Difference between revisions of "Team:UMaryland/Notebook"

 
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font-family: "Palatino Linotype", "Book Antiqua", Palatino, serif;
 +
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#cover {
 
#cover {
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−
background-image: url("https://static.igem.org/mediawiki/2015/9/97/Resultsq.png");
+
background-image: url("https://static.igem.org/mediawiki/2015/e/ed/Notebookumd.jpeg");
 
background-size: 100% ;
 
background-size: 100% ;
 
background-repeat: no-repeat;
 
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font-size:xx-large;
 
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#bar{
 
#bar{
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height:200px;
 
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position:absolute;
 
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−
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+
bottom:0px;
 
text-shadow: 4px 4px 4px white;
 
text-shadow: 4px 4px 4px white;
 +
z-index:3;
 
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−
ul.a {list-style-type: circle;font-size:24px;color:black}
+
ul.a {list-style-type: circle;font-size:18px;color:black}
 
</style>
 
</style>
  
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<p style="font-size:64px"><b>Notebook</b>
 
<p style="font-size:64px"><b>Notebook</b>
 
</div>
 
</div>
 +
</div>
 +
 +
<div id='yolo'>
 
<div id='sidemenu'>
 
<div id='sidemenu'>
 
<ul>
 
<ul>
−
<li><a href="#Week1" class="smoothScroll"><span>Week 1</span></a></li>
+
 
 
<li><a href="#Week2" class="smoothScroll"><span>Week 2</span></a></li>
 
<li><a href="#Week2" class="smoothScroll"><span>Week 2</span></a></li>
−
<li><a href="#Week3" class="smoothScroll"><span>Week 3</span></a></li>
+
 
 
<li><a href="#Week4" class="smoothScroll"><span>Week 4</span></a></li>
 
<li><a href="#Week4" class="smoothScroll"><span>Week 4</span></a></li>
−
<li><a href="#Week5" class="smoothScroll"><span>Week 5</span></a></li>
+
 
 
<li><a href="#Week6" class="smoothScroll"><span>Week 6</span></a></li>
 
<li><a href="#Week6" class="smoothScroll"><span>Week 6</span></a></li>
−
<li><a href="#Week7" class="smoothScroll"><span>Week 7</span></a></li>
+
 
 
<li><a href="#Week8" class="smoothScroll"><span>Week 8</span></a></li>
 
<li><a href="#Week8" class="smoothScroll"><span>Week 8</span></a></li>
−
<li><a href="#Week9" class="smoothScroll"><span>Week 9</span></a></li>
+
 
 
<li><a href="#Week10" class="smoothScroll"><span>Week 10</span></a></li>
 
<li><a href="#Week10" class="smoothScroll"><span>Week 10</span></a></li>
−
<li><a href="#Week11" class="smoothScroll"><span>Week 11</span></a></li>
+
 
 
<li><a href="#Week12" class="smoothScroll"><span>Week 12</span></a></li>
 
<li><a href="#Week12" class="smoothScroll"><span>Week 12</span></a></li>
−
<li><a href="#Week13" class="smoothScroll"><span>Week 13</span></a></li>
+
 
 
<li><a href="#Week14" class="smoothScroll"><span>Week 14</span></a></li>
 
<li><a href="#Week14" class="smoothScroll"><span>Week 14</span></a></li>
−
<li><a href="#Week15" class="smoothScroll"><span>Week 15</span></a></li>
+
 
 
<li><a href="#Week16" class="smoothScroll"><span>Week 16</span></a></li>
 
<li><a href="#Week16" class="smoothScroll"><span>Week 16</span></a></li>
 
</ul>
 
</ul>
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−
</div>
 
−
</div>
 
  
  
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<b>Week 1</b></a>
 
<b>Week 1</b></a>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
−
Miraculin:
+
Project Preparations: Miraculin, SRNBC (Hok/Sok homolog), Lutein pathway genes
−
<br>
+
<ul>
−
<ul class="a">
+
<li>- Prepared a bacterial culture containing constitutive GFP (K584001) to grow overnights (E. coli in LB with chloramphenicol)</li>
−
  <li>- ligated the pBAD promoter in PSB1C3 with the miraculin gene in PSB1C3 using 3A assembly</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>- plate had colonies</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>
 +
<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>
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<b>Week 2</b></a>
 
<b>Week 2</b></a>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
−
Miraculin:
+
Miraculin
 
