Difference between revisions of "Team:UMaryland/Notebook"

 
(46 intermediate revisions by 6 users not shown)
Line 1: Line 1:
−
{{UMaryland}}
+
{{Template_All_Teams}}
−
{{Team:UMaryland/UMD15_CSSJS}}
+
<html>
 +
</div></div></div></div></div></div></div></div></div></div>
 +
</html>
 +
{{Team:UMarylandClasses}}
 +
{{Team:UMarylandMenu}}
 
<html>
 
<html>
  
−
<style>  
+
<!--Attention! If you are not part of the website team, you are NOT allowed to touch anything above this line without the express permission of Best Kohai.-->
  
−
body{
+
<style type="text/css">
−
    overflow:hidden;
+
 
 +
h1,h2,h3,h4,p,a {
 +
text-decoration: none;
 
}
 
}
 +
h1 {
 +
font-size: 250%;
 +
}
 +
h2 {
 +
font-size: 200%;
 +
}
 +
h3 {
 +
font-size: 150%;
 +
}
 +
h4 {
 +
font-size: 125%;
 +
}
 +
p {
 +
font-size: 100%;
 +
}
 +
body {
 +
background-color:#ffffff;
  
−
#flipbook{
+
}
−
    width:400px;
+
#sidemenu {
−
    height:300px;
+
position:fixed;
−
    margin-left: 75px;
+
float:left;
 +
width:200px;
 +
top:200px;
 +
z-index:2;
 
}
 
}
  
−
#flipbook .page{
+
#sidemenu,
−
    width:400px;
+
#sidemenu ul,
−
    height:300px;
+
#sidemenu ul li,
−
    background-color:white;
+
#sidemenu ul li a {
−
    line-height:300px;
+
  margin: 0;
−
    font-size:20px;
+
  padding: 0;
−
    text-align:center;
+
  border: 0;
 +
  list-style: none;
 +
  line-height: 1;
 +
  display: block;
 +
  -webkit-box-sizing: border-box;
 +
  -moz-box-sizing: border-box;
 +
  box-sizing: border-box;
 
}
 
}
  
−
#flipbook .page-wrapper{
+
#sidemenu ul ul {
−
    -webkit-perspective:2000px;
+
  display: none;
−
    -moz-perspective:2000px;
+
}
−
    -ms-perspective:2000px;
+
.align-right {
−
    -o-perspective:2000px;
+
  float: right;
−
    perspective:2000px;
+
}
 +
#sidemenu > ul > li > a {
 +
  padding: 15px 20px;
 +
  border: 1px solid black;
 +
  cursor: pointer;
 +
  z-index: 2;
 +
  font-size: 14px;
 +
  font-weight: bold;
 +
  text-decoration: none;
 +
  color: #000000;
 +
background-image: -webkit-linear-gradient(bottom, #ffffff 0%, #2BB1FF 100%);
 +
  box-shadow: inset 0 1px 0 rgba(255, 255, 255, 0.15);
 
}
 
}
  
−
#flipbook .hard{
+
#sidemenu > ul > li > a:hover,
−
    background:#ccc !important;
+
#sidemenu > ul > li.active > a,
−
    color:#333;
+
#sidemenu > ul > li.open > a {
−
    -webkit-box-shadow:inset 0 0 5px #666;
+
  color: #000000;
−
    -moz-box-shadow:inset 0 0 5px #666;
+
background-image: -webkit-linear-gradient(top, #C9C9C9 0%, #FFFFFF 100%);
−
    -o-box-shadow:inset 0 0 5px #666;
+
−
    -ms-box-shadow:inset 0 0 5px #666;
+
−
    box-shadow:inset 0 0 5px #666;
+
−
    font-weight:bold;
+
 
