 +
}
 +
 
 +
table.dotted img {
 +
display: block;
 +
margin: 0 auto; /* !important;*/
 +
}
 +
 
 +
table.dotted th {
 +
background: gray;
 +
border: 1px solid black !important;
 +
}
 +
 
 +
table.dotted td {
 +
border: 1px dotted black !important;
 +
vertical-align: top;
 +
}
 +
 
 +
table.abstract td {
 +
width: 700px;
 +
padding: 0 0px 25px 0px !important;
 +
}
 +
 
 +
table.parts td {
 +
vertical-align: middle;
 +
}
 +
 
 +
table.accomp td {
 +
width: 430px;
 +
padding: 0 0px 25px 0px !important;
 +
}
 +
table.accomp td img {
 +
width: 250px;
 +
}
 +
 
 +
table.attrib td {
 +
width: 700px;
 +
padding: 10px !important;
 +
text-align: left;
 +
}
 +
table.attrib td img {
 +
float: left;
 +
margin: 10px;
 +
}
 +
table.attrib td p {
 +
padding: 10px;
 +
}
 +
 
 +
/*.h2wide h2 {
 +
display: inline-block;
 +
font-family: Arial, Helvetica, sans-serif;
 +
height: 34px;
 +
padding: 0;
 +
margin: 25px auto;
 +
}
 +
.h2wide {
 +
}
 +
.h2left {
 +
display: inline-block;
 +
width: 0;
 +
height: 0;
 +
border-top: 3px solid transparent;
 +
border-right: 150px solid red;
 +
-webkit-filter: drop-shadow(0 0 1px red);
 +
filter: drop-shadow(0 0 1px red);
 +
}
 +
.h2right {
 +
display: inline-block;
 +
width: 0;
 +
height: 0;
 +
border-top: 3px solid transparent;
 +
border-left: 150px solid red;
 +
-webkit-filter: drop-shadow(0 0 1px red);
 +
filter: drop-shadow(0 0 1px red);
 +
}*/
 +
 
 +
.no-flexbox .support {
 +
display: block;
 +
}
 +
 
 +
.container > section {
 +
/*padding: 5em 0;*/
 +
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<h2>The Next Step in DNA Synthesis</h2>
 
<h2>The Next Step in DNA Synthesis</h2>
  
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<div class="fp"><p>We can only synthesize about 150 bases at a time, so new genes have to be stitched together from smaller strands, which adds a lot to the <a href="http://blog.ginkgobioworks.com/2012/01/14/commercial-gene-synthesis/">time</a> and <a href="http://synbiobeta.com/time-new-dna-synthesis-sequencing-cost-curves-rob-carlson/">cost</a> required. This needs to change.</p></div>
 
<div class="fp"><p>We can only synthesize about 150 bases at a time, so new genes have to be stitched together from smaller strands, which adds a lot to the <a href="http://blog.ginkgobioworks.com/2012/01/14/commercial-gene-synthesis/">time</a> and <a href="http://synbiobeta.com/time-new-dna-synthesis-sequencing-cost-curves-rob-carlson/">cost</a> required. This needs to change.</p></div>
  
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<h2>A Primer on DNA Synthesis</h2>
 
<h2>A Primer on DNA Synthesis</h2>
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<div class="fp"><p>Currently we use organic (anhydrous) solvents and reactive amidites to synthesize all custom DNA sequences, whether for antibody/enzyme engineering, multi-gene pathway building, or even <a href="http://syntheticyeast.org/sc2-0/introduction/">genome construction</a>. The process looks something like this:</p></div>
 
<div class="fp"><p>Currently we use organic (anhydrous) solvents and reactive amidites to synthesize all custom DNA sequences, whether for antibody/enzyme engineering, multi-gene pathway building, or even <a href="http://syntheticyeast.org/sc2-0/introduction/">genome construction</a>. The process looks something like this:</p></div>
  
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<div class="fp"><p>This is one cycle in the process of DNA Synthesis. A solution of blocked nucleotides (whichever A, C, G, or T comes next in the sequence) is added to a group of immobilized DNA strands, and a single nucleotide is incorporated at the end of each strand. The trouble with this highly unnatural method is that <a href="http://en.wikipedia.org/wiki/Oligonucleotide_synthesis#Synthetic_cycle">each of the chemicals depicted</a> <small>(trichloroacetic acid, 2-benzylthiotetrazole, acetonitrile)</small> causes some <a href="http://www.glenresearch.com/GlenReports/GR21-211.html">harm</a> to the chemical structure of DNA, and this will limit the yield in a way that gets exponentially worse when building longer and longer pieces of DNA.</p></div>
 