<ul class="a">
 
<ul class="a">
−
   <li>- The pBAD +miraculin construct was moved back into psb1C3 backbone and sent for sequencing</li>
+
   <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>
 
</ul>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
−
Designing gBlocks:
+
Designing gBlocks
 
<ul class="a">
 
<ul class="a">
−
   <li>- Designed gblocks for epsilon-cyclase (from arabidopsis), pyocin and Hok/Sok</li>
+
   <li>- Designed gblocks for epsilon-cyclase (from arabidopsis), pyocin and Hok/Sok, and ordered them</li>
 
</ul>
 
</ul>
 
<br>
 
<br>
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<div id='contentbox'>
 
<div id='contentbox'>
−
<a name="Week 3">
+
<a name="Week3">
 
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 
<p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
−
<b>Week3</b></a>
+
<b>Week 3</b></a>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
−
Miraculin:
+
Miraculin
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- Sequence of pBAD + Miraculin confirmed through sequencing </li>
−
   <li>- failed to extract using French press, FPLC and SDS-Page </li>
+
   <li>- Culture of pBAD + Miraculin was induced with 0.1% arabinose at OD of 1</li>
−
   <li>- could be due to high arabinose induction (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>
 
</ul>
−
 
 
<br>
 
<br>
 
<br>
 
<br>
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<b>Week 4</b></a>
 
<b>Week 4</b></a>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
−
Miraculin:
+
Miraculin
 
<ul class="a">
 
<ul class="a">
−
   <li>- induced 3 test cultures with 0%, 0.05%, 0.1%, 0.2% arabinose with no significant results </li>
+
   <li>- Made overnights of pBAD+Miraculin culture to prepare for test inductions tomorrow</li>
−
   <li>- will perform procedure again using bl21 instead of Dh5a  </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>
 
</ul>
−
 
 
<br>
 
<br>
 
<br>
 
<br>
 
</div>
 
</div>
−
 
−
 
  
 
<div id='contentbox'>
 
<div id='contentbox'>
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<b>Week 5</b></a>
 
<b>Week 5</b></a>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
−
Miraculin:
+
Miraculin
 
<ul class="a">
 
<ul class="a">
 
   <li>- transformation in BL21 strain was successful </li>
 
   <li>- transformation in BL21 strain was successful </li>
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<br>
 
<br>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
−
Hok/Sok:
+
Hok/Sok
 
<ul class="a">
 
<ul class="a">
−
   <li>- inserted Hok/Sok gblock into PSB1C3 backbone using Gibson assembly</li>
+
   <li>- inserted Hok/Sok gblock into pSB1C3 backbone using Gibson assembly</li>
 
   <li>- construct sent for sequencing </li>
 
   <li>- construct sent for sequencing </li>
 
</ul>
 
</ul>
 
<br>
 
<br>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
−
Interlab Study:
+
Interlab Study
 
<ul class="a">
 
<ul class="a">
 
   <li>- transformed parts into dh5a: 3 Anderson promoters, GFP, and RFP</li>
 
   <li>- transformed parts into dh5a: 3 Anderson promoters, GFP, and RFP</li>
 
</ul>
 
</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>
 
<br>
 
<br>
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<b>Week 6</b></a>
 
<b>Week 6</b></a>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
−
Hok/Sok:
+
Hok/Sok
 
<ul class="a">
 
<ul class="a">
 
   <li>- transformed unstable GFP and RFP with PBAD into dh5a  </li>
 
   <li>- transformed unstable GFP and RFP with PBAD into dh5a  </li>
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<br>
 
<br>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
−
Interlab:
+
Interlab
 
<ul class="a">
 
<ul class="a">
−
   <li>- performed a 3A assembly of each promoter + GFP in PSB1K3 (GFP in PSB1A3 and promoters in PSB1C3) </li>
+
   <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>
 
</ul>
−
 
 
<br>
 
<br>
 
<br>
 
<br>
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<b>Week 7</b></a>
 
<b>Week 7</b></a>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
−
Hok/Sok:
+
Hok/Sok
 
<ul class="a">
 
<ul class="a">
 
   <li>- construct created through 3A assembly of quick degrading RFP and constitutive promoter +RBS was proven incorrect using sequencing </li>
 
   <li>- construct created through 3A assembly of quick degrading RFP and constitutive promoter +RBS was proven incorrect using sequencing </li>
Line 411: Line 448:
 
