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Team:UCSF/Willis Wong Notebook
From 2008.igem.org
Willis Wong
Notebook
Overall Objective
Devise an array of inputs that can be linked to regulation of silencing.
Milestones
1. Define minimal fragment for light inducible dimerization.
2. Define the configuration of dimerization/regulatory domains that will yield desired function. (Silencing/Unsilencing)
Work Done
Week 1-2: Design AB and BD parts for combinatorial cloning (includes PhyB and Pif3 parts).
- Designed primers with their respective A/B or B/D ends to each of the genes.
- PCR'ed genes from Saccharomyces cerevisiae genome.
- TOPO cloned.
- Selected clones for minipreps, submitted for sequencing.
- Identified good clones from sequence alignments.
Week 3-4: Ligate AB + BD parts into 315 vectors.
- Aar1 digests of good topo clones and 315 high copy vectors.
- Ligation of parts into 315 high copy vectors.
- Transformation into E. coli.
- Test digest and ran diagnostic gels.
- Sent for sequencing.
Week 4-5: Subclone Pif3 constructs into 303 vectors for intregration into a yeast reporter strain [B3(H)].
- Digestion of Pif3 constructs and 303 integration vector for subcloning into 303 integration vectors.
- Ligation of cut Pif3 constructs into cut 303 integration vectors.
- Integration of 303 vectors with Pif3 constructs into GFP reporter strain B3(H).
Week 7-9: Transform the PhyB constructs into the yeast strains that were integrated with Pif3.
- Transform each strain with Pif3 constructs with PhyB constructs in 315 vectors.
Week 10-11: In the dark, added PCB, pulsed with respective light, ran FACs.
- During dilution in the morning, add the PCB in the dark. Pulse with 720nm light for about two minutes to separate PhyB and Pif3 constructs.
- Pulse with 660nm light for about two minutes to bring together the Pif3 and PhyB constructs.
- Pulse again wit 660nm light every two hours as PhyB and Pif3 separate in roughly three hours.
- Run FACs after growth.
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