<div class="fp"><p>This is one cycle in the process of DNA Synthesis. A solution of blocked nucleotides (whichever A, C, G, or T comes next in the sequence) is added to a group of immobilized DNA strands, and a single nucleotide is incorporated at the end of each strand. The trouble with this highly unnatural method is that <a href="http://en.wikipedia.org/wiki/Oligonucleotide_synthesis#Synthetic_cycle">each of the chemicals depicted</a> <small>(trichloroacetic acid, 2-benzylthiotetrazole, acetonitrile)</small> causes some <a href="http://www.glenresearch.com/GlenReports/GR21-211.html">harm</a> to the chemical structure of DNA, and this will limit the yield in a way that gets exponentially worse when building longer and longer pieces of DNA.</p></div>
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<div class="fp"><p>The process pictured above typically causes enough side-reactions to chemically ruin 1.5% of the existing DNA strands in each and every cycle (leaving 98.5% of them intact and with the correct sequence). This concept is called <a href="http://www.lifetechnologies.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/technical-resources-for-oligonucleotides/dna-oligo-faq.html#7">coupling efficiency</a>, and it's the reason we can only synthesize up to <a href="http://www.sigmaaldrich.com/life-science/custom-oligos/custom-dna/learning-center/faqs.html#11">130</a>-150 bases at a time (or even <a href="http://www.idtdna.com/pages/products/dna-rna/ultramer-oligos">200</a> bases if you're willing to pay a whole lot extra).</p></div>
 
<div class="fp"><p>The process pictured above typically causes enough side-reactions to chemically ruin 1.5% of the existing DNA strands in each and every cycle (leaving 98.5% of them intact and with the correct sequence). This concept is called <a href="http://www.lifetechnologies.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/technical-resources-for-oligonucleotides/dna-oligo-faq.html#7">coupling efficiency</a>, and it's the reason we can only synthesize up to <a href="http://www.sigmaaldrich.com/life-science/custom-oligos/custom-dna/learning-center/faqs.html#11">130</a>-150 bases at a time (or even <a href="http://www.idtdna.com/pages/products/dna-rna/ultramer-oligos">200</a> bases if you're willing to pay a whole lot extra).</p></div>
  
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<h2>Our Idea: Use Enzymes</h2>
 
<h2>Our Idea: Use Enzymes</h2>
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<div class="fp"><p>This process simplification could be beneficial on two counts: less complexity in required machinery would reduce capital costs, and less material requirements would reduce operational costs.</p></div>
 
<div class="fp"><p>This process simplification could be beneficial on two counts: less complexity in required machinery would reduce capital costs, and less material requirements would reduce operational costs.</p></div>
  
−
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<div class="fp"><p>This is the enzymatic process that our team has envisioned. The enzyme is a template-independent polymerase called <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2846215/">Terminal deoxynucleotidyl Transferase</a> (TdT). It favors single-stranded 3' ends when it adds whatever dNTP's are available. Using the same principles as today's synthesis strategies, it could serve to catalyze the coupling step in each cycle. Upon inspection of this polymerase's binding pocket, it is apparent that even large blocking groups could fit snuggly without hindering the catalytic machinery, but we decided to test a small, simple blocking group for this project to begin with: the acetyl group.</p></div>
 
<div class="fp"><p>This is the enzymatic process that our team has envisioned. The enzyme is a template-independent polymerase called <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2846215/">Terminal deoxynucleotidyl Transferase</a> (TdT). It favors single-stranded 3' ends when it adds whatever dNTP's are available. Using the same principles as today's synthesis strategies, it could serve to catalyze the coupling step in each cycle. Upon inspection of this polymerase's binding pocket, it is apparent that even large blocking groups could fit snuggly without hindering the catalytic machinery, but we decided to test a small, simple blocking group for this project to begin with: the acetyl group.</p></div>
 
