Team:UCSF/Leeza Sergeeva Notebook



      Elizaveta Sergeeva



  Leeza    Sergeeva  </b>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> <span style='font-size:16.0pt;font-family:"Segoe Script"'>Notebook 6/18/08 – 10/24/08</o:p> </b>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> <span style='font-size:16.0pt;font-family:"Segoe Script"'><span style='text-decoration:none'> </o:p> </b>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> <span style='font-size:16.0pt;font-family:"Segoe Script"'>Milestones:</o:p> </b>

<p class=MsoNormal> </o:p>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> Objective </b>: Disrupt silencing to activate a gene.</o:p>

<p class=MsoNormal>Remodel chromatin so that heterochromatin can be uncoiled and opened into euchromatin so they can be activated and freely available.</o:p>

<p class=MsoNormal> </o:p>

<p class=MsoNormal>We’re <i style='mso-bidi-font-style:normal'>turning on</i> 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.</o:p>

<p class=MsoNormal> </o:p>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'>Rationale</b> - In some cases, we will want differentiation to lead to gene expression (instead of gene silencing).</o:p>

<p class=MsoNormal> </o:p>

<p class=MsoNormal> Milestone : Create multiple fusions of DNA binding domains and activations domains.</o:p>

<p class=MsoNormal> </o:p>

<p class=MsoNormal> More specifically…  </o:p>

<p class=MsoNormal>We are creating our anti-silencing circuits using various DNA Binding Domains and activation domains. (As shown below)</o:p>

<p class=MsoNormal> </o:p>

<table class=MsoNormalTable border=1 cellspacing=0 cellpadding=0 width=676 style='width:507.2pt;border-collapse:collapse;border:none;mso-border-alt:solid windowtext .5pt; mso-padding-alt:0in 5.4pt 0in 5.4pt;mso-border-insideh:.5pt solid windowtext; mso-border-insidev:.5pt solid windowtext'> <tr style='mso-yfti-irow:0;height:6.1pt'> <td width=184 valign=top style='width:138.3pt;border:solid windowtext 1.0pt; mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:6.1pt'> <p class=MsoNormal><o:p> </o:p> <td width=492 colspan=3 valign=top style='width:368.9pt;border:solid windowtext 1.0pt; border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt: solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:6.1pt'> <p class=MsoNormal align=center style='text-align:center'>DNA Binding Domains<o:p></o:p> <tr style='mso-yfti-irow:1;height:18.6pt'> <td width=184 valign=top style='width:138.3pt;border:solid windowtext 1.0pt; border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:solid windowtext .5pt; padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal>Activation domains |<br style='mso-special-character:line-break'> <![if !supportLineBreakNewLine]><br style='mso-special-character:line-break'> <![endif]><o:p></o:p> <td width=164 valign=top style='width:122.95pt;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal> LexA PEG 202 (Full Length) NLS<o:p></o:p> <td width=164 valign=top style='width:122.95pt;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal> LexA 1-261 (DBD-short length) NLS<o:p></o:p> <td width=164 valign=top style='width:123.0pt;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal> LexA 1-261 mCherry NLS<o:p></o:p> <tr style='mso-yfti-irow:2;height:18.6pt'> <td width=184 valign=top style='width:138.3pt;border:solid windowtext 1.0pt; border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:solid windowtext .5pt; padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal align=center style='text-align:center'>Sas2<o:p></o:p> <td width=164 valign=top style='width:122.95pt;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal align=center style='text-align:center'>+<o:p></o:p> <td width=164 valign=top style='width:122.95pt;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal align=center style='text-align:center'>+<o:p></o:p> <td width=164 valign=top style='width:123.0pt;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal align=center style='text-align:center'>+<o:p></o:p> <tr style='mso-yfti-irow:3;height:18.6pt'> <td width=184 valign=top style='width:138.3pt;border:solid windowtext 1.0pt; border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:solid windowtext .5pt; padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal align=center style='text-align:center'>Esa1<o:p></o:p> <td width=164 valign=top style='width:122.95pt;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal align=center style='text-align:center'>+<o:p></o:p> <td width=164 valign=top style='width:122.95pt;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal align=center style='text-align:center'>+<o:p></o:p> <td width=164 valign=top style='width:123.0pt;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal align=center style='text-align:center'>+<o:p></o:p> <tr style='mso-yfti-irow:4;height:18.6pt'> <td width=184 valign=top style='width:138.3pt;border:solid windowtext 1.0pt; border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:solid windowtext .5pt; padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal align=center style='text-align:center'>VP64<o:p></o:p> <td width=164 valign=top style='width:122.95pt;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal align=center style='text-align:center'><o:p> </o:p> <td width=164 valign=top style='width:122.95pt;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal align=center style='text-align:center'><o:p> </o:p> <td width=164 valign=top style='width:123.0pt;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal align=center style='text-align:center'><o:p> </o:p> <tr style='mso-yfti-irow:5;mso-yfti-lastrow:yes;height:18.6pt'> <td width=184 valign=top style='width:138.3pt;border:solid windowtext 1.0pt; border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:solid windowtext .5pt; padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal align=center style='text-align:center'> mCherry <o:p></o:p> <td width=164 valign=top style='width:122.95pt;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal align=center style='text-align:center'>+<o:p></o:p> <td width=164 valign=top style='width:122.95pt;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal align=center style='text-align:center'><o:p> </o:p> <td width=164 valign=top style='width:123.0pt;border-top:none;border-left: none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt; mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt; mso-border-alt:solid windowtext .5pt;padding:0in 5.4pt 0in 5.4pt;height:18.6pt'> <p class=MsoNormal align=center style='text-align:center'>+<o:p></o:p>

