Difference between revisions of "Team:UMaryland/Notebook"

   <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>

 

   <li>-  ordered pAC-LYC which encodes 3 enzymes from E. Herbicola to produce basal levels of lycopene </li>

 

   <li>- code made and proven to properly cycle machine </li>

   <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>

   <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>

 

   <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>

Hok-Sok:

   <li>- created construct with RFP and hok/sok </li>

   <li>- Pcr of the construct failed so the construct will be sequenced to check </li>

 

 

PCR machine:

   <li>- cycle data was taken and it exhibits consistency </li>

Hok/Sok:

   <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 w/ DMSO which yielded product </li>

   <li>- Ran a PCR of unstable RFP using the  3 rxn buffers above  </li>

 

   <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>

 

 

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>

 

   <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>

   <li>- Ran a Gibson Assembly of Hok/Sok + RFP </li>

   <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>

   <li>- ordered primers for Gibson Assembly </li>

   <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>

Hok/Sok:

   <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>

Interlab:

   <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>

   <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>- 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 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>

   <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>

   <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>

   <li>- Tested generation delta using DH5alpha </li>

   <li>- contained groups A-E </li>