<div class="fp"><p>We found that the acetyl group undergoes a bit of background hydrolysis, especially at high pH, so it wouldn't be the ideal blocking group. It does, however, perform well enough to make benchmarking possible so that we can establish a proof of principle for this strategy. We ran many assays, outlined below.</p></div>
 
<div class="fp"><p>We found that the acetyl group undergoes a bit of background hydrolysis, especially at high pH, so it wouldn't be the ideal blocking group. It does, however, perform well enough to make benchmarking possible so that we can establish a proof of principle for this strategy. We ran many assays, outlined below.</p></div>
  
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<h2>Accomplishments</h2>
 
<h2>Accomplishments</h2>
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<li>The lanes are labeled with how many minutes the primers were incubated with TdT and dTTP.</li>
 
<li>The lanes are labeled with how many minutes the primers were incubated with TdT and dTTP.</li>
 
<li>This assay established that TdT will add ordinary (not blocked) thymidine triphosphate to the ends of the single-stranded primers. The 30-minute result has the longest oligos (having traveled the least distance), but it appears more condensed because it had less distance to effectively resolve the differing oligo lengths. In later assays we let the gels run as far as possible to avoid the bunching-up of long oligos.</li>
 
<li>This assay established that TdT will add ordinary (not blocked) thymidine triphosphate to the ends of the single-stranded primers. The 30-minute result has the longest oligos (having traveled the least distance), but it appears more condensed because it had less distance to effectively resolve the differing oligo lengths. In later assays we let the gels run as far as possible to avoid the bunching-up of long oligos.</li>
−
<li>Based on data in the literature, we estimate that a few hundred bases were added to each oligo (on average) under these reaction conditions (detailed in the Notebook->Protocols section). A DNA ladder would unfortunately be pretty expensive to create for these short, single-stranded, fluorescent primers, so we only interpret the relative activity of the enzyme between different lanes. This assay method doesn't allow us to quantify activity.</li>
+
<li>Based on data in the literature, we estimate that a few hundred bases were added to each oligo (on average) under these reaction conditions (detailed in the Notebook-&gt;Protocols section). A DNA ladder would unfortunately be pretty expensive to create for these short, single-stranded, fluorescent primers, so we only interpret the relative activity of the enzyme between different lanes. This assay method doesn't allow us to quantify activity.</li>
 
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<ul class="reglist">
 
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<li><strong>Tested the effect of pH on acetylated thymidine incorporation</strong></li>
 
<li><strong>Tested the effect of pH on acetylated thymidine incorporation</strong></li>
−
<li>The lanes are labeled with a number representing the pH that the reaction was run in. Different MOPS buffers were used for the different pH's, except the last lane, which used NEB's Tris buffer (pH 7.9). Each reaction was carried out for 10 minutes with primers, TdT, and acetylated thymidine monomers (specific conditions can be found in the Notebook->Protocols section). The last lane used unmodified thymidine triphosphate, as a reference. </li>
+
<li>The lanes are labeled with a number representing the pH that the reaction was run in. Different MOPS buffers were used for the different pH's, except the last lane, which used NEB's Tris buffer (pH 7.9). Each reaction was carried out for 10 minutes with primers, TdT, and acetylated thymidine monomers (specific conditions can be found in the Notebook-&gt;Protocols section). The last lane used unmodified thymidine triphosphate, as a reference. </li>
 
<li>This assay demonstrates that TdT adds less 3'-acetylated ("blocked") nucleotides at a lower pH. Aside from lane "7.0" (the anomaly), there is a clean step-ladder effect where longer oligos are produced at higher pH. There is still a significant spread of differing oligo lengths in the pH 6.5 lane, which indicates that enough hydrolysis occurs (of the acetyl blocking group) to allow for multiple additions. This conflicts with the findings of the later assays (below) that show no such spread of oligo lengths after reaction at pH 6.5.</li>
 
<li>This assay demonstrates that TdT adds less 3'-acetylated ("blocked") nucleotides at a lower pH. Aside from lane "7.0" (the anomaly), there is a clean step-ladder effect where longer oligos are produced at higher pH. There is still a significant spread of differing oligo lengths in the pH 6.5 lane, which indicates that enough hydrolysis occurs (of the acetyl blocking group) to allow for multiple additions. This conflicts with the findings of the later assays (below) that show no such spread of oligo lengths after reaction at pH 6.5.</li>
 