<p class=MsoNormal>(+ - indicates what I’ve finally got)<o:p></o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal> The function of each is such :<o:p></o:p>

<p class=MsoNormal>1. We’re using various<span lang=ES-VE style='mso-ansi-language: ES-VE'> LexA domains to compete for the<span lang=EN-GB style='mso-ansi-language:EN-GB'> 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 <span class=SpellE>LexA binding site and therefore inhibit the DNA from becoming silenced.<o:p></o:p>

<p class=MsoNormal>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.<o:p></o:p>

<p class=MsoNormal>3. We’re using VP64 - as a reader to attract <span class=SpellE>HATs (writers) to they will acetylate for us and open the heterochromatin for us indirectly.<o:p></o:p>

<p class=MsoNormal>4. We’re using mCherry just as a reporter of expression level so we can run a FACs on it.<o:p></o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal><b style='mso-bidi-font-weight:normal'> <span style='font-size:18.0pt;font-family:"Segoe Script"'>Actual work done:<o:p></o:p> </b>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal><b> Week 1-2:<o:p></o:p> </b>

<p class=MsoNormal>Sequencing VP64<o:p></o:p>

<p class=MsoNormal>PCR VP64 <o:p></o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal><b> Week 3:<o:p></o:p> </b>

<p class=MsoNormal>TOPO clone VP64<o:p></o:p>

<p class=MsoNormal>Digest PCR product of VP64 with EcoRI - run gel - VP64 failed<o:p></o:p>

<p class=MsoNormal>Bacterial transformation of pFig1<o:p></o:p>

<p class=MsoNormal> Miniprep more of p315CYC<o:p></o:p>

<p class=MsoNormal> AarI digestion of p315CYC<o:p></o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal><b> Week 4:<o:p></o:p> </b>

<p class=MsoNormal>Run gel to check if AarI digestion of p315CYC worked ( success )<o:p></o:p>

<p class=MsoNormal>TOPO clone mCherry <o:p></o:p>

<p class=MsoNormal> Miniprep mCherry <o:p></o:p>

<p class=MsoNormal>Sequence mCherry <o:p></o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal><b> Week 5:<o:p></o:p> </b>

<p class=MsoNormal> EcoRI Diagnostic Digest of <span class=SpellE>mCherry <o:p></o:p>

