Team:UCSF/Jimmy Huang Notebook

JIMMY HUANG's UCSF Notebook
SubTeam: Regional Silencing

Overall Objective 1: Demonstrate that we can silence two or more genes with one operator.

Overall Objective 2: Create a ‘Stay Off’ system with Gal80.

Milestones 1: Regional Silence

1)Create 4 different directional variations of RFP and GFP reporters with a 8xLexAOps operator in the, plasmid/constructs.

2)Test for Silencing of both fluorescent reporters.

Milestones 2: BAH Hypermorphs

1) Create variant of BAH for Andrew.

Milestones 3: Gal80 1.0/2.0/3.0

1)Create Left Homology-LexAOps-GFP-NatRCassette-Right Homology.

2)Create LexAOps-Cyc1P-GFP at 3’ end of Gal80 -> Test (Failed)

3)Create Gal80 cassette, endo and adh1t versions, with operator sites in LEU plasmid.

4)Integrate into Yeast with LexA-Sir2-mCherry(his) AH81.

5)Screen for mCherry expression

6)Screen pAH32 URA for GFP expression

Week 1: 6/16-20

1.Help others with their experiments

2.Amplification of TdTomato, mCherry, pRS306.

Week 2: 6/23-27

1.Prepared parts for experiment.

2.PCR out TdTomato/mCherry with specific ends for ligations.

3.Ligate TdTomato/mCherry into respective vectors.

4.Quick Change BAH for Hypermorph Version.

Week 3: 6/30-7/4

1.Sequencing and Analysis of transformants: TdTomato/mCherry.

2.All failed, repeat PCRs.

3.Sequencing and Analysis of transformants: BAH Hypermorphs

4.Success, frozen.

Week 4: 7/7-11

1.PCRs all failed for TdTomato/mCherry.

2.Repeat. Repeat. Repeat.

3.Sequencing and Analysis of TdTomato/mCherry.

4.PCR for Gal80 1.0

5.Amplification of Gal80 1.0 materials.

Week 5: 7/14-18

1.Ligations for BAH shuffle.

2.Ligations for TdTomato/mCherry repeats.

3.Ligations for Gal80 EM2 right/left homology cassette.

4.PCR mCherry/TdTomato w/ proper restriction sites.

5.Integrate Gal10P-LexA-Sir2 into SF992.

Week 6: 7/21-25

1.PCR TdTomato/mCherry solved, Elongase was destroyed. All 3 tubes.

2.Drop TdTomato because it’s a double mCherry. Causes sequencing headache.

3.Ligated, Amplified, Digested proper mCherry cassette.

4.Quick Change XCM1 site out of mCherry for Yeast Integration.

5.Create LexA-Sir-mCherry SF992 Strain.

6.Test on FACs for mCherry expression.

Week 7: 7/28-8/1

1.Test Regional Silence constructs of mCherry for directionality via digests.

2.Ligate 8xLexAOps into confirmed directional constructs.

3.Amplify mCherry plasmid and prepare for Integration into Gal10P-LexA-Sir2.

4.Integrate Gal80 1.0 into LexA-Sir2-mCherry strain to test for ‘Stay Off’

5.Gal80 1.0 failed. Start picking up Alex’s Gal80 2.0

Week 8: 8/4-8

1.Integrate mCherry construct for Regional Silence into Gal10P-LexA-Sir2

2.In preparation for Regional Silence FACs run, plate onto 2% Gal plates.

3.Ligate broken ended 8xLexAOps into pAH32.

4.Ran FACs on mCherry SAC restriction sites.

5.Ligate 8xLexAOps into KPN restriction sites of mCherry cassette.

Week 9: 8/11-15

1.Analyze FACs data for SAC ends mCherry cassette in Regional Silence (Beautiful)

2.Hypermorph BAH AarI shuffle ligations and transformations.

3.Integrate KPN restriction sites of mCherry cassette into Gal10P-LexA-Sir2.

4.Pick up Gal80 2.0 from Alex. He is leaving.

5.Prepare KPN mCherry integrations for FACs, plate on 2% Gal.

Week 10: 8/18-22

1.Picked up Gal80 2.0 from Alex. Begin creating constructs he began.

2.Regional Silence 2/3rds done, data is beautiful. Can conclude with completion of KPN version of mCherry cassette.

3.Digest + Ligate Alex’s pAH28 to fix bad Gal80 in another construct/plasmid.

Week 11: 8/25-29

1.Amplification, Digestion, Ligation of Gal80 fragment to create desired construct.

2.Create pRS304 plasmid with PspOMI restriction sites of LexAOps.

3.Gal80 Fragment showing incorrect Gel resolutions.

4.Confirm by asking Alex about his part.

5.Also confirm by sequencing.

Week 12: 9/1-5

1.Yeast Colony PCR to confirm presence of KPN mCherry construct. Negative.

2.Re-do integration of KPN mCherry construct into Gal10P-LexAOps.

3.Meanwhile, sequencing KPN mCherry construct to be doubly sure.

4.Sequencing of Gal80 fragment from pAN28 confirmed that the desired fragment is not present. Must re-do from scratch; pull Gal80 from Yeast Genomic Template.

Week 13: 9/8-12

1.PCR, Digestion, Gel Extraction for Gal80. Multiple Failures. Big fragment hard to get. Must confirm by Topo Cloning and Sequencing.

2.KPN mCherry construct integrated into Gal10P-LexA-Sir2.

3.Ran FACs, showed both GFP and RFP present in Minus Gal. Shut off in presence of Gal.

Week 14: 9/15-19

1.Gal80 Endogenous fragment PCRs and Sequencing. Very hard to get from YGD.

2.Analysis of everything. Regional Silence constructs, Gal80 1.0/2.0

3.Completed Regional Silence.

4.Gal80 Stay Off, did not fully complete. Although it did show ways that it wouldn’t work.