<li>In other (unlisted) assays, we established that TdT gets progressively less active at pH's lower than 7.9. The "dTTP+NEB Buffer" lane shows the activity of TdT with unmodified nucleotides at pH 7.9. Comparing the activity in this lane with the others shows that acetylated nucleotides cause some truncation throughout the distribution of oligo lengths. Another (unlisted) assay shows that the difference in TdT's activity between NEB's Tris buffer and a homemade MOPS buffer (both at pH 7.9) was indiscernible.</li>
 
<li>In other (unlisted) assays, we established that TdT gets progressively less active at pH's lower than 7.9. The "dTTP+NEB Buffer" lane shows the activity of TdT with unmodified nucleotides at pH 7.9. Comparing the activity in this lane with the others shows that acetylated nucleotides cause some truncation throughout the distribution of oligo lengths. Another (unlisted) assay shows that the difference in TdT's activity between NEB's Tris buffer and a homemade MOPS buffer (both at pH 7.9) was indiscernible.</li>
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<h2>Parts</h2>
 
<h2>Parts</h2>
 
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<small style="font-size: 8px">This part was submitted late, and is not eligible for medals and awards.</small>
 
<small style="font-size: 8px">This part was submitted late, and is not eligible for medals and awards.</small>
 
 
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<h2>Students</h2>
 
<h2>Students</h2>
 
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<p>Kenny Kostenbader</p>
 
<p>Kenny Kostenbader</p>
 
<p>Chem Eng</p>
 
<p>Chem Eng</p>
 
</td>
 
</td>
 
<td>
 
<td>
−
<img src="https://static.igem.org/mediawiki/2014/8/83/Rutgers_SL.png" />
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<p>Scott Lazaro</p>
 
<p>Scott Lazaro</p>
 
<p>Cell Bio &amp; Neurosci</p>
 
<p>Cell Bio &amp; Neurosci</p>
 
</td>
 
</td>
 
<td>
 
<td>
−
<img src="https://static.igem.org/mediawiki/2014/4/42/Rutgers_WW.png" />
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<img src="./Team_Rutgers - 2014.igem.org_files/Rutgers_WW.png">
 
<p>Wilson Wong</p>
 
<p>Wilson Wong</p>
 
<p>Mol Bio</p>
 
<p>Mol Bio</p>
 
</td>
 
</td>
 
<td>
 
<td>
−
<img src="https://static.igem.org/mediawiki/2014/1/13/Rutgers_JP.png" />
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<img src="./Team_Rutgers - 2014.igem.org_files/Rutgers_JP.png">
 
<p>Jay Patel</p>
 
<p>Jay Patel</p>
 
<p>Chem Eng</p>
 
<p>Chem Eng</p>
 
</td>
 
</td>
 
</tr>
 
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<h2>Faculty</h2>
 
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<p>Sagar Khare</p>
 
<p>Sagar Khare</p>
 
<p>P.I.</p>
 
<p>P.I.</p>
 
</td>
 
</td>
 
<td>
 
<td>
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<p>Andrew Laudisi</p>
 
<p>Andrew Laudisi</p>
 
<p>Lab Manager</p>
 
<p>Lab Manager</p>
 
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<h2>Attributions</h2>
 
<h2>Attributions</h2>
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The description of each project must clearly attribute work done by the Students and distinguish it from work done by others, including host labs, Advisors, Instructors, technicians, sponsors, professional website designers, artists, and commercial services.-->
 
The description of each project must clearly attribute work done by the Students and distinguish it from work done by others, including host labs, Advisors, Instructors, technicians, sponsors, professional website designers, artists, and commercial services.-->
 
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<img src="https://static.igem.org/mediawiki/2014/5/5f/Rutgers_RJ.png" width="130" /> <p><strong>Dr Jones</strong> is a professor in the Rutgers Chemistry Department who researches modified nucleotides. He gave valuable feedback on our initial project ideas, and suggested a way to create (and characterize) 3'-acetylated thymidine triphosphate in our lab. We attempted this synthesis (involving pyridine and acetic anhydride) and got promising results (via LC/MS), but then we found out that TriLink (below) could simply synthesize and purify it for us for free.</p>
+
<img src="./Team_Rutgers - 2014.igem.org_files/Rutgers_RJ.png" width="130"> <p><strong>Dr Jones</strong> is a professor in the Rutgers Chemistry Department who researches modified nucleotides. He gave valuable feedback on our initial project ideas, and suggested a way to create (and characterize) 3'-acetylated thymidine triphosphate in our lab. We attempted this synthesis (involving pyridine and acetic anhydride) and got promising results (via LC/MS), but then we found out that TriLink (below) could simply synthesize and purify it for us for free.</p>
 