   <li>- Re- ran 3A assembly but the transformation failed </li>
 
   <li>- Re- ran 3A assembly but the transformation failed </li>
 
<li>- could be due to contaminated SOC media </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>
 
</ul>
  
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   <li>- const_promoter + RBS + RFP were replated from last week but produced no colonies </li>
 
   <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>- re-transformed original and new 3A assembly which produced the correct sequence  </li>
−
   <li>- ligated const_promoter:QD-RFP in PSB1C3 </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>- 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>
 
   <li>- site directed mutagenesis of quick degrading GFP (Bba_K750000)  failed </li>
 
<ul class="a">
 
<ul class="a">
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</ul>
 
</ul>
  
−
 
+
</ul>
 
<br>
 
<br>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
Line 442: Line 489:
 
Interlab study
 
Interlab study
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- 3A assembly attempts for the promoters and GFP failed </li>
−
   <li>- failed to extract using French press, FPLC and SDS-Page </li>
+
   <li>- Gibson Assemblies produced a vast amount of colonies </li>
−
   <li>- could be due to high arabinose induction (OD of 1) </li>
+
   <li>- questionable success because the amount of colonies may indicate false positives or contamination </li>
 
</ul>
 
</ul>
−
3A assembly attempts for the promoters and GFP failed
+
 
−
<br>
+
−
Gibson Assemblies produced a vast amount of colonies
+
−
<br>
+
−
questionable success because the amount of colonies may indicate false positives or contamination
+
−
<br>
+
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
  
 
Lutein  
 
Lutein  
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>-  ordered pAC-LYC which encodes 3 enzymes from E. Herbicola to produce basal levels of lycopene </li>
−
  <li>- failed to extract using French press, FPLC and SDS-Page </li>
+
−
  <li>- could be due to high arabinose induction (OD of 1) </li>
+
 
</ul>
 
</ul>
−
ordered pAC-LYC which encodes 3 enzymes from E. Herbicola to produce basal levels of lycopene
+
 
 
<br>
 
<br>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
PCR  
 
PCR  
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- Peltier's were still in shipping however work continued on the software
−
   <li>- failed to extract using French press, FPLC and SDS-Page  </li>
+
   <li>- designed relay configuration to both heat and cool Peltier's with an unidirectional current flow
−
  <li>- could be due to high arabinose induction (OD of 1) </li>
+
 
</ul>
 
</ul>
−
code made and proven to properly cycle machine
 
  
 
<br>
 
<br>
Line 484: Line 522:
 
Hok/Sok
 
Hok/Sok
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- previous 3A assembly from last week showed no colony growth on kanamycin plate  </li>
−
   <li>- failed to extract using French press, FPLC and SDS-Page </li>
+
   <li>- transformed RFP into C3 backbone last week  </li>
−
   <li>- could be due to high arabinose induction (OD of 1) </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>
 
</ul>
−
previous 3A assembly from last week showed no colony growth on kanamycin plate
 
−
<br>
 
−
transformed RFP into C3 backbone last week
 
−
<br>
 
−
colonies were produced that were not distinctively red but with the correct sequence
 
−
<br>
 
−
3A assembly with the RFP + Hok/Sok failed
 
−
<br>
 
−
Created primers for Gibson assembly to create Hok/Sok +const_promoter::RBS::unstable RFP
 
 
<br>  
 
<br>  
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
Interlab Study
 
Interlab Study
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- replated last week’s constructs with GFP at lower concentration selected for chloramphenicol which was successful </li>
−
   <li>- failed to extract using French press, FPLC and SDS-Page </li>
+
   <li>- K08  and K13 constructs grew colonies </li>
−
   <li>- could be due to high arabinose induction (OD of 1) </li>
+
   <li>- Miniprepped constructs and only promoter K08+GFP had the correct sequence </li>
 
</ul>
 
</ul>
−
replated last week’s constructs with GFP at lower concentration selected for chloramphenicol which was successful
+
 
−
<br>
+
−
K08  and K13 constructs grew colonies
+
−
<br>
+
−
Miniprepped constructs and only promoter K08+GFP had the correct sequence
+
 
