Difference between revisions of "Team:UMaryland/Notebook"

Revision as of 03:13, 19 September 2015

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

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

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

@@ Line 256: / Line 256: @@
 <p style="font-size:32px;text-align:left;font-family:Verdana, Geneva, sans-serif;">
 <b>Week 1</b></a>
-<p style="font-size:24px;text-align:left;text-decoration: underline;">
-First week review
-<ul>
 <p style="font-size:24px;text-align:left;text-decoration: underline;">
 Project Preparations: Miraculin, SRNBC (Hok/Sok homolog), Lutein pathway genes
-<ul class="a">
+<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>