</td>
 
</td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td>
 
<td>
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<img src="https://static.igem.org/mediawiki/2014/f/fc/Rutgers_AN.png" width="130" /> <p><strong>Arun Nayar</strong> was on last year's Rutgers iGEM team, so he helped train us with various lab protocols.</p>
+
<img src="./Team_Rutgers - 2014.igem.org_files/Rutgers_AN.png" width="130"> <p><strong>Arun Nayar</strong> was on last year's Rutgers iGEM team, so he helped train us with various lab protocols.</p>
 
</td>
 
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</tr>
 
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<img src="https://static.igem.org/mediawiki/2014/4/4b/Rutgers_Trilink.jpg" width="200" /> <p><strong>Trilink Biotech</strong> supported the project by custom-synthesizing acetylated thymidine triphosphate, and supplying it free of charge. Thanks TiLink!</p>
+
<img src="./Team_Rutgers - 2014.igem.org_files/Rutgers_Trilink.jpg" width="200"> <p><strong>Trilink Biotech</strong> supported the project by custom-synthesizing acetylated thymidine triphosphate, and supplying it free of charge. Thanks TiLink!</p>
 
</td>
 
</td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td>
 
<td>
−
<img src="https://static.igem.org/mediawiki/2014/e/e9/Rutgers_NEB.jpeg" width="200" /> <p><strong>NEB Inc</strong> supported the project by supplying a Terminal Transferase enzyme kit and a dNTP set, both free of charge. Thanks NEB!</p>
+
<img src="./Team_Rutgers - 2014.igem.org_files/Rutgers_NEB.jpeg" width="200"> <p><strong>NEB Inc</strong> supported the project by supplying a Terminal Transferase enzyme kit and a dNTP set, both free of charge. Thanks NEB!</p>
 
</td>
 
</td>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 
<td>
 
<td>
−
<img src="https://static.igem.org/mediawiki/2014/f/f7/Rutgers_Gen9.png" width="200" /> <p><strong>Gen9</strong> supported the project with a monetary donation. Thanks Gen9!</p>
+
<img src="./Team_Rutgers - 2014.igem.org_files/Rutgers_Gen9.png" width="200"> <p><strong>Gen9</strong> supported the project with a monetary donation. Thanks Gen9!</p>
 
</td>
 
</td>
 
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<h2>Protocols</h2>
 
<h2>Protocols</h2>
 
<p>Click to download a copy of each protocol that we followed and/or wrote this summer:</p>
 
<p>Click to download a copy of each protocol that we followed and/or wrote this summer:</p>
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<ul class="reglist">
 
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<li><a href="https://static.igem.org/mediawiki/2014/2/23/Rutgers_a2.pdf">Testing the extent of hydrolysis of incorporated acetylated thymidines</a></li>
 
<li><a href="https://static.igem.org/mediawiki/2014/2/23/Rutgers_a2.pdf">Testing the extent of hydrolysis of incorporated acetylated thymidines</a></li>
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<li><a href="https://static.igem.org/mediawiki/2014/f/fe/Rutgers_NEB_gibson.pdf">NEB's Gibson Assembly protocol</a></li>
 
<li><a href="https://static.igem.org/mediawiki/2014/f/fe/Rutgers_NEB_gibson.pdf">NEB's Gibson Assembly protocol</a></li>
 
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<br><br>
 
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<h2>Project phases</h2>
 
<h2>Project phases</h2>
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<ul class="reglist">
 
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<li>In <strong>May</strong> and <strong>June</strong> we brainstormed and investigated ways to create acetylated thymidine in lab. After much much research into the matter, we decided it would require far to many resources to synthesize acetylated nucleoside monomers (without phosphates present) followed by difficult and expensive triphosphorylation (the stepwise installation of the triphosphate moiety). It would be far too expensive. Instead, we tried Dr Jones' suggestion of mixing acetic anhydride with thymidine triphosphate in pyridine, and this seemed to work (there was a peak at the right molecular weight on our LC/MS run :). This phase of the project ended when we discovered that TriLink could custom-synthesize acetylated thymidine triphosphate, and they would do it for free :)</li>
 
