Side Project 1--Toggle Switch

Objective 1:  Determine if we can use heterochromatin to build a functional toggle switch with memory.

Milestones:

A)     Obtain all the parts needed to build the toggle switch.

B)      Build toggle switch using the parts.

C)      Integrate toggle switch into a yeast strain with LexA-SIR2.

Rationale: Demonstrate the modularity of the chromatin bit by building a toggle switch using chromatin.

Steps needed to accomplish objective.

1.      PCR parts from …….

a.       Topo Clone.

b.      Sequence Topo Clone

2.      Ligate parts into PAH32 and PAH77.

3.      Integrate GAL1P*/ADH1P*LexA-SIR2-mCherry and LexAop-ADH1P-TetR-yEGFP into a yeast strain (SV992).

4.      Test toggle switch using FACS.

5.      GIVE PROJECT TO LINGLI/TIFFANY.

Week 1 (7/7/08 – 7/13/08)

Discuss with Andrew about the project.

- PCR’ed up using ADH1P* and GAL1* plasmid from Collins paper as the templates.

• Parts:
• TetR with Xhol/Bamh1 ends.
• TetR with AB ends for AaR1 digest.
• yEGFP with XhoI/BamhI ends.
• yEGFP with BamhI/NotI ends.
• Gal10/1* with PspomI/XhoI ends.
• Adh1p* with PspomI/XhoI ends.
• Cyc1t with NotI/Sac1 ends.

- Dpn1 treated the PCR reaction.

- Topo cloned

- Topo cloned failed. There were 0 to 3 colonies on each plate.

 

Week 2 (7/14/08 – 7/20/08)

- Gel purified PCR product from last week PCR reaction.

- Topo cloned again.

- Topo cloned failed again. Zero colonies on the plates. The reason why Topo clone failed could’ve been that I didn’t dry the X-gal +IPTG plate before plating the cells.

- Topo cloned again using the same PCR product. This time the plates are dried before cells are plated onto.

- Digest PAH32 with XhoI & Bamh1, PAH32 with Xho1 & NotI, and AH81 AlexA with PspomI & XhoI.

- Gel purified vector PAH32, PAH32 and AH81 AlexA.

- Topo cloned failed again. Zero colonies on the plates.

- PCR’ed toggle switch parts again using the same protocol.

- I saved 4.5ul of the PCR reaction and Dpn1 treated the rest of the reaction.

- Topo cloned both Dpn1 treated and not treated.

- Topo clone is successful.

- Colony PCR’ed selected Topo clones from Dpn1 treated and not treated.

- Ran Colony PCR on a gel. Everything looks fine expect for Gal10/1* (Dpn1 treated) and Adh1p* (not Dpn1 treated). They don’t any PCR product.

-Selected clones for minipreps, submitted them for sequencing using M13 REV primer.

• Selected clones Name (restriction ends) Clone’s label:
• Dpn1 Treated
• TetR (x/b) A to C
• TetR (AB) A to C
• yEGFP (x/b) A to C
• yEGFP (b/n) A to C
• Adh1p* (p/x) A to C
• Cyc1t (n/s) A to C
• Gal10/1* (p/x) C
• Not treated with Dpn1
• Gal10/1* (p/x) A & B

 

Week 3 (7/21/08 – 7/27/08)

-Verified the Topo clones’ sequence and identified the good and bad clones.

-         Good clones (all clones)

o       TetR (x/b)

o       Gal10/1* (p/x)

o       Cyc1t (n/s)

-         Bad clones

o       TetR (AB) ALL clones.

o       yEGFP (x/b) ALL clones.

o       yEGFP (b/n) ALL clones.

o       Adh1p* (p/x) ALL clones.

-Repeated PCR on the bad clones expect TetR (AB) with exact extension time to prevent mutation. Dpn1 treated reaction

- Repeated the same PCR reaction as before.

- Ran the PCR reaction on gel. There were no bands on the gel. Expect for a Adh1p*, but faint.

- Repeated the same PCR reaction again. I diluted the template 20 fold.

- Ran 2ul of the PCR reaction and the template on a gel. Everything looks ok.

- Dpn1 treated the PCR reaction

- Topo cloned.

- Selected clones from yEGFP (x/b), yEGFP (b/n), and Adh1p* (p/x) for miniprep and submitted them for sequencing using M13 REV primer.

- Ran the PCR reactions that were Topo cloned on a gel to see if the Dpn1 treatment did anything to the PCR fragment. The fragments looked fine expect there are unknown bands.

- Resent TetR (AB) clones for sequencing.

- Verified TetR (AB) sequence. Every clone has 1 mutation.