<br>
 
<br>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
PCR
 
PCR
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- received high rated peltier units that did not break </li>
−
  <li>- failed to extract using French press, FPLC and SDS-Page  </li>
+
  <li>- took ~31 minutes to complete 1 cycle but insulated heating dropped the time to 16 minutes </li>
−
  <li>- could be due to high arabinose induction (OD of 1) </li>
+
  <li>- Took apart a hair dryer for heating element </li>
−
</ul>
+
  <li>- heating went to >95 in ~2 seconds, cooled to 50 in ~8 seconds </li>
−
received high rated peltier units that did not break
+
  <li>- The heating element is not very precise so it resulted in a lot of overshoot in temperature </li>
−
<br>
+
</ul>
−
took ~31 minutes to complete 1 cycle but insulated heating dropped the time to 16 minutes
+
−
<br>
+
−
Took apart a hair dryer for heating element  
+
−
<br>
+
−
heating went to >95 in ~2 seconds, cooled to 50 in ~8 seconds
+
−
<br>  
+
−
The heating element is not very precise so it resulted in a lot of overshoot in temperature  
+
  
 
<br>
 
<br>
Line 541: Line 561:
 
Hok-Sok
 
Hok-Sok
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- created construct with RFP and hok/sok </li>
−
   <li>- failed to extract using French press, FPLC and SDS-Page </li>
+
   <li>- Pcr of the construct failed so the construct will be sequenced to check </li>
−
  <li>- could be due to high arabinose induction (OD of 1) </li>
+
 
</ul>
 
</ul>
−
created construct with RFP and hok/sok
+
 
−
<br>
+
 
−
Pcr of the construct failed so the construct will be sequenced to check
+
−
<br>
+
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
−
PCR /Gibson Check
+
PCR  
−
<ul class="a">
+
−
  <li>- sequence confirmed through sequencing </li>
+
−
  <li>- failed to extract using French press, FPLC and SDS-Page  </li>
+
−
  <li>- could be due to high arabinose induction (OD of 1) </li>
+
−
</ul>
+
−
performed a series of check in order to ensure that all the reagents are active
+
−
<br>
+
−
everything seems to be working
+
−
<br>
+
−
<p style="font-size:24px;text-align:left;text-decoration: underline;">
+
−
PCR machine
+
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- cycle data was taken and it exhibits consistency </li>
−
  <li>- failed to extract using French press, FPLC and SDS-Page  </li>
+
−
  <li>- could be due to high arabinose induction (OD of 1) </li>
+
 
</ul>
 
</ul>
−
cycle data was taken and it exhibits consistency
 
  
 
<br>
 
<br>
Line 582: Line 585:
 
Hok/Sok
 
Hok/Sok
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- 3A assembly of H/S + RFP failed </li>
−
   <li>- failed to extract using French press, FPLC and SDS-Page </li>
+
   <li>- ran a PCR of pSB1C3+H/S+RFP using 3 different rxn buffers  </li>
−
   <li>- could be due to high arabinose induction (OD of 1) </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>
 
</ul>
−
3A assembly of H/S + RFP failed
+
 
−
<br>
+
−
ran a PCR of PSB1C3+H/S+RFP using 3 different rxn buffers
+
−
<br>
+
−
HF rxn buffer
+
−
<br>
+
−
GC rxn buffer
+
−
<br>
+
−
GC rxn buffer w/ DMSO which yielded product
+
−
<br>
+
−
Ran a PCR of unstable RFP using the  3 rxn buffers above
+
−
<br>
+
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
Interlab Study
 
Interlab Study
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- used Phusion instead of Q5 for PCR  </li>
−
   <li>- failed to extract using French press, FPLC and SDS-Page </li>
+
   <li>- ran PCR for GFP 1, GFP 3, and IL1 which produced the correct sized products </li>
−
   <li>- could be due to high arabinose induction (OD of 1) </li>
+
   <li>- Gibson Assembly of GFP + IL1 produced colonies </li>
 +
  <li>- pSB1C3 - IL3 did not have bands in gel of appropriate size  </li>
 +
 
 
</ul>
 
</ul>
−
used Phusion instead of Q5 for PCR
+
 
−
<br>
+
−
ran PCR for GFP 1, GFP 3, and IL1 which produced the correct sized products
+
−
<br>
+
−
Gibson Assembly of GFP + IL1 produced colonies
+
−
<br>
+
−
PSB1C3 - IL3 did not have bands in gel of appropriate size
+
−
<br>
+
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
−
Lutein  
+
Lutein
−
<br>
+
<ul class="a">
−
RE digests of pLAC-RFP in PSB1C3
+
  <li>- RE digests of pLAC-RFP in pSB1C3  </li>
−
<br>
+
  <li>- ran a Gibson Assembly of E-cyclase/C3 and E-hydroxylase/C3 which produced colonies </li>
−
ran a Gibson Assembly of E-cyclase/C3 and E-hydroxylase/C3 which produced colonies
+
</ul>
−
<br>
+
 