<li>In <strong>May</strong> and <strong>June</strong> we brainstormed and investigated ways to create acetylated thymidine in lab. After much much research into the matter, we decided it would require far to many resources to synthesize acetylated nucleoside monomers (without phosphates present) followed by difficult and expensive triphosphorylation (the stepwise installation of the triphosphate moiety). It would be far too expensive. Instead, we tried Dr Jones' suggestion of mixing acetic anhydride with thymidine triphosphate in pyridine, and this seemed to work (there was a peak at the right molecular weight on our LC/MS run :). This phase of the project ended when we discovered that TriLink could custom-synthesize acetylated thymidine triphosphate, and they would do it for free :)</li>
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<li><strong>September</strong> and <strong>October</strong> saw the bulk of our assaying. We had, however, neglected to move the mouse TdT gene from our pET-29b+ plasmid over to the standard iGEM pSB1C3 plasmid until early October. Our first Gibson Assembly did not work well (transformation yielded no colonies), so we ended up submitting our part late. That was a bummer.</li>
 
<li><strong>September</strong> and <strong>October</strong> saw the bulk of our assaying. We had, however, neglected to move the mouse TdT gene from our pET-29b+ plasmid over to the standard iGEM pSB1C3 plasmid until early October. Our first Gibson Assembly did not work well (transformation yielded no colonies), so we ended up submitting our part late. That was a bummer.</li>
 
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<li>Appropriate precautions were taken when casting and using the Polyacrylamide Electrophoresis gels for our various assays. Used gels were disposed of properly in acrylamide waste bins.</li>
 
<li>Appropriate precautions were taken when casting and using the Polyacrylamide Electrophoresis gels for our various assays. Used gels were disposed of properly in acrylamide waste bins.</li>
 
<li>Our team's safety form can be found <a href="https://igem.org/Safety/Safety_Form?team_id=1556">here</a>.</li>
 
<li>Our team's safety form can be found <a href="https://igem.org/Safety/Safety_Form?team_id=1556">here</a>.</li>
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Revision as of 19:56, 21 August 2015

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"> Team:Rutgers - 2014.igem.org

 

Team:Rutgers

From 2014.igem.org

The Next Step in DNA Synthesis

De novo DNA Synthesis is extremely important to the world of Synthetic Biology, but the low efficiency of today's DNA Synthesis technology limits us.

We can only synthesize about 150 bases at a time, so new genes have to be stitched together from smaller strands, which adds a lot to the time and cost required. This needs to change.

A Primer on DNA Synthesis

Currently we use organic (anhydrous) solvents and reactive amidites to synthesize all custom DNA sequences, whether for antibody/enzyme engineering, multi-gene pathway building, or even genome construction. The process looks something like this:

This is one cycle in the process of DNA Synthesis. A solution of blocked nucleotides (whichever A, C, G, or T comes next in the sequence) is added to a group of immobilized DNA strands, and a single nucleotide is incorporated at the end of each strand. The trouble with this highly unnatural method is that each of the chemicals depicted (trichloroacetic acid, 2-benzylthiotetrazole, acetonitrile) causes some harm to the chemical structure of DNA, and this will limit the yield in a way that gets exponentially worse when building longer and longer pieces of DNA.

The process pictured above typically causes enough side-reactions to chemically ruin 1.5% of the existing DNA strands in each and every cycle (leaving 98.5% of them intact and with the correct sequence). This concept is called coupling efficiency, and it's the reason we can only synthesize up to 130-150 bases at a time (or even 200 bases if you're willing to pay a whole lot extra).

Our Idea: Use Enzymes

We believe that both cost and efficiency could be vastly improved by using enzymes for de novo DNA Synthesis. Polymerases have evolved alongside nucleic acids for billions of years and developed beautiful two-metal-ion machinery that makes phosphate diester formation a piece of cake. In theory, this machinery could be applied to de novo DNA Synthesis to eliminate side-reactions and significantly simplify the process overall. A more natural, aqueous synthesis environment would lend itself to higher efficiency, too.

This process simplification could be beneficial on two counts: less complexity in required machinery would reduce capital costs, and less material requirements would reduce operational costs.