<p class=MsoNormal> AarI digestion of <span class=SpellE>LexA DBD, LexA FL, <span class=SpellE>LexA mCherry ,p315ADH, p305GAL and VP16<o:p></o:p>

<p class=MsoNormal>Run gel on AarI digestion (success)<o:p></o:p>

<p class=MsoNormal>Gel purify LexA DBD, <span class=SpellE>LexA FL, LexA mCherry , p315ADH, p305GAL and VP16<o:p></o:p>

<p class=MsoNormal> Ligations - failed <o:p></o:p>

<p class=MsoNormal>           <span class=SpellE>Cyc-LexA DBD; Cyc-LexA FL; <span class=SpellE>Cyc-mCherry ; Cyc –VP16<o:p></o:p>

<p class=MsoNormal>           Gal-<span class=SpellE>LexA DBD; Gal- LexA FL; Gal-<span class=SpellE>mCherry ; Gal-VP16<o:p></o:p>

<p class=MsoNormal> AarI  <span class=GramE> digestion of LexA DBD, VP16, <span class=SpellE>LexA mCherry, Sas2, <span class=SpellE>pGal, pAdh , pCyc. <o:p></o:p>

<p class=MsoNormal>Rung gel - digestion failed<o:p></o:p>

<p class=MsoNormal> Ligations - success <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal-LexADBD-VP16<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA FL-VP16<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA mCherry-VP16 <o:p></o:p>

<p class=MsoNormal>Bacterial Transformation of successful ligations <o:p></o:p>

<p class=MsoNormal style='margin-left:.5in'><o:p> </o:p>

<p class=MsoNormal><b> Week 6:<o:p></o:p> </b>

<p class=MsoNormal> AarI  <span class=GramE> digestion of VP16, Esa1, pGal, <span class=SpellE>pCyc, Sas2, LexA <span class=SpellE>mCherry. <o:p></o:p>

<p class=MsoNormal>Run Gel - VP16 (failed)<o:p></o:p>

<p class=MsoNormal>Colony PCR of ligations <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal-LexADBD-VP16<span style='mso-tab-count:2'>                    Success <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA FL-VP16                       Success <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA mCherry-VP16 Success <o:p></o:p>

<p class=MsoNormal>Yeast transformation of successful ligations into silenced strains:<o:p></o:p>

<p class=MsoNormal>           - Adh-LexA-Sir2 / Cyc-5’-GFP<o:p></o:p>

<p class=MsoNormal>           - Cyc-LexA-Sir2 / Cyc-5’-GFP<o:p></o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal> Ligations - success <o:p></o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal><b> Week 7:<o:p></o:p> </b>

<p class=MsoNormal>Cut p305Gal with XcmI <o:p></o:p>

<p class=MsoNormal>Bacterial Transformation of ligations <o:p></o:p>

<p class=MsoNormal>Colonies PCR- run gel - unclear results<o:p></o:p>

<p class=MsoNormal> Ligations – success - Bacterial Transformations - success <o:p></o:p>

<p class=MsoNormal> Miniprep colonies of Gal, <span class=SpellE> Adh and Cyc ligations <o:p></o:p>

<p class=MsoNormal>Diagnostic digest of Gal, <span class=GramE>Adh and Cyc <span class=SpellE>ligations with PspOMI &amp; <span class=SpellE>SacI - success <o:p></o:p>

<p class=MsoNormal> Miniprep Fig ligations <o:p></o:p>

<p class=MsoNormal>Diagnostic digest of Fig ligations with XhoI &amp; NotI - success <o:p></o:p>

<p class=MsoNormal>Repeat of diagnostic digest of Gal, <span class=GramE>Adh and Cyc <span class=SpellE>ligations with PspOMI &amp; <span class=SpellE>SacI - success <o:p></o:p>

<p class=MsoNormal> AarI digestions of <span class=SpellE>LexA FL, ADH, VP16, mCherry <o:p></o:p>

