Difference between revisions of "Team:UMaryland/Notebook"

Line 382: Line 382:
 
<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>Week 6</b></a>
 
<b>Week 6</b></a>
−
<p style="font-size:24px;text-align:left;">
+
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
Hok/Sok:
 
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>
 
<br>
−
Transformed unstable GFP and RFP with PBAD into dh5a
+
<p style="font-size:24px;text-align:left;text-decoration: underline;">
−
<br>
+
−
Transformed unstable RFP and inducible lac promoter (k1399011) and const_promoter+RBS (K081005) into dh5a
+
−
<br>
+
−
ligated const_promoter + RBS + RFP
+
−
<br>
+
 
Interlab:
 
Interlab:
−
<br>
+
<ul class="a">
−
performed a 3A assembly of each promoter + GFP in PSB1K3 (GFP in PSB1A3 and promoters in PSB1C3)
+
  <li>- performed a 3A assembly of each promoter + GFP in PSB1K3 (GFP in PSB1A3 and promoters in PSB1C3) </li>
−
 
+
</ul>
  
 
<br>
 
<br>
Line 404: Line 404:
 
<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>Week 7</b></a>
 
<b>Week 7</b></a>
−
<p style="font-size:24px;text-align:left;">
+
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
Hok/Sok:
 
Hok/Sok:
−
<br>
+
<ul class="a">
−
construct created through 3A assembly of quick degrading RFP and constitutive promoter +RBS was proven incorrect using sequencing
+
  <li>- construct created through 3A assembly of quick degrading RFP and constitutive promoter +RBS was proven incorrect using sequencing </li>
−
<br>
+
  <li>- Sequencing showed a 3A assembly of quick degrading RFP and a "leaky" lactose promoter </li>
−
Sequencing showed a 3A assembly of quick degrading RFP and a "leaky" lactose promoter
+
  <li>- Re- ran 3A assembly but the transformation failed </li>
−
<br>
+
<li>- could be due to contaminated SOC media </li>
−
Re- ran 3A assembly but the transformation failed
+
</ul>
−
<br>
+
−
could be due to contaminated SOC media
+
  
 
<br>
 
<br>
Line 425: Line 423:
 
<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>Week 8</b></a>
 
<b>Week 8</b></a>
−
<p style="font-size:24px;text-align:left;">
+
<p style="font-size:24px;text-align:left;text-decoration: underline;">
  
 
Hok/Sok  
 
Hok/Sok  
−
<br>
+
<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>
 
const_promoter + RBS + RFP were replated from last week but produced no colonies
 
const_promoter + RBS + RFP were replated from last week but produced no colonies
 
<br>
 
<br>
Line 441: Line 443:
 
there was no change from the original sequence
 
there was no change from the original sequence
 
<br>
 
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
 
Interlab study
 
Interlab study
−
<br>
+
<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>
 
3A assembly attempts for the promoters and GFP failed
 
3A assembly attempts for the promoters and GFP failed
 
<br>
 
<br>
Line 449: Line 457:
 
questionable success because the amount of colonies may indicate false positives or contamination
 
questionable success because the amount of colonies may indicate false positives or contamination
 
<br>
 
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 +
 
Lutein  
 
Lutein  
−
<br>
+
<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>
 
  ordered pAC-LYC which encodes 3 enzymes from E. Herbicola to produce basal levels of lycopene
 
  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;">
 
PCR  
 
PCR  
−
<br>
+
<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>
 
code made and proven to properly cycle machine  
 
code made and proven to properly cycle machine  
  
Line 467: Line 486:
 
<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>Week 9</b></a>
 
<b>Week 9</b></a>
−
<p style="font-size:24px;text-align:left;">
+
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
Hok/Sok
 
Hok/Sok
−
<br>
+
<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>
 
previous 3A assembly from last week showed no colony growth on kanamycin plate  
 
previous 3A assembly from last week showed no colony growth on kanamycin plate  
 
<br>
 
<br>
Line 480: Line 503:
 
Created primers for Gibson assembly to create Hok/Sok +const_promoter::RBS::unstable RFP
 
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;">
 
Interlab Study
 
Interlab Study
−
<br>
+
<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>
 
replated last week’s constructs with GFP at lower concentration selected for chloramphenicol which was successful
 
replated last week’s constructs with GFP at lower concentration selected for chloramphenicol which was successful
 
<br>
 
<br>
Line 488: Line 516:
 
Miniprepped constructs and only promoter K08+GFP had the correct sequence
 
Miniprepped constructs and only promoter K08+GFP had the correct sequence
 
<br>
 
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
PCR
 
PCR
−
<br>
+
<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>
 
received high rated peltier units that did not break
 
received high rated peltier units that did not break
 
<br>
 
<br>
Line 510: Line 543:
 
<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>Week 10</b></a>
 
<b>Week 10</b></a>
−
<p style="font-size:24px;text-align:left;">
+
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
Hok-Sok
 
Hok-Sok
−
<br>
+
<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>
 
created construct with RFP and hok/sok
 
created construct with RFP and hok/sok
 
<br>
 
<br>
 
Pcr of the construct failed so the construct will be sequenced to check
 
Pcr of the construct failed so the construct will be sequenced to check
 
<br>
 
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
PCR /Gibson Check
 
PCR /Gibson Check
−
<br>
+
<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
 
performed a series of check in order to ensure that all the reagents are active
 
<br>
 
<br>
 
everything seems to be working
 
everything seems to be working
 
<br>
 
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
PCR machine
 
PCR machine
−
<br>
+
<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>
 
cycle data was taken and it exhibits consistency
 
cycle data was taken and it exhibits consistency
  
Line 537: Line 584:
 
