Team:UCSF/Leeza Sergeeva Notebook

Leeza Sergeeva

Notebook 6/18/08 – 10/24/08

 

Milestones:

 

Objective: Disrupt silencing to activate a gene.

Remodel chromatin so that heterochromatin can be uncoiled and opened into euchromatin so they can be activated and freely available.

 

We’re turning on the already silenced genes to produce the desired response (shift the activated genes towards silencing) by opening heterochromatin and acetylating them to become euchromatin and open.

 

Rationale - In some cases, we will want differentiation to lead to gene expression (instead of gene silencing).

 

Milestone: Create multiple fusions of DNA binding domains and activations domains.

 

More specifically…

We are creating our anti-silencing circuits using various DNA Binding Domains and activation domains. (As shown below)

 

	DNA Binding Domains
Activation domains |	LexA PEG 202 (Full Length) NLS	LexA 1-261 (DBD-short length) NLS	LexA 1-261 mCherry NLS
Sas2	+	+	+
Esa1	+	+	+
VP64
mCherry	+		+

(+ - indicates what I’ve finally got)

 

The function of each is such:

1. We’re using various LexA domains to compete for the LexA binding site. This won’t necessarily open heterochromatin, but it will prevent DNA from becoming heterochromatin because it will block and HDAC from binding to the LexA binding site and therefore inhibit the DNA from becoming silenced.

2. We’re using HATs such as Sas2 and Esa1 because they will cause an oozing model where we end up acetylating the heterochromatin and “open” it into euchromatin.

3. We’re using VP64 - as a reader to attract HATs (writers) to they will acetylate for us and open the heterochromatin for us indirectly.

4. We’re using mCherry just as a reporter of expression level so we can run a FACs on it.

 

Actual work done:

 

Week 1-2:

Sequencing VP64

PCR VP64

 

Week 3:

TOPO clone VP64

Digest PCR product of VP64 with EcoRI - run gel - VP64 failed

Bacterial transformation of pFig1

Miniprep more of p315CYC

AarI digestion of p315CYC

 

Week 4:

Run gel to check if AarI digestion of p315CYC worked (success)

TOPO clone mCherry

Miniprep mCherry

Sequence mCherry

 

Week 5:

EcoRI Diagnostic Digest of mCherry

AarI digestion of LexA DBD, LexA FL, LexA mCherry,p315ADH, p305GAL and VP16

Run gel on AarI digestion (success)

Gel purify LexA DBD, LexA FL, LexA mCherry, p315ADH, p305GAL and VP16

Ligations - failed

Cyc-LexA DBD; Cyc-LexA FL; Cyc-mCherry; Cyc–VP16

Gal-LexA DBD; Gal-LexA FL; Gal-mCherry; Gal-VP16

AarI digestion of LexA DBD, VP16, LexA mCherry, Sas2, pGal, pAdh, pCyc.

Rung gel - digestion failed

Ligations - success

-         Gal-LexADBD-VP16

-         Gal-LexA FL-VP16

-         Gal-LexA mCherry-VP16

Bacterial Transformation of successful ligations

 

Week 6:

AarI digestion of VP16, Esa1, pGal, pCyc, Sas2, LexA mCherry.

Run Gel - VP16 (failed)

Colony PCR of ligations

-         Gal-LexADBD-VP16 Success

-         Gal-LexA FL-VP16 Success

-         Gal-LexA mCherry-VP16 Success

Yeast transformation of successful ligations into silenced strains:

- Adh-LexA-Sir2 / Cyc-5’-GFP

- Cyc-LexA-Sir2 / Cyc-5’-GFP

 

Ligations - success

Gal1-LexAmCherry-Esa1

Gal1-LexAmCherry-Sas2

Gal1-LexA PEG 202 (FL)-Esa1

Gal1-LexA PEG 202 (FL)-Sas2

Gal1-LexA DBD-Esa1

Gal1-LexA DBD-Sas2

Adh1-LexAmCherry-Esa1

Adh1-LexAmCherry-Sas2

Adh1-LexA PEG 202 (FL)-Esa1

Adh1-LexA PEG 202 (FL)-Sas2

Adh1-LexA DBD-Esa1

Adh1-LexA DBD-Sas2

Cyc1-LexAmCherry-Esa1

Cyc1-LexAmCherry-Sas2

Cyc1-LexA PEG 202 (FL)-Esa1

Cyc1-LexA PEG 202 (FL)-Sas2

Cyc1-LexA DBD-Esa1

Cyc1-LexA DBD-Sas2

 

