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Team OUTPUTS
From 2008.igem.org
Subteam:
Output AKA DEATH team
Objective:
Connect silencing/unsilencing to two distinct biological functions (aka
"memory readouts") - cell differentiation and immune response.
Rationale - Demonstrate the modularity of the chromatin bit - i.e. that it can be used with multiple outputs. Demonstrate that silencing or unsilencing can initiate differentiation.
Milestones:
1. Connect silencing/unsilencing to the filamentation pathway ("differentiation"):
a. Check if deletion of DIG1 signaling leads to filamentation.
b. Demonstrate that silencing of DIG1 leads to filamentation.
2. Connect silencing/unsilencing to antimicrobial peptide secretion by yeast
a. Research literature for antibacterial peptides
b. Make sure peptides can be secreted by yeast and determine which strains are viable
c. Test yeast supernatant effects on bacterial growth
Week 1 (6/23/08 – 6/29/08)
- Transformed DIG1 and TEC1.
- Selected colonies and miniprep.
Week 2 (6/30/08 – 7/6/08)
- Double Digested PRS304 and DIG1 plasmid with PspomI and BamHI.
- Gel purified.
- Ligated DIG1 into PRS304 vector.
- Stick Ended PCR’ed LexA.
- Gel purified.
- Selected colonies and minprep.
- Combined Sticky ened fragments together.
- Digested PRS304+DIG1 with BamhI and PspomI.
- Gel purified.
- Ligated sticky ended PCR into PRS304+DIG1 vector.
- Selected colonies for miniprep.
- Repeated ligation with BamhI LexA Sticky ened PCR.
- Test digested with PspomI.
- Ran a gel. No bands
Week 3 (7/7/08 – 7/13/08)
- Test PCR’ed miniprep.
- Ran a gel. Things look good.
- Selected Colonies for miniprep.
- Colony PCR’ed.
- Ran a gel. No bands.
- Repeated test digest PRS304+DIG1 with BamhI and PspomI.
- Ran a gel. Forgot to load a ladder and there were weird bands.
- Test PCR’ed LexA+PRS304+DIG1.
- Ran a gel. Everything looks great expect there were two weird bands for two clones.
- Repeated test digest with BamhI and PspomI to make sure that the insert got ligated.
- PCR’ed PNC1 from genome DNA for iGEM preproject.
- Ran a gel.
- PCR purified.
- Ran a gel for test digest. No band.
- Repeated Test PCR, but with different primer.
- Digested PRS315 Cyc1p and Adh1p with Xho1 and Not1.
- Gel purified.
- Ligated PNC1 into PRS315 Cyc1p and Adh1p vector.. The ligation didn’t work because I forgot to digest PNC1
- Double digested PNC1 with Xho1 + Not1.
- Gel purified
- Repeated ligation, PNC1 into PRS315 Cyc1p and Adh1p vector.
- Ran a gel for the test PCR. The PCR looks wrong. There are too much bands that are wrong size.
- Colony PCR’ed LexA+PRS304+DIG1 (BamhI and PspomI), but with miniprep as template.
- Ran a gel. PCR didn’t work. There are no bands.
- PCR’ed FRE from a FRE plasmid. FRE is filamentous growth response element that detects filamentous growth.
- Gel purified.
- Repeated ligation, PNC1 into PRS315 Cyc1p and Adh1p vector.
- Digest PAW157 and PAN006 with PspomI and XhoI. Also digested FRE with PspomI.
- PCR purified FRE.
- Digest FRE with SalI.
- Gel purified PAW157, PAN006, and FRE.
- Ligated FRE –URA and FRE –LEU into PAW157 and PAN006 vector.
- Selected colonies for miniprep.
- Test digested PNC1+ PRS315 Cyc1p & Adh1p with Xho1 and Not1.
Week 4 (7/14/08 – 7/20/08)
- Transformed PNC1+ PRS315 Cyc1p & Adh1p into AH77-110 yeast strain.
- Ligated mcherry into PJAC4 vector and pFRE-GFP into PRS306 vector.
- Repeated ligation, mcherry into PJAC4 vector and pFRE-GFP into PRS306 vector.
- Verified sequence of PNC1. The quality of the miniprep was poor so it they weren’t able to sequence very well.
Week 5 (7/21/08 – 7/27/08)
- Digest PNH059 with XhoI and BamhI.
- Gel purified.
- Repeated ligation, pFRE-GFP into PRS306 vector and mcherry into PJAC5 vector.
- Selected colonies for miniprep.
Week 5 (7/28/08 – 8/3/08)
- Test digested with Xho1 and BamhI.
- Ran a gel. All the sample of the top of the gel looks fine, but the bottom part looks funny. Maybe it’s because I force the combs into the gel when its starting to harden.
- Double digested PJAC 7 with Xho1 and BamHI.
- Ran a gel and gel purified the vector.
- Ligated mcherry into PJAC7 vector.
- Set up FACS to see if PNC1 would help silencing with SD -/+ 2% gal.
- Digested PJAC4, 6 and & with Xcm1.
- Ran FACS to see if PNC1 would help silencing with SD -/+ 2% gal. FACS failed because I used SD, when I need to use Sraf.
- Transform PJAC4, 6, and 7 into CB008 yeast strain.
- PCR’ed DIG! KO NAT casset.
- Digested PJAC7 with Xcm1.
- Transformed PAC7 into LexA-Sir2 and LexA-Sir2-PCAT1 yeast strain. Also transformed DIG1 KO NAT casset into CB008 and LexA-Sir2 yeast strain.
- Replicated plated CB008 and LexA-Sir2 yeast strain DIG1 KO transformation onto NAT plates.
- Minipreped PNC1+ PRS315 Cyc1p & Adh1p from yeast and transform the miniprep into TG1 cells.
Week 6 (8/4/08 – 8/10/08)
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Week 7 (8/11/08 – 8/17/08)
- Digested PJAC2, PJAC3, and PJAC7 with Xcm1.
- Transformed PJAC2, 3 and 7 into DIG1 KO CB008 and DIG1 KO LexA-Sir2 yeast strain.
- Tried Filamentous growth assay.
