Team:UCSF/Alex Ng Notebook

Alex Ng’s Notebook Week one 5/27 – 5/30

•	Obtained plasmids that had necessary parts for constructing the matrix

•	Digested plasmids and purified the parts we needed (promoters, silencers, and reporters)

•	PCRed a KPN1 flanked 8xLexA operator repeat

•	Ligated and transformed all combinations of promoter/silencers and promoters/operator sites with GFP

•	Ligated pAH32 with 8xLexAops

•	Mini-prepped and test digested a few of the ligations and kept the ones that worked

•	PCRed modifiers the high school students couldn’t get. Using Aar1 stitch method (failed, not enough cycles)

-	reran with 26 cycles (failed)

-	reran with 30 cycles (most worked, verified on gel), purified the ones that did work

Week two 6/2 - 6/6

•	Finished the test digestions of the mini-prepped plasmids done last week (only a couple transformations didn’t work)

-	Ligated and transformed the ones that didn’t work

•	TOPO cloned parts for the Aar1 stitch (modifiers)

•	Mini-prepped transformations and test digested.

•	PCRed the rest of the modifiers that didn’t work last week (Sir 1 5’, Sir3 3’, Sir4 5’, and Sir4 3’)

-	Used different method this time. Used Phusion PCR and HiFi buffer and GC buffer. (two different reactions)

•	Digested most reporter plasmids (promoter w/ 5’ or 3’ operator site and GFP) for yeast transformation.

-	Transformed 8 out of 12 reporters into yeast (sv992 strain)

-	The ones not transformed are: Cyc1p w/ 5’ ops and GFP, Gal10p w/ 5’ ops and GFP, Adh1p w/ 3’ ops and GFP, Gal10p w/ 3’ ops and GFP.

•	Mini-prepped TOPO clones and sent for sequencing

•	Digested plasmid with Kpn1 to purify Gal 80 (failed because Gal80 contains a Kpn1 site)

-	Quick Change PCR of Gal 80 to delete the KPN1 site.

•	Yeast transformations from earlier in the week worked except for Prm2p with 3’ops GFP

-	Restreaked the ones that did work

Week three 6/9 – 6/13

•	Took out yeast restreaks from last week

•	DPN treated the Gal80 Kpn1 delete quick change PCR

-	Transformed into bacteria after it was finished.

•	Transformed 315 Adh and 315 Cyc plasmids for amplification

•	Looked at sequencing for Aar1 stitch of modifiers

-	All had mutations but going ahead with a select few that may be okay. (Sas3, Dot4, Sas1, and Set1)

-	Digested the ones that we’re continuing with using Aar1

•	Did yeast transformations

-	Transformed: Cyc1p w/ 5’ ops and GFP, Gal10p w/ 5’ops and GFP, Prm2p w/ 3’ ops and GFP, Adh1p w/ 3’ ops and GFP, Gal10p w/ 3’ops and GFP

-	Looked good

•	Set up a block to run on FACS (reporters only)

•	Digested vectors (315 cyc and 315 adh) with Aar1

-	Purified and digested with Xma1

•	Ran FACS

-	All were done in sRaf Comp media except for the ones w/ Gal promoters. Added Gal to the ones with the Gal promoters.

-	Added alpha factor to the ones with alpha factor inducible promoters (Prm2p and Fig1p)

•	Digested plasmid with Kpn1 (to obtain Gal80 with the Kpn1 site delete.)

-	Gel purified

•	PCRed Sir1, Sir2, and Sir3

-	Template was ordered from a company.

-	Sir1 did not work, going to continue without it. Sir3 and Sir4 worked.

-	TOPO cloned Sir3 and Sir4

•	Ligated Dot4, Sas3, Set1, and Sas1 into the 315Adh and 315Cyc vectors. (Aar1 stitch)

•	Ligated Gal80 into a plasmid with Cyc1p w/ 5’ ops and GFP

•	Did yeast transformations

-	All reporter plasmids were transformed into yeast (cb008 strain) except for the ones using Prm2p. We are continuing the project without this promoter because it is too weak.