<p class=MsoNormal> Run Gel - LexA FL and ADH failed<o:p></o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal><b> Week 8:<o:p></o:p> </b>

<p class=MsoNormal>Yeast transformation of Gal ligations into silenced strains:<o:p></o:p>

<p class=MsoNormal>Diagnostic digest of Gal Ligations with XhoI &amp; NotI - success <o:p></o:p>

<p class=MsoNormal> Restrick Gal yeasts on <span class=SpellE>SRaf + 2% Gal and SD- Leu <o:p></o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal><b> Week 9:<o:p></o:p> </b>

<p class=MsoNormal>Prepare Gal yeasts for FACs in SD + 2% Gal and SD- Leu medium.<o:p></o:p>

<p class=MsoNormal> Successful FACs results that shows unsilencing of silenced gene:<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA FL- EsaI on Gal10-LexA-Sir2 / Cyc -GFP<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA FL-Sas2 on Gal10-LexA-Sir2 / Cyc -GFP<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA MC- EsaI on Gal10-LexA-Sir2 / Cyc -GFP<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA FL- EsaI on Cyc1-LexA-Sir2 / Cyc-5'opr-GFP<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA MC- EsaI on Cyc1-LexA-Sir2 / Cyc-5'opr-GFP<o:p></o:p>

<p class=MsoNormal>Prepare successful FACs results for repeat FACs experiment in –gal and +gal medium. (<span class=SpellE>SRaf +2% gal and SDComp )<o:p></o:p>

<p class=MsoNormal> Successful FACs results that show strong unsilencing :<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA MC- EsaI on Gal10-LexA-Sir2 / Cyc -GFP<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA FL-Sas2 on Gal10-LexA-Sir2 / Cyc -GFP<o:p></o:p>

<p class=MsoNormal>Set up FACs experiment <span class=SpellE>unsilencing strain on silencing strain <o:p></o:p>

<p class=MsoNormal>Adh-LexA-Sir2/Cyc-5’-GFP:<o:p></o:p>

<p class=MsoNormal> FACs results:<o:p></o:p>

<p class=MsoNormal>The control strain (silenced strain wasn’t fully silenced)<o:p></o:p>

<p class=MsoNormal> Discovery - The low concentration of <span class=SpellE>EsaI induces silencing, but the high concentration unsilence the silenced gene.<o:p></o:p>

<p class=MsoNormal>Set up cultures for FACs  <span class=GramE> : <o:p></o:p>

<p class=MsoNormal style='margin-left:40.5pt;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .5in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> <span style='mso-fareast-language: ZH-CN'>Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p>

<p class=MsoNormal style='margin-left:40.5pt;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .5in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> <span style='mso-fareast-language: ZH-CN'>Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p>

<p class=MsoNormal style='margin-left:40.5pt;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .5in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA PEG 202 (FL)-Esa1 on silenced strain Gal10-LexA-Sir2/Cyc-5’-GFP<o:p></o:p>

<p class=MsoNormal> FACs results - success - <span class=SpellE>unsilenced silenced genes<o:p></o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal><b> Week 10:<o:p></o:p> </b>

<p class=MsoNormal>Transform <i> Adh1-LexAmCherry-Esa1 (wrong plasmid) </i> and Cyc1-LexA PEG 202 (FL)-Esa1 again into yeast silenced strain Adh-LexA-Sir2/Cyc-5’-GFP. (FAILURE!!! MISTAKE!!! <span class=GramE>TRANSFORMED WRONG PLASMID!!!) <o:p></o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal>Prepare Quick Lysates for Western Blot of:<o:p></o:p>

<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .5in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> <span style='mso-fareast-language: ZH-CN'>Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p>

<p class=MsoNormal style='margin-left:.5in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .5in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> <span style='mso-fareast-language: ZH-CN'>Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p>

