Team:UCSF/Ryan Quan Notebook

Subteam: Silencing

Overall Objective: The objective of the silencing team was to create yeast constructs containing different promoters driving the expression of Sir2 production and a reporter (GFP). Using these constructs, we wanted to see the minimal requirement of Sir2 production needed to silence a gene Rationale: Our main goal was to generate a response in our yeast that yielded a “poised” silencing state that could be altered by various inputs – that is, a yeast strain in which the target gene was neither silenced nor activated.

Milestones: To accomplish all of this, we first had to: 1)	Create the different promoter/silencer and promoter/reporter combinations in bacteria. 2)	Transform all of the possible combinations into yeast strains. 3)  Test all the yeast strains we constructed for silencing.

Week 1 June 18, 2008 ~ June 20, 2008
Verified Sequences for Ssn8 gene in TOPO driven by Cyc1p and Adh1p (IG20 or IG21 both 100%, IG23 99%)

Ran FACs for yeast strains with GFP

Integrated Cyc1p driving GFP from the 5’ end into strains containing Sir2 production driven by different promoters

Week 2 June 23, 2008 ~ June 27, 2008
-Prepared FACs block for iGEM pre-project

-Ran FACs for iGem pre-project

-Prepared FACs block

-Xho1/Not1 Digests

-Intergrated Adh1p and Cyc1p driving GFP from the 3’ end into strains containing Sir2 production driven by different promoters

-Verified Sir3 and Sir4 sequences that Alex cloned (Sir3-2 and Sir4-3)

-Aar1 digests of Sir3-2 and Sir4-3

-Ran gel and purified Sir3-2 and Sir4-3 fragments

-Aar1 digested Sir3-2 again with more precise ratios of DNA to enzyme

-Re-ran and gel purified Sir3-2

-Prepared iGem pre-project FACs block

Week 3 June 30, 2008 ~ July 4, 2008
-Integrated Fig1p driving GFP from the 3’ end into strains containing Sir2 production driven by different promoters

-Ligated Sir3-2 into 315 vectors with Cyc1p and Adh1p promoters and transformed into bacteria

-Set up colony PCR to check Sir3-2 and Sir4-3 fragments

-Integrated Gal10p driving GFP into strains containing Sir2 production driven by different promoters

-Ran colony PCR on a gel (none looked ok)

-Re-colony PCR’d for Sir3-2 and Sir4-3

-Set up FACs block for iGEM pre-project

-Ran gel for colony PCR and chose one Sir3-2 cyc1p and one Sir3-2 Adh1p that was good

-Nhe1 digests

-Ran FACs for iGEM pre-project

-Set up PCR to check Sir3-2 in 315 vectors

-Ran gel to check PCR of Sir3-2 in 315 vecotr (all looked good)

-Set up digets of Sir4-3 in TOPO

-Set up Aar1 digets for Sir4-3

-Integrated Gal1 driving GFP from the 3’ end into strains containing Sir2 production driven by different promoters

-Ran Gel for Aar1 digests of Sir4-3 and gel purified

Week 4 July 7, 2008 ~ July 11, 2008
-Ligated Sir4-3 fragment into 315 vectors with Cyc1p and Adh1p promoters and transformed into bacteria

-Set up colony PCR for Sir4-3 in 315 vectors -Transformed Ssn8 and Sir3-2 in 315 vectors driven by Cyc1p and Adh1p into an assay strain from Andrew

-Ran Gel for Sir4-3 colony PCR and chose two clones of Sir4-3 Adh1p that were good

-Set up colony PCR for Sir4-3 Cyc1p

-Ran Gel for colony PCR ( no good clones so I decided to re-ligate Sir4-3 into the 315 vectors)

-Ligated Sir4-3 into 315 vectors and transformed into bacteria

-Set up colony PCR for Sir4-3 ligations

-Ran colony PCR on a gel (none worked so I decided to start again with the Sir4-3 TOPOs and re-cut)

-Set up digests for Sir4-3 in TOPO

-Aar1 digests 315 vector containing Cyc1p

-Aar1 digests for Sir4-3 in TOPO

Week 5 July 14, 2008 ~ July 18, 2008
-Ran gel for Sir4-3 digests and 315 Cyc1p vector and gel purified

-Set up FACs in tubes for constitutively driven Sir2 production vs constitutively driven GFP

-Set up ligations for Sir4-3 in 315 Cyc1p and transformed into bacteria

-Re-did digests for Sir4-3 and 315 Cyc1p vector (to try and get better yields)