Week 7:

Cut p305Gal with XcmI

Bacterial Transformation of ligations

Colonies PCR- run gel - unclear results

Ligations – success - Bacterial Transformations - success

Fig1-LexAmCherry-Esa1

Fig1-LexAmCherry-Sas2

Fig1-LexA PEG 202 (FL)-Esa1

Fig1-LexA PEG 202 (FL)-Sas2

Fig1-LexA DBD-Esa1

Fig1-LexA DBD-Sas2

Miniprep colonies of Gal, Adh and Cyc ligations

Diagnostic digest of Gal, Adh and Cyc ligations with PspOMI & SacI - success

Miniprep Fig ligations

Diagnostic digest of Fig ligations with XhoI & NotI - success

Repeat of diagnostic digest of Gal, Adh and Cyc ligations with PspOMI & SacI - success

AarI digestions of LexA FL, ADH, VP16, mCherry

Run Gel - LexA FL and ADH failed

 

Week 8:

Yeast transformation of Gal ligations into silenced strains:

Gal1-LexA-Sir2 / Cyc-GFP

Adh1-LexA-Sir2 / Cyc-5'opr-GFP

Gal10-LexA-Sir2 / Cyc-GFP

Cyc1-LexA-Sir2 / Cyc-5'opr-GFP

Diagnostic digest of Gal Ligations with XhoI & NotI - success

Restrick Gal yeasts on SRaf + 2% Gal and SD-Leu

 

 

Week 9:

Prepare Gal yeasts for FACs in SD + 2% Gal and SD-Leu medium.

Successful FACs results that shows unsilencing of silenced gene:

-         Gal-LexA FL-EsaI on Gal10-LexA-Sir2 / Cyc-GFP

-         Gal-LexA FL-Sas2 on Gal10-LexA-Sir2 / Cyc-GFP

-         Gal-LexA MC-EsaI on Gal10-LexA-Sir2 / Cyc-GFP

-         Gal-LexA FL-EsaI on Cyc1-LexA-Sir2 / Cyc-5'opr-GFP

-         Gal-LexA MC-EsaI on Cyc1-LexA-Sir2 / Cyc-5'opr-GFP

Prepare successful FACs results for repeat FACs experiment in –gal and +gal medium. (SRaf +2% gal and SDComp)

Successful FACs results that show strong unsilencing:

-         Gal-LexA MC-EsaI on Gal10-LexA-Sir2 / Cyc-GFP

-         Gal-LexA FL-Sas2 on Gal10-LexA-Sir2 / Cyc-GFP

Set up FACs experiment unsilencing strain on silencing strain

Adh-LexA-Sir2/Cyc-5’-GFP:

Adh1-LexAmCherry-Esa1

Adh1-LexAmCherry-Sas2

Cyc1-LexA PEG 202 (FL)-Esa1

Cyc1-LexA PEG 202 (FL)-Sas2

Adh1-LexA DBD-Esa1

Adh1-LexA DBD-Sas2

Fig1-LexAmCherry-Esa1

Fig1-LexAmCherry-Sas2

Fig1-LexA PEG 202 (FL)-Esa1

Fig1-LexA PEG 202 (FL)-Sas2

Adh1-LexA PEG 202 (FL)-Esa1

Adh1-LexA PEG 202 (FL)-Sas2

FACs results:

The control strain (silenced strain wasn’t fully silenced)

Discovery - The low concentration of EsaI induces silencing, but the high concentration unsilence the silenced gene.

Set up cultures for FACs :

-      Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP

-      Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP

-      Gal-LexA PEG 202 (FL)-Esa1 on silenced strain Gal10-LexA-Sir2/Cyc-5’-GFP

FACs results - success - unsilenced silenced genes

 

Week 10:

Transform Adh1-LexAmCherry-Esa1 (wrong plasmid) and Cyc1-LexA PEG 202 (FL)-Esa1 again into yeast silenced strain Adh-LexA-Sir2/Cyc-5’-GFP. (FAILURE!!! MISTAKE!!! TRANSFORMED WRONG PLASMID!!!)

