Difference between revisions of "Team:UMaryland/Notebook"

Revision as of 03:34, 19 September 2015

Week 1

Project Preparations: Miraculin, SRNBC (Hok/Sok homolog), Lutein pathway genes

- Prepared a bacterial culture containing constitutive GFP (K584001) to grow overnights (E. coli in LB with chloramphenicol)
- 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
- Performed a mini-prep on our overnights with the constitutive GFP in order to isolate plasmids and verify that they were present through spectrophotometry
- 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
- 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

- Transformed β-cyclase, AppY, and CRTBEY genes (for lutein project) in bacteria to grow on plates overnight
- Started a new 3A assembly for the SRNBC and constitutive GFP construct along with pBAD, constitutive colicinFy, and lysostaphin
- Final products were SRNBC + constitutive GFP, SRNBC + constitutive colicinFy, and pBAD + lysostaphin
- Prepared overnights for the pBAD + miraculin construct that grew on the plates and the β-cyclase, AppY, and CRTBEY genes
- 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
- Performed transformations on the SRNBC + constitutive GFP, SRNBC + constitutive colicinFy, and pBAD + lysostaphin digests and let them grow overnight

Week 2

Miraculin

- 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
- Ran gel with all parts and cut out the bands
- Mini-prepped SRNBC + constitutive colicinFy and shipped them off to be sequenced
- Performed gel purification on gels from day before but they failed; they didn’t show up in the spectrophotometer
- Performed minipreps on pBAD+Miraculin in the PSB1C3 backbone as well as the const. GFP+SRNBC
- Performed RE digests on the previous mini-preps: EcoRI and PstI on pBAD + Miraculin, and XBa1 and Pst1 on SRNBC + Constitutive GFP

Designing gBlocks

- Designed gblocks for epsilon-cyclase (from arabidopsis), pyocin and Hok/Sok, and ordered them

Week 3

Miraculin

- Sequence of pBAD + Miraculin confirmed through sequencing
- Culture of pBAD + Miraculin was induced with 0.1% arabinose at OD of 1
- 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)
- Ran the resulting 62 elutions through an SDS-PAGE
- Failed to extract Miraculin using French press, FPLC and SDS-Page
- Made overnights of pBAD + Miraculin culture to prepare for test inductions

SRNBC and Hok/Sok

- Performed a gel extraction of SRNBC, const. GFP, and pBAD which we then ligated them together to make the SRNBC construct
- Performed transformations of the constitutive GFP + SRNBC construct
- Transformations of SRNBC + Constitutive GFP failed
- Performed PCR on the PSB1C3 backbone in order to amplify it in preparation for a Gibson Assembly with the Hok/Sok gene

PCR

- Began work on Arduino code to cycle the machine
- Purchased Peltier units and lm35 temperature sensors

@@ Line 345: / Line 345: @@
 <b>Week 4</b></a>
 <p style="font-size:24px;text-align:left;text-decoration: underline;">
-Miraculin
+  <li>- Made overnights of pBAD+Miraculin culture to prepare for test inductions tomorrow</li>
-<ul class="a">
+   <li>- Inoculated 3 test cultures of pBAD+Miraculin in LB broth with Ampicillin</li>
-   <li>- induced 3 test cultures with  0%, 0.05%, 0.1%, 0.2% arabinose with no significant results </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>- will perform procedure again using bl21 instead of Dh5a  </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>
 <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>- 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>- Purchased an AC/DC power converter
 </li>
 </ul>