This is the enzymatic process that our team has envisioned. The enzyme is a template-independent polymerase called Terminal deoxynucleotidyl Transferase (TdT). It favors single-stranded 3' ends when it adds whatever dNTP's are available. Using the same principles as today's synthesis strategies, it could serve to catalyze the coupling step in each cycle. Upon inspection of this polymerase's binding pocket, it is apparent that even large blocking groups could fit snuggly without hindering the catalytic machinery, but we decided to test a small, simple blocking group for this project to begin with: the acetyl group.

We found that the acetyl group undergoes a bit of background hydrolysis, especially at high pH, so it wouldn't be the ideal blocking group. It does, however, perform well enough to make benchmarking possible so that we can establish a proof of principle for this strategy. We ran many assays, outlined below.

Accomplishments

These assays serve to elucidate the functionality of TdT in the conditions of our envisioned DNA Synthesis strategy. We used short, single-stranded primers (each composed of 15 thymidine residues) with 5' fluorescent tags (5'-FAM), and visualized the results on 22% polyacrylamide nondenaturing gels. The images are oriented with the loading wells above, so that the shortest oligos (which travel the fastest) end up furthest down in each lane. Each assay has a Control lane containing only the 15-length primer for reference. All lanes are labeled with neon-green text superimposed over the strange gray blotches of loading dye.


  • Tested the extension of single-stranded primers with TdT
  • The lanes are labeled with how many minutes the primers were incubated with TdT and dTTP.
  • This assay established that TdT will add ordinary (not blocked) thymidine triphosphate to the ends of the single-stranded primers. The 30-minute result has the longest oligos (having traveled the least distance), but it appears more condensed because it had less distance to effectively resolve the differing oligo lengths. In later assays we let the gels run as far as possible to avoid the bunching-up of long oligos.
  • Based on data in the literature, we estimate that a few hundred bases were added to each oligo (on average) under these reaction conditions (detailed in the Notebook->Protocols section). A DNA ladder would unfortunately be pretty expensive to create for these short, single-stranded, fluorescent primers, so we only interpret the relative activity of the enzyme between different lanes. This assay method doesn't allow us to quantify activity.
  • Tested the effect of pH on acetylated thymidine incorporation
  • The lanes are labeled with a number representing the pH that the reaction was run in. Different MOPS buffers were used for the different pH's, except the last lane, which used NEB's Tris buffer (pH 7.9). Each reaction was carried out for 10 minutes with primers, TdT, and acetylated thymidine monomers (specific conditions can be found in the Notebook->Protocols section). The last lane used unmodified thymidine triphosphate, as a reference.
  • This assay demonstrates that TdT adds less 3'-acetylated ("blocked") nucleotides at a lower pH. Aside from lane "7.0" (the anomaly), there is a clean step-ladder effect where longer oligos are produced at higher pH. There is still a significant spread of differing oligo lengths in the pH 6.5 lane, which indicates that enough hydrolysis occurs (of the acetyl blocking group) to allow for multiple additions. This conflicts with the findings of the later assays (below) that show no such spread of oligo lengths after reaction at pH 6.5.
  • In other (unlisted) assays, we established that TdT gets progressively less active at pH's lower than 7.9. The "dTTP+NEB Buffer" lane shows the activity of TdT with unmodified nucleotides at pH 7.9. Comparing the activity in this lane with the others shows that acetylated nucleotides cause some truncation throughout the distribution of oligo lengths. Another (unlisted) assay shows that the difference in TdT's activity between NEB's Tris buffer and a homemade MOPS buffer (both at pH 7.9) was indiscernible.
  • Tested the extent of hydrolysis of incorporated acetylated thymidines
  • The lanes are labeled according to their respective pH of reaction. Labels ending in "a" were carried out with acetylated thymidine for 10 minutes, before splitting the reaction volume in half and halting the reaction in the first half. The second half continues to react, with more unmodified thymidine added to the mixture. The two lanes labeled "ccc" were similar reactions, except that they had unmodified thymidine in the first reaction ("a"). These "ccc" reactions were carried out at a pH of 6.5 to show how active the enzyme is at that pH (with unmodified nucleotides).
  • This assay attempted to show that 3'-blocked nucleotides can effectively halt multiple additions, because of the terminal acetyl group. There is clearly a large amount of blocking-group-hydrolysis in the pH 7.9 reactions, leading to multiple additions when the extra unmodified nucleotide was added. The hydrolysis is limited enough at a pH of 6.5 that no significant amount of multiple additions occurs, even after adding extra unmodified nucleotide. These results are promising.
  • The second "7.9a" lane in the bottom image is mislabeled, it should read "7.9b".