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
−
PCR Machine
+
PCR  
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- rebuilt top of hair dryer housing w/ soda can  </li>
−
   <li>- failed to extract using French press, FPLC and SDS-Page  </li>
+
   <li>- observed random temp spikes occurring during each run  </li>
−
   <li>- could be due to high arabinose induction (OD of 1) </li>
+
  <li>- caused by hardware issue with relays </li>
 +
   <li>- purchased new relays to test this week  </li>
 
</ul>
 
</ul>
−
rebuilt top of hair dryer housing w/ soda can
 
−
<br>
 
−
observed random temp spikes occurring during each run
 
−
<br>
 
−
caused by hardware issue with relays
 
−
<br>
 
−
purchased new relays to test this week
 
−
 
  
 
<br>
 
<br>
Line 647: Line 630:
 
<b>Week 12</b></a>
 
<b>Week 12</b></a>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
−
Hok/Sok:
+
Hok/Sok
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- Ran a Gibson Assembly of Hok/Sok + RFP </li>
−
  <li>- failed to extract using French press, FPLC and SDS-Page  </li>
+
−
  <li>- could be due to high arabinose induction (OD of 1) </li>
+
 
</ul>
 
</ul>
−
Ran a Gibson Assembly of Hok/Sok + RFP
 
 
<br>
 
<br>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
Interlab
 
Interlab
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- worked on finishing the last construct </li>
−
   <li>- failed to extract using French press, FPLC and SDS-Page </li>
+
   <li>- K13 sequenced correctly and the pcr looks good </li>
−
   <li>- could be due to high arabinose induction (OD of 1) </li>
+
   <li>- contacted W&M on possible collaboration for the last construct </li>
 
</ul>
 
</ul>
−
worked on finishing the last construct
 
−
<br>
 
−
K13 sequenced correctly and the pcr looks good
 
−
<br>
 
−
contacted W&M on possible collaboration for the last construct
 
 
<br>
 
<br>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
Lutein
 
Lutein
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- ordered primers for Gibson Assembly </li>
−
  <li>- failed to extract using French press, FPLC and SDS-Page  </li>
+
−
  <li>- could be due to high arabinose induction (OD of 1) </li>
+
 
</ul>
 
</ul>
−
ordered primers for Gibson Assembly
 
 
<br>
 
<br>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
PCR
 
PCR
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- replaced old relays with higher powered relays </li>
−
   <li>- failed to extract using French press, FPLC and SDS-Page </li>
+
   <li>- reduced mass by over 50% of the peltier machine </li>
−
   <li>- could be due to high arabinose induction (OD of 1) </li>
+
   <li>- resulted in speedier temperature changes  </li>
 
</ul>
 
</ul>
−
replaced old relays with higher powered relays
 
−
<br>
 
−
reduced mass by over 50% of the peltier machine
 
−
<br>
 
−
resulted in speedier temperature changes
 
  
 
<br>
 
<br>
Line 703: Line 670:
 
Hok/Sok
 
Hok/Sok
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- sequence of hok/sok construct created last week was incorrect  </li>
−
   <li>- failed to extract using French press, FPLC and SDS-Page </li>
+
   <li>- may put a unique RE site between hok/sok and rfp when retrying 3A assembly, RE cloning and Gibson </li>
−
  <li>- could be due to high arabinose induction (OD of 1) </li>
+
 
</ul>
 
</ul>
−
sequence of hok/sok construct created last week was incorrect
 
−
<br>
 
−
may put a unique RE site between hok/sok and rfp when retrying 3A assembly, RE cloning and Gibson
 
 
<br>
 
<br>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
Interlab
 
Interlab
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- Gibsons of IL3 (K13 + GFP) have all failed </li>
−
   <li>- failed to extract using French press, FPLC and SDS-Page </li>
+
   <li>- 3 trials of IL2 , IL1,+ control (const_promoter with GFP), - control were tested using the plate reader </li>
−
   <li>- could be due to high arabinose induction (OD of 1) </li>
+
   <li>- IL2 was a little low in fluorescence, similar to the - control </li>
 
</ul>
 
</ul>
−
Gibsons of IL3 (K13 + GFP) have all failed
 
−
<br>
 
−
3 trials of IL2 , IL1,+ control (const_promoter with GFP), - control were tested using the plate reader
 