 

Prepare Quick Lysates for Western Blot of:

-         Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP

-         Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP

LigationsàBacterial transformations - partial success

-         Adh-LexA FL-mCherry

-         Adh-LexA FL-VP64

-         Adh-LexA MC-mCherry

-         Adh-LexA DBD-VP64

-         Gal-LexA FL-mCherry

-         Gal-LexA FL-VP64

-         Gal-LexA MC- mCherry

-         Gal-LexA DBD-VP64

-         Fig-LexA FL-mCherry

-         Fig-LexA FL-VP64

-         Fig-LexA MC- mCherry

-         Fig-LexA DBD-VP64

Miniprep succeeded ligations

Cut Gal vectors with XcmI (for yeast transformation)

Yeast Transformation of succeeded ligations into new silenced strains (from Alex):

- Adh-LexA-Sir2 / Cyc-5’-GFP

- Cyc-LexA-Sir2 / Cyc-5’-GFP

Diagnostic Digest of succeeded ligations with PspOMI & SacI - partial success

Run gel - worked:

-         Adh-LexA FL-mCherry

-         Gal-LexA FL-mCherry

-         Gal-LexA MC- mCherry

-         Fig-LexA FL-mCherry

-         Fig-LexA MC- mCherry

Set up FACs experiment on

-         Adh1-LexA MC-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP

-         Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP

FACs results: New transformations didn’t work. The control didn’t work.

 

Week 11:

Set up FACs on new transformation of:

- Silenced strain Adh-LexA-Sir2/Cyc-5’-GFP (Medium: SD-Leu +0.5% Glu)

-         Adh-LexA FL-mCherry

-         Adh-LexA MC-Esa1

-         Cyc- LexA FL-Esa1

- Silenced strain Gal10-LexA-Sir2/Cyc-5’-GFP (Medium: SRaf Comp +2% Gal)

-         Adh-LexA FL-mCherry

-         Cyc- LexA FL-Esa1

FACs results: Too much background - unclear results

AarI digestion of VP64 - run gel - couldn’t cut and gel purify VP64

 

 

Week 12:

Digest Fig-LexA FL-mCherry & Fig-LexA MC-mCherry with PspOMI and SacI

Ligation into 2m plasmid:

-         Fig-LexA FL-mCherry

-         Fig-LexA MC-mCherry

Set up FACs on:

-         Adh-LexA MC-Esa1

-         Adh-LexA FL-Esa1

-         Adh-LexA DBD-Esa1

-         Adh-LexA FL-mCherry

FACs results: control wasn’t perfect - Adh-LexA MC-Esa1 and Adh-LexA FL-mCherry seems to be working.

 

Week 13:

Run Western Blot on prepared samples of

-         Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP

-         Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP

for detecting LexA and Hexokinase.

Result - partial success - western blot worked - hexokinase showed up, but the LexA protein wasn’t detected.

 

Week 14 – 18:

Repeat western blot à gel failed to transfer to the paper.

Prepare new Quick Lysates for Western Blot of:

-         Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP

-         Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP

 

Week 19:

Testing the shuttle vectors from AO

Andrew has done:

1)      Cut AO shuttle vectors with AarI, XmaI digested, CIP treated, gel purified.

2)      Cut AB and BD donors with AarI, gel purified.

3)      Ligations

-         Plate 1: AB shuttle + AB LacI - 1107bp

-         Plate 2: AB shuttle + LexA (1-261) - 261bp (Probably FAILED)

-         Plate 3: BD shuttle + RPD3cat - 1000bp

-         Plate 4: BD shuttle + LexA (1-261) - 261bp

I continued work:

Miniprep Plates 1-4

Digest vectors with EcoRI & SpeI - Run gel - digestion failed

Repeat digestion with EcoRI & SpeI - Run gel – success

 

Week 20:

Run the final gel that shows AarI cut vectors and EcoRI & SpeI Shuttle vectors - success