Team:UCSF/Willis Wong Notebook

= Willis Wong =

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.