−
<br>
 
−
IL2 was a little low in fluorescence, similar to the - control
 
 
<br>
 
<br>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
PCR
 
PCR
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- Rewired hair dryer and changed relays to handle higher wattage  </li>
−
   <li>- failed to extract using French press, FPLC and SDS-Page </li>
+
   <li>- attempted PCR cycle failed  </li>
−
   <li>- could be due to high arabinose induction (OD of 1) </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>
 
</ul>
−
Rewired hair dryer and changed relays to handle higher wattage
 
−
<br>
 
−
attempted PCR cycle failed
 
−
<br>
 
−
assumed temp sensor needed to be immersed in mineral oil to properly report temp
 
−
<br>
 
−
denaturation temp may have been too low
 
−
 
−
 
−
 
−
 
 
<br>
 
<br>
 
<br>
 
<br>
Line 755: Line 704:
 
Hok/Sok
 
Hok/Sok
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- Began testing for RFP fluorescence for Hok/Sok effectiveness as a plasmid maintenance system </li>
−
   <li>- failed to extract using French press, FPLC and SDS-Page </li>
+
   <li>- Group A: RFP coding region; constitutive promoter ; chloramphenicol in liquid culture  </li>
−
   <li>- could be due to high arabinose induction (OD of 1) </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>
 
</ul>
−
Began testing for RFP fluorescence for Hok/Sok effectiveness as a plasmid maintenance system
 
−
<br>
 
−
Group A: RFP coding region; constitutive promoter ; chloramphenicol in liquid culture
 
−
<br>
 
−
Group B: RFP coding region; constitutive promoter ; no chloramphenicol in liquid culture
 
−
<br>
 
−
Group C: RFP coding region; no promoter ; chloramphenicol in liquid culture
 
−
<br>
 
−
Group D: H/S-RFP coding region; constitutive promoter ; chloramphenicol in liquid culture
 
−
<br>
 
−
Group E: H/S-RFP coding region; constitutive promoter ; nochloramphenicol in liquid culture
 
−
<br>
 
−
Group F: H/S-RFP coding region; constitutive promoter ; chloramphenicol in liquid culture
 
 
<br>
 
<br>
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
<p style="font-size:24px;text-align:left;text-decoration: underline;">
−
Interlab:
+
Interlab
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- obtained the last construct from W&M  </li>
−
   <li>- failed to extract using French press, FPLC and SDS-Page </li>
+
   <li>- tested the last construct using the plate reader </li>
−
   <li>- could be due to high arabinose induction (OD of 1) </li>
+
   <li>- Turned in the IL study </li>
 
</ul>
 
</ul>
−
Obtained the last construct from W&M
 
 
<br>
 
<br>
−
Tested the last construct using the plate reader
+
PCR
−
<br>
+
<ul class="a">
−
Turned in the IL study
+
  <li>-began work on incubator
−
 
+
  <li>-incubator was proven to maintain 37 degrees Celsius
−
 
+
 
<br>
 
<br>
 
<br>
 
<br>
Line 801: Line 739:
 
Hok/Sok
 
Hok/Sok
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- Tested generations alpha beta and gamma in BL21 cells </li>
−
   <li>- failed to extract using French press, FPLC and SDS-Page </li>
+
   <li>- Alpha and beta contained groups A-E </li>
−
   <li>- could be due to high arabinose induction (OD of 1) </li>
+
   <li>- gamma had only group F </li>
 
</ul>
 
</ul>
−
Tested generations alpha beta and gamma in BL21 cells
 
−
<br>
 
−
alpha and beta contained groups A-E
 
−
<br>
 
−
gamma had only group F
 
−
 
  
 
<br>
 
<br>
Line 826: Line 758:
 
Hok/Sok
 
Hok/Sok
 
<ul class="a">
 
<ul class="a">
−
   <li>- sequence confirmed through sequencing </li>
+
   <li>- Tested generation delta using DH5alpha </li>
−
   <li>- failed to extract using French press, FPLC and SDS-Page </li>
+
   <li>- contained groups A-E </li>
−
  <li>- could be due to high arabinose induction (OD of 1) </li>
+
 
</ul>
 
</ul>
−
Tested generation delta using DH5alpha
 
−
<br>
 
−
contained groups A-E
 
−
 
−
 
−
 
  
 
<br>
 
<br>

Latest revision as of 23:57, 1 October 2015