Difference between revisions of "Team:UMaryland/Notebook"

 
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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;">
−
First week review
+
Project Preparations: Miraculin, SRNBC (Hok/Sok homolog), Lutein pathway genes
 
<ul>
 
<ul>
−
<li>Cleaned the lab</li>
+
<li>- Prepared a bacterial culture containing constitutive GFP (K584001) to grow overnights (E. coli in LB with chloramphenicol)</li>
−
<li>moved in iGEM materials</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>Miniprep<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>analyzed sequences</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>prepared 3A assembly clone of pBAD-miraculin</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>
 
<ul>
−
<li>3A assembly</li>
+
<li>- Transformed β-cyclase, AppY, and CRTBEY genes (for lutein project) in bacteria to grow on plates overnight</li>
−
<li>two backbones</li>
+
<li>- Started a new 3A assembly for the SRNBC and constitutive GFP construct along with pBAD, constitutive colicinFy, and lysostaphin</li>
−
<li>pSB1C3</li>
+
<li>- Final products were SRNBC + constitutive GFP, SRNBC + constitutive colicinFy, and pBAD + lysostaphin</li>
−
<li>pBAD promoter + genes for miraculin</li>
+
<li>- Prepared overnights for the pBAD + miraculin construct that grew on the plates and the β-cyclase, AppY, and CRTBEY genes</li>
−
<li>cut out pBAD with 2 enzymes (EcoRI and SpeI) and cut out miraculin (XbaI and PstI)</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>want to put pBAD in front of miraculin</li>
+
<li>- Performed transformations on the SRNBC + constitutive GFP, SRNBC + constitutive colicinFy, and pBAD + lysostaphin digests and let them grow overnight</li>
−
<li>put into backbone w/ different antibiotic resistance than original backbone: pSB1A3 (means of selection)</li>
+
−
<li>plate had colonies<li>
+
−
 
+
−
<li>Human Practices<li>
+
−
Kevin got in contact w/ Bioacademy program @ Northwest High School
+
−
Trying to set up a talk for this Wednesday
+
−
Kevin + Adam + Dylan + Juhye going
+
−
goal to target various age groups
+
−
create game
+
−
fundamentals of genetics via presentations, arts, etc.
+
−
Conference scheduled for June 25th
+
−
4 teams confirmed: UVA, UMBC, Stonybrook, Duke
+
−
contact iGEM headquarters to inform them about meetup → Robert
+
−
<p class=MsoNormal>Kevin got in contact w/ <span class=SpellE>Bioacademy</span>
+
−
program @ Northwest High School <o:p></o:p></p>
+
−
 
+
−
<p class=MsoNormal>Trying to set up a talk for this <span class=SpellE><span
+
−
class=GramE>wednesday</span></span> <o:p></o:p></p>
+
−
 
+
−
<p class=MsoNormal>Kevin + Adam + Dylan + <span class=SpellE>Juhye</span> going
+
−
<o:p></o:p></p>
+
−
 
+
−
<p class=MsoNormal><span class=GramE>goal</span> to target various age groups <o:p></o:p></p>
+
−
 
+
−
<p class=MsoNormal><span class=GramE>create</span> game <o:p></o:p></p>
+
−
 
+
−
<p class=MsoNormal><span class=GramE>fundamentals</span> of genetics via
+
−
presentations, arts, etc. <o:p></o:p></p>
+
−
 
+
−
<p class=MsoNormal>Conference scheduled for June 25th <o:p></o:p></p>
+
−
 
+
−
<p class=MsoNormal>4 teams confirmed: UVA, UMBC, <span class=SpellE>Stonybrook</span>,
+
−
<span class=GramE>Duke</span> <o:p></o:p></p>
+
−
 
+
−
<p class=MsoNormal><span class=GramE>contact</span> <span class=SpellE>iGEM</span>
+
−
headquarters to inform them about meetup &#8594; Robert</p>
+
  
  
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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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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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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>
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   <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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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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   <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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PCR  
 
PCR  
 
<ul class="a">
 
<ul class="a">
−
   <li>- code made and proven to properly cycle machine  </li>
+
   <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>
 
</ul>
  
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<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>- cycle data was taken and it exhibits consistency </li>
 
   <li>- cycle data was taken and it exhibits consistency </li>
Line 558: Line 586:
 
<ul class="a">
 
<ul class="a">
 
   <li>- 3A assembly of H/S + RFP failed </li>
 
   <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>- ran a PCR of pSB1C3+H/S+RFP using 3 different rxn buffers  </li>
 
   <li>- HF rxn buffer </li>
 
   <li>- HF rxn buffer </li>
 
   <li>- GC rxn buffer </li>
 
   <li>- GC rxn buffer </li>
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   <li>- ran PCR for GFP 1, GFP 3, and IL1 which produced the correct sized products  </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>- Gibson Assembly of GFP + IL1 produced colonies </li>
−
   <li>- PSB1C3 - IL3 did not have bands in gel of appropriate size  </li>
+
   <li>- pSB1C3 - IL3 did not have bands in gel of appropriate size  </li>
  
 
</ul>
 
</ul>
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Lutein
 
Lutein
 
<ul class="a">
 
<ul class="a">
−
   <li>- RE digests of pLAC-RFP in PSB1C3 </li>
+
   <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>
 
   <li>- ran a Gibson Assembly of E-cyclase/C3 and E-hydroxylase/C3 which produced colonies  </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;">
−
PCR Machine
+
PCR  
 
<ul class="a">
 
<ul class="a">
 
   <li>- rebuilt top of hair dryer housing w/ soda can  </li>
 
   <li>- rebuilt top of hair dryer housing w/ soda can  </li>

Latest revision as of 23:57, 1 October 2015