}
 
}
 +
#sidemenu > ul > li.open > a {
 +
  box-shadow: inset 0 1px 0 rgba(255, 255, 255, 0.15), 0 1px 1px rgba(0, 0, 0, 0.15);
 +
  border-bottom: 1px solid #1682ba;
 +
}
 +
 +
 +
#sidemenu > ul > li > a:hover > span::after,
 +
#sidemenu > ul > li.active > a > span::after,
 +
#sidemenu > ul > li.open > a > span::after {
 +
  border-color: #eeeeee;
 +
}
 +
.holder::before {
 +
  top: 18px;
 +
  border-top: 2px solid;
 +
  border-left: 2px solid;
 +
  border-top-color: inherit;
 +
  border-left-color: inherit;
 +
}
 +
#sidemenu ul ul li a {
 +
  cursor: pointer;
 +
  border-bottom: 1px solid #32373e;
 +
  border-left: 1px solid #32373e;
 +
  border-right: 1px solid #32373e;
 +
  padding: 10px 20px;
 +
  z-index: 1;
 +
  text-decoration: none;
 +
  font-size: 13px;
 +
  color: #eeeeee;
 +
  background: #49505a;
 +
  box-shadow: inset 0 1px 0 rgba(255, 255, 255, 0.1);
 +
}
 +
#sidemenu ul ul li:hover > a,
 +
#sidemenu ul ul li.open > a,
 +
#sidemenu ul ul li.active > a {
 +
  background: #424852;
 +
  color: #ffffff;
 +
}
 +
#sidemenu ul ul li:first-child > a {
 +
  box-shadow: none;
 +
}
 +
#sidemenu ul ul ul li:first-child > a {
 +
  box-shadow: inset 0 1px 0 rgba(255, 255, 255, 0.1);
 +
}
 +
#sidemenu ul ul ul li a {
 +
  padding-left: 30px;
 +
}
 +
#sidemenu > ul > li > ul > li:last-child > a,
 +
#sidemenu > ul > li > ul > li.last > a {
 +
  border-bottom: 0;
 +
}
 +
#sidemenu > ul > li > ul > li.open:last-child > a,
 +
#sidemenu > ul > li > ul > li.last.open > a {
 +
  border-bottom: 1px solid #32373e;
 +
}
 +
#sidemenu > ul > li > ul > li.open:last-child > ul > li:last-child > a {
 +
  border-bottom: 0;
 +
}
 +
#sidemenu ul ul li.has-sub > a::after {
 +
  display: block;
 +
  position: absolute;
 +
  content: "";
 +
  width: 5px;
 +
  height: 5px;
 +
  right: 20px;
 +
  z-index: 10;
 +
  top: 11.5px;
 +
  border-top: 2px solid #eeeeee;
 +
  border-left: 2px solid #eeeeee;
 +
  -webkit-transform: rotate(-135deg);
 +
  -moz-transform: rotate(-135deg);
 +
  -ms-transform: rotate(-135deg);
 +
  -o-transform: rotate(-135deg);
 +
  transform: rotate(-135deg);
 +
}
 +
#sidemenu ul ul li.active > a::after,
 +
#sidemenu ul ul li.open > a::after,
 +
#sidemenu ul ul li > a:hover::after {
 +
  border-color: #ffffff;
 +
}
 +
 +
 +
#yolo {
 +
width:100%;
 +
background-color: #89e1ff;
 +
position:relative;
 +
z-index:-1;
 +
top:-220px;
 +
}
 +
 +
#contentbox{
 +
width: 70%;
 +
float:left;
 +
left:240px;
 +
position:relative;
 +
max-width: 1900px;
 +
min-width: 1000px;
 +
text-align: justify;
 +
margin:auto;
 +
font-family: "Palatino Linotype", "Book Antiqua", Palatino, serif;
 +
font-size:18px;
 +
}
 +
#cover {
 +
overflow:hidden;
 +
position:relative;
 +
width:100%;
 +
margin:auto;
 +
padding:0px;
 +
background-image: url("https://static.igem.org/mediawiki/2015/e/ed/Notebookumd.jpeg");
 +
background-size: 100% ;
 +
background-repeat: no-repeat;
 +
height: 100%;
 +
width:100%;
 +
min-height:800px;
 +
color: #0DFF00;
 +
font-family: Verdana, Geneva, sans-serif;
 +
font-size:xx-large;
 +
top:-200px;
 +
z-index:3;
 +
}
 +
 +
#bar{
 +
display: inline-block;
 +
opacity:1;
 +
background-color: rgba(255,255,255,.5);
 +
color:black;
 +
border-top:4px solid black;
 +
border-bottom:4px solid black;
 +
width:100%;
 +
text-align:center;
 +
height:200px;
 +
position:absolute;
 +
bottom:0px;
 +
text-shadow: 4px 4px 4px white;
 +
z-index:3;
 +
}
 +
ul.a {list-style-type: circle;font-size:18px;color:black}
 +
</style>
 +
 +
<div id='cover'>
 +
<div id='bar'>
 +
<p style="font-size:64px"><b>Notebook</b>
 +
</div>
 +
</div>
 +
 +
<div id='yolo'>
 +
<div id='sidemenu'>
 +
<ul>
 +
 +
<li><a href="#Week2" class="smoothScroll"><span>Week 2</span></a></li>
 +
 +
<li><a href="#Week4" class="smoothScroll"><span>Week 4</span></a></li>
 +