<p class=MsoNormal> Ligations <span style='font-family: Wingdings;mso-ascii-font-family:"Times New Roman";mso-hansi-font-family:"Times New Roman"; mso-char-type:symbol;mso-symbol-font-family:Wingdings'><span style='mso-char-type: symbol;mso-symbol-font-family:Wingdings'>à Bacterial transformations - partial success<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Adh-LexA FL- mCherry <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Adh-LexA FL-VP64<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Adh-LexA MC- mCherry <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Adh-LexA DBD-VP64<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA FL- mCherry <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA FL-VP64<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA MC- mCherry <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA DBD-VP64<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Fig- LexA FL- mCherry <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Fig- LexA FL-VP64<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Fig- LexA MC- mCherry <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Fig- LexA DBD-VP64<o:p></o:p>

<p class=MsoNormal> Miniprep succeeded <span class=SpellE>ligations <o:p></o:p>

<p class=MsoNormal>Cut Gal vectors with XcmI (for yeast transformation)<o:p></o:p>

<p class=MsoNormal>Yeast Transformation of succeeded ligations into new silenced strains (from Alex):<o:p></o:p>

<p class=MsoNormal>           - Adh-LexA-Sir2 / Cyc-5’-GFP<o:p></o:p>

<p class=MsoNormal>           - Cyc-LexA-Sir2 / Cyc-5’-GFP<o:p></o:p>

<p class=MsoNormal>Diagnostic Digest of succeeded ligations with PspOMI &amp; SacI - partial success<o:p></o:p>

<p class=MsoNormal>Run gel - worked:<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Adh-LexA FL- mCherry <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA FL- mCherry <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Gal- LexA MC- mCherry <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Fig- LexA FL- mCherry <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Fig- LexA MC- mCherry <o:p></o:p>

<p class=MsoNormal>Set up FACs experiment on <o:p></o:p>

<p class=MsoNormal style='margin-left:49.5pt;text-indent:-.25in;mso-list:l1 level1 lfo1'><![if !supportLists]><span style='mso-font-width:0%'> -<span style='font:7.0pt "Times New Roman"'> <![endif]> <span style='mso-fareast-language: ZH-CN'>Adh1-LexA MC-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p>

<p class=MsoNormal style='margin-left:49.5pt;text-indent:-.25in;mso-list:l1 level1 lfo1'><![if !supportLists]><span style='mso-font-width:0%'> -<span style='font:7.0pt "Times New Roman"'> <![endif]> <span style='mso-fareast-language: ZH-CN'>Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p>

<p class=MsoNormal> FACs results: New transformations didn’t work. The control didn’t work.<o:p></o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal><b> Week 11: <o:p></o:p> </b>

<p class=MsoNormal>Set up FACs on new transformation of: <o:p></o:p>

<p class=MsoNormal>- Silenced strain Adh-LexA-Sir2/Cyc-5’-GFP (Medium: SD-<span class=SpellE>Leu +0.5% Glu )<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Adh-LexA FL- mCherry <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Adh-LexA MC-Esa1 <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Cyc - <span class=SpellE>LexA FL-Esa1<o:p></o:p>

<p class=MsoNormal>- Silenced strain Gal10-LexA-Sir2/Cyc-5’-GFP (Medium: <span class=SpellE>SRaf Comp +2% Gal)<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Adh-LexA FL- mCherry <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Cyc - <span class=SpellE>LexA FL-Esa1<o:p></o:p>

<p class=MsoNormal> FACs results: Too much background - unclear results<o:p></o:p>

<p class=MsoNormal> AarI digestion of VP64 - run gel - couldn’t cut and gel purify VP64<o:p></o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal><b> Week 12:<o:p></o:p> </b>

<p class=MsoNormal>Digest Fig- LexA FL-<span class=SpellE>mCherry &amp; Fig- LexA MC-<span class=SpellE>mCherry with PspOMI and <span class=SpellE>SacI <o:p></o:p>

<p class=MsoNormal> Ligation into 2m plasmid:<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Fig- LexA FL- mCherry <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Fig- LexA MC- mCherry <o:p></o:p>

<p class=MsoNormal>Set up FACs on:<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Adh-LexA MC-Esa1<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Adh-LexA FL-Esa1<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Adh-LexA DBD-Esa1<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Adh-LexA FL- mCherry <o:p></o:p>

