Notebook + Progress
|Spring||Begun weekly meetings to discuss our project and begun talks with Berkley Lab to obtain the PhyB- Pif3 system.|
|June 8th-12th||Assay for the Extraction of PCB from Ecoli: Grew a Liquid Culture of a PCB producing Ecoli (ecoli transformed with SyA plasmid. Induced the culture with IPTG and 0.002% arabinose and incubated for 5 hours in the dark. Collected the ecoli supernatant and lysate and performed a chloroform extraction, all of which will be assayed for PCB.|
|June 15th-19th||Assay for the Extraction of PCB from Ecoli: Placed the three ecoli samples in the spectrometer to determine the concentration of PCB. However, the concentration was to low to read.
Creation of the PhyB-CFP and Pif-YFP Plasmids: Transformed the Berkley Labs Pif3 and PhyB plasmids into bacteria and plated to be used as stock. Performed PCR to amplify the PhyB, CFP, Pif3 and YFP sequences (PhyB and Pif3 obtained from the Berkley Plasmids). Begun Fusion PCR to obtain the Pif3-YFP and PhyB-CFP DNA. The Pif3-YFP sequence was obtained and digested with NotI and SacII and inserted into pRS313 (a backbone with a constitutive Myo2 promotor and His gene).
|June 22nd-26th||Creation of the PhyB-CFP and Pif-YFP Plasmids: The PhyB-CFP PCR product was obtained at the end of this week and was inserted into pRS313. The localization sequence primers arrived and PRC to obtain and amplify these sequences from the bacterial genome was begun. Initial PCR resulted in the amplification of Nyv1(part of Longin localization sequence which localizes to the Vacuole) and Msp3 (a nuclear localization sequence). Esc1 (a nuclear localization sequence), SncI (the second part of the Longin sequence) and Ras2 (a plasma localization sequence) were also obtained through separate PCRs. Full oligos were ordered for Bcl2 (a mitochondrial localization sequence)as it is 66bp long, a Kinase reaction was performed to anneal the two single stranded oligos. Finally the NyvI-SncI fusion was constructed and amplified through PCR. The Mas70p sequence failed to amplify and was dropped. Note: All PCRs used Phusion Enzyme.|
|June 29th - July 3rd||Creation of the PhyB-CFP and Pif-YFP Plasmids:The PhyB-CFP and Pif3-YFP plasmids were transformed into ecoli which were subsequently mini-preped.
Insertion of the Localization sequences into the PhyB-CFP and Pif3-YFP Plasmids: Digestion, Ligation and cloning of the localization sequences and PhyB-CFP and Pif3-YFP Plasmids was begun. The Msp3 and Longin sequences were destined for insertion into the N terminus of the Pif3-YFP sequence and the Ras2, Esc1 and Bcl-2 destined for the C terminus of the PhyB-CFP plasmid. The digestion enzymes Asc1 and Pac1 failed to cut multiple times.
|July 6th-10th||Insertion of the Localization sequences into the PhyB-CFP and Pif3-YFP Plasmids: The Msp3-Pif3-YFP, Longin-Pif3-YFP, Ras2-PhyB-CFP and Bcl-2-PhyB-CFP plasmids were obtained after overnight digestion and ligation. Esc1 failed to clone.
Observation of localization of Pif3 and PhyB in Yeast: Each of the four constructs (Msp3-Pif3-YFP, Longin-Pif3-YFP, Ras2-PhyB-CFP and Bcl-2-PhyB-CFP) was individually transformed into His(-) yeast.