 +
<li><a href="#Week6" class="smoothScroll"><span>Week 6</span></a></li>
 +
 +
<li><a href="#Week8" class="smoothScroll"><span>Week 8</span></a></li>
 +
 +
<li><a href="#Week10" class="smoothScroll"><span>Week 10</span></a></li>
 +
 +
<li><a href="#Week12" class="smoothScroll"><span>Week 12</span></a></li>
 +
 +
<li><a href="#Week14" class="smoothScroll"><span>Week 14</span></a></li>
 +
 +
<li><a href="#Week16" class="smoothScroll"><span>Week 16</span></a></li>
 +
</ul>
 +
</div>
 +
 +
 +
 +
 +
 +
<div id='contentbox'>
 +
<a name="Week1">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Week 1</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Project Preparations: Miraculin, SRNBC (Hok/Sok homolog), Lutein pathway genes
 +
<ul>
 +
<li>- Prepared a bacterial culture containing constitutive GFP (K584001) to grow overnights (E. coli in LB with chloramphenicol)</li>
 +
<li>- Performed a transformation in order to replicate our DNA with the constitutive GFP, and plated half of them on the plates containing chloramphenicol and used the other half for another overnight batch</li>
 +
<li>- Performed a mini-prep on our overnights with the constitutive GFP in order to isolate plasmids and verify that they were present through spectrophotometry<li>
 +
<li>- Started two separate 3A assemblies in order to piece together the pBAD promoter with the miraculin gene and the constitutive GFP gene to the SRNBC gene</li>
 +
<li>- Performed a transformation in order to clone our new plasmids, one containing the pBAD promoter and miraculin gene and the other containing SRNBC and the constitutive GFP</li>
 +
</ul>
 +
<ul>
 +
<li>- Transformed β-cyclase, AppY, and CRTBEY genes (for lutein project) in bacteria to grow on plates overnight</li>
 +
<li>- Started a new 3A assembly for the SRNBC and constitutive GFP construct along with pBAD, constitutive colicinFy, and lysostaphin</li>
 +
<li>- Final products were SRNBC + constitutive GFP, SRNBC + constitutive colicinFy, and pBAD + lysostaphin</li>
 +
<li>- Prepared overnights for the pBAD + miraculin construct that grew on the plates and the β-cyclase, AppY, and CRTBEY genes</li>
 +
<li>- Performed minipreps on the overnights we had prepared the day before (for the β-cyclase, AppY, and CRTBEY genes) and shipped them off to be sequenced</li>
 +
<li>- Performed transformations on the SRNBC + constitutive GFP, SRNBC + constitutive colicinFy, and pBAD + lysostaphin digests and let them grow overnight</li>
 +
 +
 +
<br>
 +
<br>
 +
</div>
 +
 +
 +
 +
<div id='contentbox'>
 +
<a name="Week2">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Week 2</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Miraculin
 +
<ul class="a">
 +
  <li>- RE digest on β-cyclase, AppY, CREB, and CRTBEY genes along with the pSB1C3 backbone and the pBAD + miraculin out of the pSB1A3 backbone so that we could move the genes to the pSB1C3 backbone</li>
 +
  <li>- Ran gel with all parts and cut out the bands</li>
 +
  <li>- Mini-prepped SRNBC + constitutive colicinFy and shipped them off to be sequenced</li>
 +
  <li>- Performed gel purification on gels from day before but they failed; they didn’t show up in the spectrophotometer</li>
 +
  <li>- Performed minipreps on pBAD+Miraculin in the pSB1C3 backbone as well as the const. GFP+SRNBC</li>
 +
  <li>- Performed RE digests on the previous mini-preps: EcoRI and PstI on pBAD + Miraculin, and XBa1 and Pst1 on SRNBC + Constitutive GFP</li>
 +
</ul>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Designing gBlocks
 +
<ul class="a">
 +
  <li>- Designed gblocks for epsilon-cyclase (from arabidopsis), pyocin and Hok/Sok, and ordered them</li>
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
 +
 +
 +
 +
<div id='contentbox'>
 +
