Difference between revisions of "Team:UMaryland/Notebook"

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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>- 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>

<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>

<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>

<a name="Week3">

<b>Week 3</b></a>

<p style="font-size:24px;text-align:left;text-decoration: underline;">

Miraculin

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<ul class="a">

  <li>-  Began work on Arduino code to cycle the machine</li>

<p style="font-size:24px;text-align:left;text-decoration: underline;">

Miraculin

  <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>

<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>

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<p style="font-size:24px;text-align:left;text-decoration: underline;">

  <li>-  burned out Peltier elements and broke temperature sensor, and ordered new ones   

<p style="font-size:24px;text-align:left;text-decoration: underline;">

Hok/Sok

  <li>- performed a 3A assembly of each promoter + GFP in pSB1K3 (GFP in pSB1A3 and promoters in pSB1C3) </li>

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<ul class="a">

  <li>- received new Peltier unit and temperature sensor

<p style="font-size:24px;text-align:left;text-decoration: underline;">

Hok/Sok

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<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

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<p style="font-size:24px;text-align:left;text-decoration: underline;">

 

<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;">

<ul class="a">

  <li>- designed relay configuration to both heat and cool Peltier's with an unidirectional current flow

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<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>

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<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>

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<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>

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<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>

 

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<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>

<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>

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<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>

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<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>

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<ul class="a">

<ul class="a">

  <li>-began work on incubator

  <li>-incubator was proven to maintain 37 degrees Celsius

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<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>

<p style="font-size:24px;text-align:left;text-decoration: underline;">

<ul class="a">

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

  <li>- contained groups A-E </li>