<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>Week 11</b></a>
 
<b>Week 11</b></a>
−
<p style="font-size:24px;text-align:left;">
+
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
Hok/Sok
 
Hok/Sok
−
<br>
+
<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>
 
3A assembly of H/S + RFP failed
 
3A assembly of H/S + RFP failed
 
<br>
 
<br>
Line 552: Line 603:
 
Ran a PCR of unstable RFP using the  3 rxn buffers above  
 
Ran a PCR of unstable RFP using the  3 rxn buffers above  
 
<br>
 
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
Interlab Study
 
Interlab Study
−
<br>
+
<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>
 
used Phusion instead of Q5 for PCR  
 
used Phusion instead of Q5 for PCR  
 
<br>
 
<br>
Line 562: Line 618:
 
PSB1C3 - IL3 did not have bands in gel of appropriate size  
 
PSB1C3 - IL3 did not have bands in gel of appropriate size  
 
<br>
 
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
Lutein  
 
Lutein  
 
<br>
 
<br>
Line 568: Line 625:
 
ran a Gibson Assembly of E-cyclase/C3 and E-hydroxylase/C3 which produced colonies
 
ran a Gibson Assembly of E-cyclase/C3 and E-hydroxylase/C3 which produced colonies
 
<br>
 
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
PCR Machine
 
PCR Machine
−
<br>
+
<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>
 
rebuilt top of hair dryer housing w/ soda can  
 
rebuilt top of hair dryer housing w/ soda can  
 
<br>
 
<br>
Line 589: Line 651:
 
<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>Week 12</b></a>
 
<b>Week 12</b></a>
−
<p style="font-size:24px;text-align:left;">
+
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
Hok/Sok:
 
Hok/Sok:
−
<br>
+
<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>
 
Ran a Gibson Assembly of Hok/Sok + RFP
 
Ran a Gibson Assembly of Hok/Sok + RFP
 
<br>
 
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
Interlab
 
Interlab
−
<br>
+
<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>
 
worked on finishing the last construct
 
worked on finishing the last construct
 
<br>
 
<br>
Line 602: Line 673:
 
contacted W&M on possible collaboration for the last construct
 
contacted W&M on possible collaboration for the last construct
 
<br>
 
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
Lutein
 
Lutein
−
<br>
+
<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>
 
ordered primers for Gibson Assembly
 
ordered primers for Gibson Assembly
 
<br>
 
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
PCR
 
PCR
−
<br>
+
<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>
 
replaced old relays with higher powered relays
 
replaced old relays with higher powered relays
 
<br>
 
<br>
Line 624: Line 705:
 
<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>Week 13</b></a>
 
<b>Week 13</b></a>
−
<p style="font-size:24px;text-align:left;">
+
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
Hok/Sok
 
Hok/Sok
−
<br>
+
<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>
 
sequence of hok/sok construct created last week was incorrect  
 
sequence of hok/sok construct created last week was incorrect  
 
<br>
 
<br>
 
may put a unique RE site between hok/sok and rfp when retrying 3A assembly, RE cloning and Gibson
 
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;">
 
Interlab
 
Interlab
−
<br>
+
<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>
 
Gibsons of IL3 (K13 + GFP) have all failed
 
Gibsons of IL3 (K13 + GFP) have all failed
 
<br>
 
<br>
Line 639: Line 729:
 
IL2 was a little low in fluorescence, similar to the - control
 
IL2 was a little low in fluorescence, similar to the - control
 
<br>
 
<br>
 +
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
PCR
 
PCR
−
<br>
+
<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>
 
Rewired hair dryer and changed relays to handle higher wattage  
 
Rewired hair dryer and changed relays to handle higher wattage  
 
<br>
 
<br>
Line 662: Line 757:
 
<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>Week 14</b></a>
 
<b>Week 14</b></a>
−
<p style="font-size:24px;text-align:left;">
+
<p style="font-size:24px;text-align:left;text-decoration: underline;">
 
Hok/Sok
 
Hok/Sok
−
<br>
+
<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>
 
Began testing for RFP fluorescence for Hok/Sok effectiveness as a plasmid maintenance system
 
Began testing for RFP fluorescence for Hok/Sok effectiveness as a plasmid maintenance system
 
<br>
 
<br>
Line 679: Line 778:
 
Group F: H/S-RFP coding region; constitutive promoter ; chloramphenicol in liquid culture
 
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;">
 
Interlab:
 
Interlab:
−
<br>
+
<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>
 
Obtained the last construct from W&M  
 
Obtained the last construct from W&M  
 
<br>
 
<br>
Line 698: Line 802:
 
<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>Week 15</b></a>
 
<b>Week 15</b></a>
−
<p style="font-size:24px;text-align:left;">
+
<p style="font-size:24px;text-align:left;text-decoration: underline;">
  
 
Hok/Sok
 
Hok/Sok
−
<br>
+
<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>
 
Tested generations alpha beta and gamma in BL21 cells
 
Tested generations alpha beta and gamma in BL21 cells
 
<br>
 
<br>
Line 719: Line 827:
 
<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>Week 16</b></a>
 
<b>Week 16</b></a>
−
<p style="font-size:24px;text-align:left;">
+
<p style="font-size:24px;text-align:left;text-decoration: underline;">
  
 
Hok/Sok
 
Hok/Sok
−
<br>
+
<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>
 
Tested generation delta using DH5alpha
 
Tested generation delta using DH5alpha
 
<br>
 
<br>

Revision as of 09:30, 18 September 2015