<a name="Week3">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Week 3</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Miraculin
 +
<ul class="a">
 +
  <li>- Sequence of pBAD + Miraculin confirmed through sequencing </li>
 +
  <li>- Culture of pBAD + Miraculin was induced with 0.1% arabinose at OD of 1</li>
 +
  <li>- Attempted to purify the Miraculin out of the induction culture by first using a French Press to lyse the cells and then running it through cobalt bead column by FPLC (Fast Protein Liquid Chromatography)</li>
 +
  <li>- Ran the resulting 62 elutions through an SDS-PAGE</li>
 +
  <li>- Failed to extract Miraculin using French press, FPLC and SDS-Page</li>
 +
  <li>- Made overnights of pBAD + Miraculin culture to prepare for test inductions</li>
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
SRNBC and Hok/Sok
 +
<ul class="a">
 +
  <li>- Performed a gel extraction of SRNBC, const. GFP, and pBAD which we then ligated them together to make the SRNBC construct</li>
 +
  <li>- Performed transformations of the constitutive GFP + SRNBC construct</li>
 +
  <li>- Transformations of SRNBC + Constitutive GFP failed</li>
 +
  <li>- Performed PCR on the pSB1C3 backbone in order to amplify it in preparation for a Gibson Assembly with the Hok/Sok gene</li>
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
PCR
 +
<ul class="a">
 +
  <li>-  Began work on Arduino code to cycle the machine</li>
 +
  <li>-  Purchased Peltier units and lm35 temperature sensors </li>
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
 +
 +
 +
<div id='contentbox'>
 +
<a name="Week4">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Week 4</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Miraculin
 +
<ul class="a">
 +
  <li>- Made overnights of pBAD+Miraculin culture to prepare for test inductions tomorrow</li>
 +
  <li>- Inoculated 3 test cultures of pBAD+Miraculin in LB broth with Ampicillin</li>
 +
  <li>- Induced the tubes with 0.05%, 0.1%, and 0.2% arabinose in order to initiate production of miraculin at an OD of 0.4</li>
 +
  <li>- Prepared the induction samples for SDS-PAGE by separating out the media, supernatant after lysing with 200 uL SDS and then the supernatant after lysing with SDS again (pellet supernatant)</li>
 +
  <li>- Ran samples of 0%, 0.05%, 0.1%, and 0.2% arabinose through the gel, but no bands appeared</li>
 +
  <li>- Prepared to perform procedure again using bl21 cells instead of Dh5a cells </li>
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Hok/Sok
 +
<ul class="a">
 +
  <li>- Performed an RE digest on the pSB1C3 backbone with XbaI and SpeI and then ran it through a gel to separate it out, to prepare for the moving of Hok/Sok into pSB1C3</li>
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
PCR
 +
<ul class="a">
 +
  <li>- Received Peltier's, lm35's and began testing the code
 +
  <li>- 6 volt battery pack was found to be insufficient to run Peltier's
 +
  <li>- Purchased an AC/DC power converter 
 +
</li>
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
 +
 +
<div id='contentbox'>
 +
<a name="Week5">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Week 5</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Miraculin
 +
<ul class="a">
 +
  <li>- transformation in BL21 strain was successful </li>
 +
  <li>- SDS page showed a band at roughly 25 kDa  </li>
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Hok/Sok
 +
<ul class="a">
 +
  <li>- inserted Hok/Sok gblock into pSB1C3 backbone using Gibson assembly</li>
 +
  <li>- construct sent for sequencing </li>
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Interlab Study
 +
<ul class="a">
 +
  <li>- transformed parts into dh5a: 3 Anderson promoters, GFP, and RFP</li>
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