<p class=MsoNormal> FACs results: control wasn’t perfect - Adh- LexA MC-Esa1 and Adh-LexA FL- mCherry seems to be working.<o:p></o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal><b> Week 13:<o:p></o:p> </b>

<p class=MsoNormal>Run Western Blot on prepared samples of <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> <span style='mso-fareast-language: ZH-CN'>Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> <span style='mso-fareast-language: ZH-CN'>Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p>

<p class=MsoNormal> for detecting LexA and Hexokinase. <o:p></o:p>

<p class=MsoNormal>Result - partial success - western blot worked - <span class=SpellE>hexokinase showed up, but the LexA protein wasn’t detected.<o:p></o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal><b> Week 14 – 18:<o:p></o:p> </b>

<p class=MsoNormal>Repeat western blot <span style='font-family:Wingdings; mso-ascii-font-family:"Times New Roman";mso-hansi-font-family:"Times New Roman"; mso-char-type:symbol;mso-symbol-font-family:Wingdings'><span style='mso-char-type: symbol;mso-symbol-font-family:Wingdings'>à gel failed to transfer to the paper.<o:p></o:p>

<p class=MsoNormal>Prepare new Quick Lysates for Western Blot of:<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> <span style='mso-fareast-language: ZH-CN'>Adh1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> <span style='mso-fareast-language: ZH-CN'>Cyc1-LexA PEG 202 (FL)-Esa1 on silenced strain Adh-LexA-Sir2/Cyc-5’-GFP<o:p></o:p>

<p class=MsoNormal><o:p> </o:p>

<p class=MsoNormal><b> Week 19:<o:p></o:p> </b>

<p class=MsoNormal>Testing the shuttle vectors from AO<o:p></o:p>

<p class=MsoNormal>           Andrew has done:<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l0 level1 lfo2; tab-stops:list .75in'><![if !supportLists]> 1)<span style='font:7.0pt "Times New Roman"'>      <![endif]><span dir=LTR>Cut AO shuttle vectors with AarI, <span class=SpellE>XmaI digested, CIP treated, gel purified.<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l0 level1 lfo2; tab-stops:list .75in'><![if !supportLists]> 2)<span style='font:7.0pt "Times New Roman"'>      <![endif]><span dir=LTR>Cut AB and BD donors with AarI, gel purified.<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l0 level1 lfo2; tab-stops:list .75in'><![if !supportLists]> 3)<span style='font:7.0pt "Times New Roman"'>      <![endif]><span dir=LTR> Ligations <o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Plate 1: AB shuttle + AB <span class=SpellE>LacI - 1107bp<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Plate 2: AB shuttle + <span class=SpellE>LexA (1-261) - 261bp (Probably FAILED)<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Plate 3: BD shuttle + RPD3cat - 1000bp<o:p></o:p>

<p class=MsoNormal style='margin-left:.75in;text-indent:-.25in;mso-list:l1 level1 lfo1; tab-stops:list .75in'><![if !supportLists]> <span style='mso-list:Ignore'>- <![endif]> Plate 4: BD shuttle + <span class=SpellE>LexA (1-261) - 261bp<o:p></o:p>

<p class=MsoNormal>I continued work:<o:p></o:p>

<p class=MsoNormal> Miniprep Plates 1-4<o:p></o:p>

<p class=MsoNormal>Digest vectors with EcoRI &amp; <span class=SpellE>SpeI - Run gel - digestion failed<o:p></o:p>

<p class=MsoNormal>Repeat digestion with EcoRI &amp; <span class=SpellE>SpeI - Run gel – success<o:p></o:p>

<p class=MsoNormal> <o:p> </o:p>

<p class=MsoNormal><b> Week 20:<o:p></o:p> </b>

<p class=MsoNormal>Run the final gel that shows AarI cut vectors and EcoRI &amp; SpeI Shuttle vectors - success <o:p></o:p>

<p class=MsoNormal><o:p> </o:p>