Week four 6/16 – 6/20

•	Mini-prepped sir3 and sir4

•	Ran Phusion PCR for Gal80 knockout

•	Sent in Sir3 and Sir4 for sequencing

•	Set up for FACS

-	Reporters in sv992 (left out Prm2p and Fig1p) and cb008 (left out Prm2p)

-	Ran five colonies of each construct.

-	The ones with Gal inducible constructs didn’t work because I grew in the wrong media (Grew in SDcomp instead of SRaf +Gal)

-	Picked three colonies for each construct labeled L,M, and H. (low, medium, and high expressers)

•	Transformed Gal80 knockout PCR into yeast (sv992 strain)

-	First plated on YPD, then replica plated on to NAT plates. •	Transformed silencer plasmids into yeast (cb008) with Cyc1p w/ 5’ops and GFP low, medium, and high expressers and Adh1p w/ 5’ops and GFP low, medium, and high expressers. (total 60 transformations, 10 plasmids going into 6 different yeast strains)

-	Retransformed the plasmids that I used for the yeast transformation to amplify.

Week five 6/23 – 6/27

•	Mini-prepped cultures for plasmid amplification from last week

•	Restreaked single colonies of Gal80 KO in sv992

•	Streaked for single colonies for the 60 transformations done last week. (silencers into reporters with 5’ops and GFP using the Cyc1p and Adh1p promoter)

•	Ran FACS for Gal inducible promoters

•	Streaked single colonies from the restreaks of the 60 transformations done earlier

•	Did yeast transformations (got help from Ryan, Cathy, and Jacinto)

-	Transformed all silencing constructs into yeast strains with Cyc1p w/ 3’ops and GFP in cb008 and Adh1p w/ 3’ops and GFP in cb008 (low, medium, and high for both). Total 60 transformations. (10 constructs into 6 strains)

-	I accidentally threw away 20 of the transformations because I thought they didn’t work, but they were just growing slower than usual. I need to retransform th e ones I threw away.

•	Ryan is taking over Sir3 and Sir4 cloning project along with 315 Adh1p Sas3, 315 Adh1p Sas1, and 315 Cyc1p Sas1

•	Colony PCR checked for Gal80 KO (checked 8 colonies) (all worked)

Week six 6/30 – 7/4

•	Restreaked yeast transformations from last week.

•	Did yeast transformations

-	Transformed all silencing constructs into Fig1p w/ 5’ops and GFP in cb008 and Fig1p w/ 3’ops and GFP in cb008 (total 60 transformations, Ryan helped)

•	Ran FACS

-	Gal1p driving 5’ops GFP in sv992 and Gal10p driving 5’ops GFP in cb008 (tested each under three different galactose conditions. One that was forever induced with Gal, another that was induced overnight with Gal, and the last was induced in the morning with Gal.)

•	Did more yeast transformations

-	Did the ones that I accidentally threw away last time.

-	Also put Gal80 gene with Kpn1 site delete into the Gal80 KO strain in sv992

•	Did even more yeast transformations

-	Put all silencer constructs into yeast with Gal1p driving 3’ops with GFP in cb008 and Gal10p driving 3’ops with GFP in cb008

Week seven 7/7 – 7/11

•	Restreaked all the yeast transformations that were done last week.

•	Colony PCR checked the Gal80 transformation (Gal80 into Gal80 KO sv992)

•	Ran FACS (in block)

-	Cyc1p (silencer) vs. Cyc1p (reporter), Adh1p vs. Cyc1p, Adh1p vs. Adh1p, Cyc1p vs. Adh1p, Gal10 vs. Cyc1p, Gal1p vs. Cyc1p

•	Ran more FACS (in block)

-	Cyc1p (silencer) vs. Fig1p (reporter), Adh1p (silencer) vs. Fig1p (reporter),

•	Ran more FACS (in tubes)

-	ADh1p (silencer) vs. Cyc1p (reporter), Cyc1p vs. Adh1p, Cyc1p vs. Cyc1p, Gal1p vs. Cyc1p, Gal10p vs. Cyc1p, Gal1p vs. Adh1p, Gal10p vs. Adh1p

-	Grown in wrong growth media (grown in YPD), it might effect silencing because of the starvation condition and NAD+ concentration

-	Reran in correct media.