-Set up Colony PCR for Sir4-3 in 315 Cyc1p (older ones)

-Set up FACs block for iGEM pre-project

-Ran FACs for samples in tubes

-Ran gel for colony PCRs (none worked)

-Ran gel and gel purified Sir4-3 and 315 Cyc1p digests

-Ligated Sir4-3 into 315 Cyc1p vector and transformed into bacteria

-Set up colony PCR for ligations

-Set up Aar1 digests for Sir2 and mCherry AB BD parts and also 305 vector with Gal1p promoter

-Transformed Sir3-2 in 315 vectors with Cyc1p and Adh1p promoters into our two best strains (Adh1p driving Sir2 production with Cyc1p driving GFP from the 5’ end and Adh1p driving Sir2 production with Adh1p driving GFP from the 3’ end)

-Set up iGEM FACs block

-Ran gel for colony PCR of Sir4-3 in 315 Cyc1p (none worked)

-Set up test digests for Sir4-3 in 315 Cyc1p

-Ran gel for tests digests (some possibly working clones)

-Ligated the Sir2 and mCherry AB BD parts into the 315 vector with Gal1p promoter and transformed into bacteria

-Suspected 315 vectors to be the wrong ones so I test digested them all

-Ran FACs for iGEM pre-project (Ssn8 and Sir3 seemed to improve silencing)

-Ran gel for test digests of 315 vectors

-Transformed Sir3-2 in Cyc1p and Adh1p and Ssn8 in Cyc1p and Adh1p vectors into our two top silencing strains (Adh1p driving Sir2 production with Cyc1p driving GFP from the 5’ end and Adh1p driving Sir2 production with Adh1p driving GFP from the 3’ end)

-Sent in vectors for sequencing

Week 6 July 21, 2008 ~ July 25, 2008
-Transformed Sir2/mCherry constructs in 305 Gal1p vectors into our two top silencing strains (Adh1p driving Sir2 production with Cyc1p driving GFP from the 5’ end and Adh1p driving Sir2 production with Adh1p driving GFP from the 3’ end)

-Set up cultures for FACs for Gal80 stuff

-Set up digests for Alex’s Gal80 construct

-Set up digests for Sir2/mCherry in 305 vector (forgot to digest when I did the transformations before)

-Ran gel for Alex’s Gal80 construct

-Transformed Sir2/mCherry in 305 vector into our two top strains (Adh1p driving Sir2 production with Cyc1p driving GFP from the 5’ end and Adh1p driving Sir2 production with Adh1p driving GFP from the 3’ end)

-Set up digests for Noah’s plasmid with his terminator for a synthetic Gal80 we wanted to make

-Ran gel for digests of Noah’s plasmid (looked good)

-Gel purified digests of Noah’s plasmid

-Set up tubes for FACs for Ssn8, Sir3 and Sir4 in 315 vectors all in one of our top silencing strains (Adh1p driving Sir2 production with Cyc1p driving GFP from the 5’ end)

-Ligated Adh1p into one of Noah’s plasmids and Cyc1p into one of Noah’s plasmids

-Digested out Cyc1p! and Adh1p! out of Noah’s plasmids

-Ran FACs for Ssn8, Sir3 and Sir4 in 315 vectors all in one of our top silencing strains (Adh1p driving Sir2 production with Cyc1p driving GFP from the 5’ end) Ssn8 seemed to work the best as a modifier

Week 7 July 28, 2008 ~ August 1, 2008
-Ran Cyc1p! and Adh1p! on a gel and gel purified

-Set up FACs blocks to test modifiers

-Ligated Sir3 and Sir4 into Adh1p! and transformed into bacteria

-Set up Colony PCR for Sir3 and Sir4 in Adh1p!

-Set up digests for Sir2/mCherry in 305 vectors

-Ran FACs for modifiers

-Transformed Sir2/mCherry in 305 vector into our two top strains (Adh1p driving Sir2 production with Cyc1p driving GFP from the 5’ end and Adh1p driving Sir2 production with Adh1p driving GFP from the 3’ end)

-Alex ran gel for my colony PCRs of Sir3 and Sir4 in Adh1p! (Sir4 worked but I used the wrong primer for Sir3)

-Set up test PCR for Sir3

-Ran gel for Sir3 PCR tests ( all worked)

-Set up FACs block for modifiers

-Ligated Sir3 and Sir4 into Cyc1p!

-Set up colony PCR for Sir3 and Sir4 in Cyc1p!