PCR
 +
<ul class="a">
 +
  <li>-  Peltier units heated and cooled to proper temperatures however took 30 minutes to cycle properly
 +
  <li>-  started construction of housing to insulate the element to cycle temperature faster and reduced time to 15 minutes 
 +
  <li>-  burned out Peltier elements and broke temperature sensor, and ordered new ones   
 +
</li>
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
 +
 +
<div id='contentbox'>
 +
<a name="Week6">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Week 6</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Hok/Sok
 +
<ul class="a">
 +
  <li>- transformed unstable GFP and RFP with PBAD into dh5a  </li>
 +
  <li>- transformed unstable RFP and inducible lac promoter (k1399011) and const_promoter+RBS (K081005) into dh5a  </li>
 +
  <li>- ligated const_promoter + RBS + RFP </li>
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Interlab
 +
<ul class="a">
 +
  <li>- performed a 3A assembly of each promoter + GFP in pSB1K3 (GFP in pSB1A3 and promoters in pSB1C3) </li>
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
PCR
 +
<ul class="a">
 +
  <li>- received new Peltier unit and temperature sensor
 +
  <li>- began milling on metal PCR tube holder
 +
</li>
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
 +
 +
<div id='contentbox'>
 +
<a name="Week7">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Week 7</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Hok/Sok
 +
<ul class="a">
 +
  <li>- construct created through 3A assembly of quick degrading RFP and constitutive promoter +RBS was proven incorrect using sequencing </li>
 +
  <li>- Sequencing showed a 3A assembly of quick degrading RFP and a "leaky" lactose promoter  </li>
 +
  <li>- Re- ran 3A assembly but the transformation failed </li>
 +
<li>- could be due to contaminated SOC media </li>
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
PCR
 +
<ul class="a">
 +
  <li>-  received milled PCR tube holder
 +
  <li>- embedded temperature sensors into holder reran the machine and again burned out Peltier's
 +
  <li>- spoke with Peltier manufacturers and Open PCR staff and found the Peltier's used for PCR were specialized and more expensive
 +
  <li>- bought higher grade Peltier's to handle high voltages
 +
</li>
 +
</ul>
 +
 +
<br>
 +
<br>
 +
</div>
 +
 +
 +
 +
<div id='contentbox'>
 +
<a name="Week8">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Week 8</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
 +
Hok/Sok
 +
<ul class="a">
 +
  <li>- const_promoter + RBS + RFP were replated from last week but produced no colonies </li>
 +
  <li>- re-transformed original and new 3A assembly which produced the correct sequence  </li>
 +
  <li>- ligated const_promoter:QD-RFP in pSB1C3 </li>
 +
  <li>- performed a 3A assembly of Hok/Sok plasmid with QD-RFP downstream in pSB1K3  </li>
 +
  <li>- site directed mutagenesis of quick degrading GFP (Bba_K750000)  failed </li>
 +
<ul class="a">
 +
  <li>- there was no change from the original sequence </li></li>
 +
</ul>
 +
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
 +
Interlab study
 +
<ul class="a">
 +
  <li>- 3A assembly attempts for the promoters and GFP failed </li>
 +
  <li>- Gibson Assemblies produced a vast amount of colonies  </li>
 +
  <li>- questionable success because the amount of colonies may indicate false positives or contamination </li>
 +
</ul>
 +
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
 +
Lutein
 +
<ul class="a">
 +
  <li>-  ordered pAC-LYC which encodes 3 enzymes from E. Herbicola to produce basal levels of lycopene </li>
 +
</ul>
 +
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
PCR
 +
<ul class="a">
 +
  <li>- Peltier's were still in shipping however work continued on the software