-	Ran one colony in three different medias (SRaf, SDcomp, and YPD) to see its effects on silencing.

•	Did yeast transformations

-	Gal10p LexA-Sir contruct into Gal80 KO in sv992 with Gal80 gene with Kpn1 site delete

Week eight 7/14 – 7/18

•	Restreaked yeast transformation from last week (Gal10p driven silencer construct into Gal80KO in sv992 with Gal80 gene with Kpn1 site delete.)

-	Restreaked onto plus and minus Gal plates.

•	Ran FACS (in tubes)

-	Constitutive vs. Constitutive, Constitutive vs. Alpha factor inducible.)

-	All were done in SRaf media.

-	One yeast colony was done in three different medias (YPD, SDcomp, and SRaf)

•	Ran more FACS (in tubes)

-	Ran top two prospects from last FACS run.

-	The two prospects were: Adh1p LexA Sir2 vs. Cyc1p w/ 5’ops with GFP (high expresser) and Adh1p LexA Sir2 vs. Adh1p w/ 3’ops with GFP (low expresser)

•	Ryan did yeast transformations

-	Sir3 into top prospects

•	Digested Sir2 and mCherry with Aar1 (to try sir2 over expression)

•	Ran FACS (in tubes)

-	Ran the top prospects in .5% Glucose media. (To try to help silencing)

-	Ran Gal80 feedback yeast strain in minus and plus Galactose

-	Results of Gal80 feedback was opposite of what we expected

•	Ligated and transformed sir2 Aar1 part into different backbones.

Week nine 7/21 – 7/25

•	Ran FACS for Gal80 feedback again

-	Picked five new colonies and all in both plus and minus galactose media.

-	The old ones from last time were switched to the opposite plates. Example: the ones that were plated on +Gal were plated on –Gal and ran on FACS.

-	Got the same unexpected results, Ran the parent strain for control and GFP was off (no expression)

•	Ran test digests to check promoter of Gal80 construct (making sure it was the right promoter) and also to check the orientation of the Gal80 gene.

-	Also retransformed the construct into the yeast strain as a backup.

•	Ryan is starting to build the synthetic Gal80 construct using Noah’s plasmids.

-	Using a different terminator, and also going to try to use two different promoters (adh1p and cyc1p)

Week ten 7/28 – 8/1

•	Yeas Transformation that was done last week did not work, they looked extremely weird. (they were EXTREMELY pink/red)

•	PCRed Gal80 w/ Xho1 and Not1 ends to build the synthetic Gal80 construct

•	Ran FACS (in block)

-	Constitutive vs. Constitutive, Constitutive vs. alpha factor inducible.

•	TOPO cloned Gal80 PCR

-	Mini-prepped

-	Sent in for sequencing

•	Digested Noah’s plasmid to prepare for the synthetic Gal80 cassette.

Week eleven 8/4 – 8/8

•	Restreaked plates for heterochromatin cooperativity experiment

•	TOPO cloned synthetic Gal80 cassette

-	Sent out for sequencing.

-	Digested out and transformed into my plasmid

•	Ran FACS for cooperativity experiment

-	All variations of silencing constructs into Cyc1p w/ 5’ops with GFP

•	Ligated and transformed the full Gal80 construct.

Week twelve 8/11 – 8/15

•	Test digested synthetic Gal80 construct for correct

•	Snap froze the important constructs

•	Started handing over projects to other people

•	Transformed the Gal80 construct and Gal10p LexA-Sir2 mCherry into yeast.