-Ran FACs for modifiers

-Ran gel for colony PCR of Sir3 and Sir4 in Cyc1p (chose tow clones to move on with)

-TOPO cloned Gal80 grafment with a HA tag

-Transformed Sir3 Cyc1p!, Sir3 Adh1p!, Sir4 Cyc1p! and Sir4 Adh1p! into yeast

Week 8 August 4, 2008 ~ August 8, 2008
-Transformed Sir3 Adh1p, Sir3 Adh1p!, Sir4 Adh1p! and Ssn8 Adh1p into our two top silencing strains (Adh1p driving Sir2 production with Cyc1p driving GFP from the 5’ end and Adh1p driving Sir2 production with Adh1p driving GFP from the 3’ end)

-Analyzed FACs data and concluded:

-Sir3, Sir4 and Ssn8 seemed to improve silencing

-I will use Sir3, Sir4 and Ssn8 to test in Adh1p driving Sir2 production with Adh1p driving GFP from the 3’ end and Adh1p driving Sir2 production with Fig1p driving GFP from the 5’ end

-I will re-transform Sir3, Sir4 and Ssn8 all at the same time

-I will test in tubes for a cleaner experiment

-Set up digests for Adh1p/Cyc1p + Gal80 + Noah’s terminator

-Ran gel for digests of Adh1p/Cyc1p + Gal80 + Noah’s terminator (all worked except Adh1p + Gal80 + Noah’s terminator)

-Set up digests for Adh1p/Cyc1p + Gal80 + Noah’s terminator

-Set up FACs cultures for Sir3 Adh1p, Sir3 Adh1p!, Sir4 Adh1p!, Ssn8 Adh1p all in two of our silencing strains (Adh1p driving Sir2 production with Adh1p driving GFP from the 3’ end and Adh1p driving Sir2 production with Fig1p driving GFP from the 5’ end)

-Ran gel for digest of Adh1p/Cyc1p + Gal80 + Noah’s terminator (didn’t seem to cut well, decided to try different enzymes)

-Set up FACs block for Sir2/mCherry in 305 vectors

-Ligated Cyc1p + Gal80 + Noahs terminator into Alex’s PAN028 vector and Alex transformed them

-Set up digest for Adh1p + Gal80 + Noah’s terminator with different enzymes this time

-Prepared FACs plate for Sir3 Adh1p, Sir3 Adh1p!, Sir4 Adh1p!, Ssn8 Adh1p all in two of our silencing strains (Adh1p driving Sir2 production with Adh1p driving GFP from the 3’ end and Adh1p driving Sir2 production with Fig1p driving GFP from the 5’ end)

Week 9 August 11, 2008 ~ August 15, 2008
-Sent in sequencing for Adh1p/Cyc1p + Gal80 + Noah’d Terminator in TOPO

-Ligated Adh1p + Gal80 + Noah’s Terminator into Alex’s vector PAN028 and transformed into bacteria

-Set up cultures for FACs for Sir3 Adh1p, Sir3 Adh1p!, Sir4 Adh1p!, Ssn8 Adh1p all in two of our silencing strains (Adh1p driving Sir2 production with Adh1p driving GFP from the 3’ end and Adh1p driving Sir2 production with Fig1p driving GFP from the 5’ end) because last time the FACs machine had problems so I couldn’t run them

-Set up test digests for Adh1p/Cyc1p + Gal80 + Noah’s Terminator with HA tag

-Ran gel for test digests (all worked)

-Ran gel to purify Adh1p/Cyc1p + Gal80 + Noah’s Terminator with Kpn1 ends

-Ran FACs for Sir3 Adh1p, Sir3 Adh1p!, Sir4 Adh1p!, Ssn8 Adh1p all in two of our silencing strains (Adh1p driving Sir2 production with Adh1p driving GFP from the 3’ end and Adh1p driving Sir2 production with Fig1p driving GFP from the 5’ end) and got weird scatter, needed to run again

-Set up FACs for Sir3 Adh1p, Sir3 Adh1p!, Sir4 Adh1p!, Ssn8 Adh1p all in two of our silencing strains (Adh1p driving Sir2 production with Adh1p driving GFP from the 3’ end and Adh1p driving Sir2 production with Fig1p driving GFP from the 5’ end)

-Set up FACs plate for Sir3 Adh1p, Sir3 Adh1p!, Sir4 Adh1p!, Ssn8 Adh1p all in two of our silencing strains (Adh1p driving Sir2 production with Adh1p driving GFP from the 3’ end and Adh1p driving Sir2 production with Fig1p driving GFP from the 5’ end)

-Froze yeast strains (refer to Alex’s notebook for strains)