Team OUTPUTS



       Subteam: Output AKA DEATH team



 Subteam: Output AKA DEATH team

 Objective: Connect silencing/unsilencing to two distinct biological functions (aka &quot;memory readouts&quot;) - cell differentiation and immune response.</o:p></b>

<p class=listparagraph style='margin-left:0in'>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.

<p class=listparagraph style='margin-left:0in'>Milestones:

1. Connect silencing/unsilencing to the filamentation pathway (&quot;differentiation&quot;):

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

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<p class=listparagraph style='margin-left:0in'>Week 1 (6/23/08 &#8211; 6/29/08)</b>

<p class=listparagraph style='margin-left:0in'>- Transformed DIG1 and TEC1.

<p class=listparagraph style='margin-left:0in'>- Selected colonies and miniprep.

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<p class=listparagraph style='margin-left:0in'>Week 2 (6/30/08 &#8211; 7/6/08)</b>

<p class=listparagraph style='margin-left:0in'>- Double Digested PRS304 and DIG1 plasmid with PspomI and BamHI.

<p class=listparagraph style='margin-left:0in'>- Gel purified.

<p class=listparagraph style='margin-left:0in'>- Ligated DIG1 into PRS304 vector.

<p class=listparagraph style='margin-left:0in'>- Stick Ended PCR’ed LexA.

<p class=listparagraph style='margin-left:0in'>- Gel purified.

<p class=listparagraph style='margin-left:0in'>- Selected colonies and minprep.

<p class=listparagraph style='margin-left:0in'>- Combined Sticky ened fragments together.

<p class=listparagraph style='margin-left:0in'>- Digested PRS304+DIG1 with BamhI and PspomI.

<p class=listparagraph style='margin-left:0in'>- Gel purified.

<p class=listparagraph style='margin-left:0in'>- Ligated sticky ended PCR into PRS304+DIG1 vector.

<p class=listparagraph style='margin-left:0in'>- Selected colonies for miniprep.

<p class=listparagraph style='margin-left:0in'>- Repeated ligation with BamhI LexA Sticky ened PCR.

<p class=listparagraph style='margin-left:0in'>- Test digested with PspomI.

<p class=listparagraph style='margin-left:0in'>- Ran a gel. No bands

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<p class=listparagraph style='margin-left:0in'>Week 3 (7/7/08 &#8211; 7/13/08)</b>

<p class=listparagraph style='margin-left:0in'>- Test PCR’ed miniprep.

<p class=listparagraph style='margin-left:0in'>- Ran a gel. Things look good.

<p class=listparagraph style='margin-left:0in'>- Selected Colonies for miniprep.

<p class=listparagraph style='margin-left:0in'>- Colony PCR’ed.

<p class=listparagraph style='margin-left:0in'>- Ran a gel. No bands.

<p class=listparagraph style='margin-left:0in'>- Repeated test digest PRS304+DIG1 with BamhI and PspomI.

<p class=listparagraph style='margin-left:0in'>- Ran a gel. Forgot to load a ladder and there were weird bands.

<p class=listparagraph style='margin-left:0in'>- Test PCR’ed LexA+PRS304+DIG1.

<p class=listparagraph style='margin-left:0in'>- Ran a gel. Everything looks great expect there were two weird bands for two clones.

<p class=listparagraph style='margin-left:0in'>- Repeated test digest with BamhI and PspomI to make sure that the insert got ligated.

<p class=listparagraph style='margin-left:0in'>- PCR’ed PNC1 from genome DNA for iGEM preproject.

<p class=listparagraph style='margin-left:0in'>- Ran a gel.

<p class=listparagraph style='margin-left:0in'>- PCR purified.

<p class=listparagraph style='margin-left:0in'>- Ran a gel for test digest. No band.

<p class=listparagraph style='margin-left:0in'>- Repeated Test PCR, but with different primer.

<p class=listparagraph style='margin-left:0in'>- Digested PRS315 Cyc1p and Adh1p with Xho1 and Not1.

<p class=listparagraph style='margin-left:0in'>- Gel purified.

<p class=listparagraph style='margin-left:0in'>- Ligated PNC1 into PRS315 Cyc1p and Adh1p vector.. The ligation didn’t work because I forgot to digest PNC1

<p class=listparagraph style='margin-left:0in'>- Double digested PNC1 with Xho1 + Not1.

<p class=listparagraph style='margin-left:0in'>- Gel purified

<p class=listparagraph style='margin-left:0in'>- Repeated ligation, PNC1 into PRS315 Cyc1p and Adh1p vector.

<p class=listparagraph style='margin-left:0in'>- Ran a gel for the test PCR. The PCR looks wrong. There are too much bands that are wrong size.

<p class=listparagraph style='margin-left:0in'>- Colony PCR’ed LexA+PRS304+DIG1 (BamhI and PspomI), but with miniprep as template.

<p class=listparagraph style='margin-left:0in'>- Ran a gel. PCR didn’t work. There are no bands.