Parts

Name

Type

Decription

Length
  • BBa_K1556000

coding

Mouse TdT

1590
This part was submitted late, and is not eligible for medals and awards.

Students

Kenny Kostenbader

Chem Eng

Scott Lazaro

Cell Bio & Neurosci

Wilson Wong

Mol Bio

Jay Patel

Chem Eng

Faculty

Sagar Khare

P.I.

Andrew Laudisi

Lab Manager

Attributions

Dr Jones is a professor in the Rutgers Chemistry Department who researches modified nucleotides. He gave valuable feedback on our initial project ideas, and suggested a way to create (and characterize) 3'-acetylated thymidine triphosphate in our lab. We attempted this synthesis (involving pyridine and acetic anhydride) and got promising results (via LC/MS), but then we found out that TriLink (below) could simply synthesize and purify it for us for free.

Arun Nayar was on last year's Rutgers iGEM team, so he helped train us with various lab protocols.

All of the pictured results on the Project page represent assays that were designed jointly by the students and Dr Khare, and carried out completely by the undergraduate team members in the Khare lab. Additional help was provided by other Rutgers students as well, namely: Diego Barreto, Wesley Okwemba, Neil Patel, Harsh Patel, and Samantha Ashley. The fluorescent gels were imaged using the "BioRad Gel Doc" machine in the Kalodimos lab (next door). Kenny did the web design and coding.

Trilink Biotech supported the project by custom-synthesizing acetylated thymidine triphosphate, and supplying it free of charge. Thanks TiLink!

NEB Inc supported the project by supplying a Terminal Transferase enzyme kit and a dNTP set, both free of charge. Thanks NEB!

Gen9 supported the project with a monetary donation. Thanks Gen9!

Protocols

Click to download a copy of each protocol that we followed and/or wrote this summer:



Project phases

  • In May and June we brainstormed and investigated ways to create acetylated thymidine in lab. After much much research into the matter, we decided it would require far to many resources to synthesize acetylated nucleoside monomers (without phosphates present) followed by difficult and expensive triphosphorylation (the stepwise installation of the triphosphate moiety). It would be far too expensive. Instead, we tried Dr Jones' suggestion of mixing acetic anhydride with thymidine triphosphate in pyridine, and this seemed to work (there was a peak at the right molecular weight on our LC/MS run :). This phase of the project ended when we discovered that TriLink could custom-synthesize acetylated thymidine triphosphate, and they would do it for free :)
  • In July we expressed TdT in E. coli using the trusty pET-29b+ vector. Expression level was low (but still something!), so we were relieved when we found out that NEB would be happy to supply us with free a free TdT enzyme kit.
  • In August we ordered fluorescent primers to begin assaying with. It took the full month to test different methods for every step of the designed assay (especially fluorescence visualization) until we finally settled on the protocol(s) that we've uploaded (above)
  • September and October saw the bulk of our assaying. We had, however, neglected to move the mouse TdT gene from our pET-29b+ plasmid over to the standard iGEM pSB1C3 plasmid until early October. Our first Gibson Assembly did not work well (transformation yielded no colonies), so we ended up submitting our part late. That was a bummer.
  • All lab members underwent Biosafety Level 2 training with the Rutgers Environmental Health & Safety department.
  • We utilized only one organism: Escherichia coli K-12 DH5a
  • Public health and safety would be under little conceivable risk in the event of an accidental release of our biobrick part into the wild (in the form DNA or a transformed colony of cells), because the TdT enzyme expression is very low, and the enzyme is only capable of adding deoxynucleoside triphosphates to the 3' ends of DNA strands. Given that very little TdT would be produced, and that free 3' DNA ends are rare in living systems, we believe that the risk posed by our biobrick part is minimal.
  • Appropriate precautions were taken when casting and using the Polyacrylamide Electrophoresis gels for our various assays. Used gels were disposed of properly in acrylamide waste bins.
  • Our team's safety form can be found here.

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