 +
  <li>- designed relay configuration to both heat and cool Peltier's with an unidirectional current flow
 +
</ul>
 +
 +
<br>
 +
<br>
 +
</div>
 +
 +
 +
 +
<div id='contentbox'>
 +
<a name="Week9">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Week 9</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Hok/Sok
 +
<ul class="a">
 +
  <li>- previous 3A assembly from last week showed no colony growth on kanamycin plate  </li>
 +
  <li>- transformed RFP into C3 backbone last week  </li>
 +
  <li>- colonies were produced that were not distinctively red but with the correct sequence  </li>
 +
  <li>- 3A assembly with the RFP + Hok/Sok failed </li>
 +
  <li>- Created primers for Gibson assembly to create Hok/Sok +const_promoter::RBS::unstable RFP  </li>
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Interlab Study
 +
<ul class="a">
 +
  <li>- replated last week’s constructs with GFP at lower concentration selected for chloramphenicol which was successful </li>
 +
  <li>- K08  and K13 constructs grew colonies  </li>
 +
  <li>- Miniprepped constructs and only promoter K08+GFP had the correct sequence </li>
 +
</ul>
 +
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
PCR
 +
<ul class="a">
 +
  <li>- received high rated peltier units that did not break </li>
 +
  <li>- took ~31 minutes to complete 1 cycle but insulated heating dropped the time to 16 minutes  </li>
 +
  <li>- Took apart a hair dryer for heating element </li>
 +
  <li>- heating went to >95 in ~2 seconds, cooled to 50 in ~8 seconds </li>
 +
  <li>- The heating element is not very precise so it resulted in a lot of overshoot in temperature  </li>
 +
</ul>
 +
 +
<br>
 +
<br>
 +
</div>
 +
 +
 +
 +
<div id='contentbox'>
 +
<a name="Week10">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Week 10</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Hok-Sok
 +
<ul class="a">
 +
  <li>- created construct with RFP and hok/sok </li>
 +
  <li>- Pcr of the construct failed so the construct will be sequenced to check  </li>
 +
</ul>
 +
 +
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
PCR
 +
<ul class="a">
 +
  <li>- cycle data was taken and it exhibits consistency </li>
 +
</ul>
 +
 +
<br>
 +
<br>
 +
</div>
 +
 +
 +
 +
<div id='contentbox'>
 +
<a name="Week11">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Week 11</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Hok/Sok
 +
<ul class="a">
 +
  <li>- 3A assembly of H/S + RFP failed </li>
 +
  <li>- ran a PCR of pSB1C3+H/S+RFP using 3 different rxn buffers  </li>
 +
  <li>- HF rxn buffer </li>
 +
  <li>- GC rxn buffer </li>
 +
  <li>- GC rxn buffer w/ DMSO which yielded product  </li>
 +
  <li>- Ran a PCR of unstable RFP using the  3 rxn buffers above  </li>
 +
</ul>
 +
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Interlab Study
 +
<ul class="a">
 +
  <li>- used Phusion instead of Q5 for PCR  </li>
 +
  <li>- ran PCR for GFP 1, GFP 3, and IL1 which produced the correct sized products  </li>
 +
  <li>- Gibson Assembly of GFP + IL1 produced colonies </li>
 +
  <li>- pSB1C3 - IL3 did not have bands in gel of appropriate size  </li>
 +
 +
</ul>
 +
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Lutein
 +
<ul class="a">
 +
  <li>- RE digests of pLAC-RFP in pSB1C3  </li>
 +
  <li>- ran a Gibson Assembly of E-cyclase/C3 and E-hydroxylase/C3 which produced colonies  </li>
 +
</ul>
 +
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
PCR
 +
<ul class="a">
 +
  <li>- rebuilt top of hair dryer housing w/ soda can  </li>
 +
  <li>- observed random temp spikes occurring during each run  </li>
 +
  <li>- caused by hardware issue with relays </li>
 +
  <li>- purchased new relays to test this week  </li>
 +
</ul>
 +
 +
<br>
 +
<br>
 +
</div>
 +
</div>
 +
  