- PCR’ed TetR (AB).

- Verified sequences for yEGFp (x/b), yEGFP (b/n), and Adh1p* (p/x). There are no good clones. However, I found that the mutations are all in the same spot. So I decided to proceed with Adh1p* (p/x) clone B and yEGFP (b/n) clone A.

- Digest PAH 32 and Topo TetR (x/b) clone A with XhoI & BamhI, PAH32 and Topo yEGFP (b/n) with BamhI & NotI, PAH32 with XhoI & NotI, AH81 AlexA, Topo Gal10/1* (p/x) clone A, and Topo Adh1p* (p/x) with PspomI & XhoI.

- Ran a gel and gel purified the vector and insert above.

 

Week 4 (7/28/08 – 8/3/08)

-Repeated digest Topo Adh1p* (p/x) with PspomI and XhoI again because yield was low, so I believe that I got the wrong gel slice.

- Ran a gel for Adh1p* (p/x) and gel purified.

- Repeated PCR for for yEGFp (x/b), yEGFP (b/n), and Adh1p* (p/x) to prove my theory that the template contains the mutation and that it wasn’t caused by the PCR reaction.

- Ran a gel for TetR (x/b), yEGFp (x/b), yEGFP (b/n), and Adh1p* (p/x). Everything looks fine expect Adh1p* (p/x) fragment isn’t the right size. So I ran the gel longer and found that there are two bands on each lane. Jimmy said that someone moved the gel, so that could be the cause of the double bands. Also the ladder looks kind of squash together.

- Ligated TetR (x/b) into PAH32 vector, yEGFP (b/n) into PAH32 vector, Gal10/1*P (p/x) into AH81 AlexA vector, Adh1p* (p/x) into AH81 AlexA vector, and TetR (x/b) and yEGFP (b/n) into PAH32 vector.

- Topo cloned TetR (AB), yEGFP (b/n), and yEGFP (x/b).

- Selected clones from TetR (x/b) + PAH32, yEGFP (b/n) + PAH32, Gal10/1*P (p/x) + AH81 AlexA, Ahd1p* (p/x) + AH81 AlexA, and TetR (x/b) and yEGFP (b/n) + PAH32 for miniprep and then test digest.

- Ran a gel for the test digest. Everything looks fine. Even the 3 part ligation looks correct.

- Repeated test digest on TetR (x/b) and yEGFP (b/n) + PAH32. I just wanted to make sure it ligated correctly.

- Ran a gel for the test digest. Everything looks correct.

- Selected clones from Topo clone TetR (AB), yEGFP (b/n), and yEGFP (x/b) for miniprep.

- Digest PJAC9 (TetR (x/b) and yEGFP (b/n) + PAH32) with Xcm1.

- Grow overnight culture, SV992 yeast strain for integration.

- Digest PJAC9 with Xcm1 and more DNA and PJAC10 (Ahd1p* (p/x) + AH81 AlexA) and PJAC11 (Gal10/1*P (p/x) + AH81 AlexA) with Nhe1.

- Transform PJAC9 PJAC10, PJAC11, PJAC9/PJAC10, and PJAC9/PJAC11 into SV992 yeast strain. PJAC9 is a PRS306 vector and PJAC10 and PJAC11 is a PRS303 vector. PRS306 vector are –URA and PRS303 is –HIS. For double transformation I plated them onto SD –HIS –URA plates.

 

Week 5 (8/4/08 – 8/10/08)

- Transformation efficiency was low so I place the cells in the incubator for an extra day.

- Made ATC plates. Then restreak PJAC9/PJAC10 and PJAC9/PJAC11 onto the ATC plates.

- Restreak PJAC9 onto SD –URA, PJAC10 and PJAC11 onto SD –HIS.

- Setup culture for FACs on the Toggle switch stuff.

- Ran FACs in block.

 

Week 6 (8/11/08 – 8/17/08)

- Digest PJAC9, PAN023, and PAN0027 with PspomI & XhoI.

- Ran a gel for the digest.

- Repeated digest. (I believe that I forgot to add one enzyme)

- Ran a gel for the digest and then gel purify.

- Ligated Adh1p into PJAC 9 vector and Gal1 into PJAC9 vector.

- Discuss the project with Lingli, so I can continue it.

 

Week 7 (8/18/08 – 8/24/08)

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Week 8 (8/25/08 – 8/31/08)

- Digest PJAC9, PJAC39 (Adh1p-TetR-GFP-Adh1T-LexOP), and PJAC40 (LexOP-Cyc1p-TetR-GFP-Adh1T) with NdeI and PJAC10 and PJAC11 with Xcm1