<p class=listparagraph style='margin-left:0in'>- PCR’ed FRE from a FRE plasmid. FRE is filamentous growth response element that detects filamentous growth.

<p class=listparagraph style='margin-left:0in'>- Gel purified.

<p class=listparagraph style='margin-left:0in'>- Repeated ligation, PNC1 into PRS315 Cyc1p and Adh1p vector.

<p class=listparagraph style='margin-left:0in'>- Digest PAW157 and PAN006 with PspomI and XhoI. Also digested FRE with PspomI.

<p class=listparagraph style='margin-left:0in'>- PCR purified FRE.

<p class=listparagraph style='margin-left:0in'>- Digest FRE with SalI.

<p class=listparagraph style='margin-left:0in'>- Gel purified PAW157, PAN006, and FRE.

<p class=listparagraph style='margin-left:0in'>- Ligated FRE &#8211;URA and FRE &#8211;LEU into PAW157 and PAN006 vector.

<p class=listparagraph style='margin-left:0in'>- Selected colonies for miniprep.

<p class=listparagraph style='margin-left:0in'>- Test digested PNC1+ PRS315 Cyc1p &amp; Adh1p with Xho1 and Not1.

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<p class=listparagraph style='margin-left:0in'>Week 4 (7/14/08 &#8211; 7/20/08)

<p class=listparagraph style='margin-left:0in'>- Transformed PNC1+ PRS315 Cyc1p &amp; Adh1p into AH77-110 yeast strain.

<p class=listparagraph style='margin-left:0in'>- Ligated mcherry into PJAC4 vector and pFRE-GFP into PRS306 vector.

<p class=listparagraph style='margin-left:0in'>- Repeated ligation, mcherry into PJAC4 vector and pFRE-GFP into PRS306 vector.

<p class=listparagraph style='margin-left:0in'>- Verified sequence of PNC1. The quality of the miniprep was poor so it they weren’t able to sequence very well.

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<p class=listparagraph style='margin-left:0in'>Week 5 (7/21/08 &#8211; 7/27/08)</b>

<p class=listparagraph style='margin-left:0in'>- Digest PNH059 with XhoI and BamhI.

<p class=listparagraph style='margin-left:0in'>- Gel purified.

<p class=listparagraph style='margin-left:0in'>- Repeated ligation, pFRE-GFP into PRS306 vector and mcherry into PJAC5 vector.

<p class=listparagraph style='margin-left:0in'>- Selected colonies for miniprep.

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<p class=listparagraph style='margin-left:0in'>Week 5 (7/28/08 &#8211; 8/3/08)</b>

<p class=listparagraph style='margin-left:0in'>- Test digested with Xho1 and BamhI.

<p class=listparagraph style='margin-left:0in'>- 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.

<p class=listparagraph style='margin-left:0in'>- Double digested PJAC 7 with Xho1 and BamHI.

<p class=listparagraph style='margin-left:0in'>- Ran a gel and gel purified the vector.

<p class=listparagraph style='margin-left:0in'>- Ligated mcherry into PJAC7 vector.

<p class=listparagraph style='margin-left:0in'>- Set up FACS to see if PNC1 would help silencing with SD -/+ 2% gal.

<p class=listparagraph style='margin-left:0in'>- Digested PJAC4, 6 and &amp; with Xcm1.

<p class=listparagraph style='margin-left:0in'>- 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.

<p class=listparagraph style='margin-left:0in'>- Transform PJAC4, 6, and 7 into CB008 yeast strain.

<p class=listparagraph style='margin-left:0in'>- PCR’ed DIG! KO NAT casset.

<p class=listparagraph style='margin-left:0in'>- Digested PJAC7 with Xcm1.

<p class=listparagraph style='margin-left:0in'>- Transformed PAC7 into LexA-Sir2 and LexA-Sir2-PCAT1 yeast strain. Also transformed DIG1 KO NAT casset into CB008 and LexA-Sir2 yeast strain.

<p class=listparagraph style='margin-left:0in'>- Replicated plated CB008 and LexA-Sir2 yeast strain DIG1 KO transformation onto NAT plates.

<p class=listparagraph style='margin-left:0in'>- Minipreped PNC1+ PRS315 Cyc1p &amp; Adh1p from yeast and transform the miniprep into TG1 cells.

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<p class=listparagraph style='margin-left:0in'>Week 6 (8/4/08 &#8211; 8/10/08)</b>

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<p class=listparagraph style='margin-left:0in'>Week 7 (8/11/08 &#8211; 8/17/08)</b>

<p class=listparagraph style='margin-left:0in'>- Digested PJAC2, PJAC3, and PJAC7 with Xcm1.

<p class=listparagraph style='margin-left:0in'>- Transformed PJAC2, 3 and 7 into DIG1 KO CB008 and DIG1 KO LexA-Sir2 yeast strain.

<p class=listparagraph style='margin-left:0in'>- Tried Filamentous growth assay.

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