 +
<div id='contentbox'>
 +
<a name="Week12">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Week 12</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Hok/Sok
 +
<ul class="a">
 +
  <li>- Ran a Gibson Assembly of Hok/Sok + RFP </li>
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Interlab
 +
<ul class="a">
 +
  <li>- worked on finishing the last construct </li>
 +
  <li>- K13 sequenced correctly and the pcr looks good  </li>
 +
  <li>- contacted W&M on possible collaboration for the last construct </li>
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Lutein
 +
<ul class="a">
 +
  <li>- ordered primers for Gibson Assembly </li>
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
PCR
 +
<ul class="a">
 +
  <li>- replaced old relays with higher powered relays </li>
 +
  <li>- reduced mass by over 50% of the peltier machine  </li>
 +
  <li>- resulted in speedier temperature changes  </li>
 +
</ul>
  
−
</style>  
+
<br>
 +
<br>
 +
</div>
  
−
<head>
 
  
  
 +
<div id='contentbox'>
 +
<a name="Week13">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Week 13</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Hok/Sok
 +
<ul class="a">
 +
  <li>- sequence of hok/sok construct created last week was incorrect  </li>
 +
  <li>- may put a unique RE site between hok/sok and rfp when retrying 3A assembly, RE cloning and Gibson  </li>
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Interlab
 +
<ul class="a">
 +
  <li>- Gibsons of IL3 (K13 + GFP) have all failed </li>
 +
  <li>- 3 trials of IL2 , IL1,+ control (const_promoter with GFP), - control were tested using the plate reader  </li>
 +
  <li>- IL2 was a little low in fluorescence, similar to the - control </li>
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
PCR
 +
<ul class="a">
 +
  <li>- Rewired hair dryer and changed relays to handle higher wattage  </li>
 +
  <li>- attempted PCR cycle failed  </li>
 +
  <li>- assumed temp sensor needed to be immersed in mineral oil to properly report temp </li>
 +
<li>- denaturation temp may have been too low </li>
 +
<li>- increased the denaturation temperature, attained an amplified product  </li>
 +
</ul>
 +
<br>
 +
<br>
 +
</div>
  
−
</head>
 
  
−
<body>
 
  
 +
<div id='contentbox'>
 +
<a name="Week14">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Week 14</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Hok/Sok
 +
<ul class="a">
 +
  <li>- Began testing for RFP fluorescence for Hok/Sok effectiveness as a plasmid maintenance system </li>
 +
  <li>- Group A: RFP coding region; constitutive promoter ; chloramphenicol in liquid culture  </li>
 +
  <li>- Group B: RFP coding region; constitutive promoter ; no chloramphenicol in liquid culture </li>
 +
  <li>- Group C: RFP coding region; no promoter ; chloramphenicol in liquid culture </li>
 +
  <li>- Group D: H/S-RFP coding region; constitutive promoter ; chloramphenicol in liquid culture  </li>
 +
  <li>- Group E: H/S-RFP coding region; constitutive promoter ; nochloramphenicol in liquid culture </li>
 +
  <li>- Group F: H/S-RFP coding region; constitutive promoter ; chloramphenicol in liquid culture </li>
 +
</ul>
 +
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
Interlab
 +
<ul class="a">
 +
  <li>- obtained the last construct from W&M  </li>
 +
  <li>- tested the last construct using the plate reader  </li>
 +
  <li>- Turned in the IL study </li>
 +
</ul>
 +
<br>
 +
PCR
 +
<ul class="a">
 +
  <li>-began work on incubator
 +
  <li>-incubator was proven to maintain 37 degrees Celsius
 +
<br>
 +
<br>
 +
</div>
  
−
<div>
 
−
<div id="flipbook">
 
  
−
<div class="hard"><p> Notebook </p></div>
 
−
<div class="hard"></div>
 
−
<div style="background-image:url(https://static.igem.org/mediawiki/2015/e/ea/Notebook1.png)"></div>
 
−
<div style="background-image:url(https://static.igem.org/mediawiki/2015/b/bd/Notebook2.png)"></div>
 
−
<div class="hard"></div>
 
−
<div class="hard"></div>
 
  
 +
<div id='contentbox'>
 +
<a name="Week15">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Week 15</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
  
 +
Hok/Sok
 +
<ul class="a">
 +
  <li>- Tested generations alpha beta and gamma in BL21 cells </li>
 +
  <li>- Alpha and beta contained groups A-E  </li>
 +
  <li>- gamma had only group F </li>
 +
</ul>
  
−
<script type="text/javascript">
+
<br>
 +
<br>
 +
</div>
  
  
−
$("#flipbook").turn({
 
−
    width: 800,
 
−
    height: 600,
 
−
    autoCenter: false,
 
−
    elevation: 50
 
−
});
 
  
 +
<div id='contentbox'>
 +
<a name="Week16">
 +
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 +
<b>Week 16</b></a>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
  
−
</script>  
+
Hok/Sok
 +
<ul class="a">
 +
  <li>- Tested generation delta using DH5alpha </li>
 +
  <li>- contained groups A-E  </li>
 +
</ul>
  
 +
<br>
 +
<br>
  
−
</body>  
+
</div>
  
 +
</div>
  
 
</html>
 
</html>

Latest revision as of 23:57, 1 October 2015