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| | {{UChicago/Header}} | | {{UChicago/Header}} |
| | | | |
| − | <html xmlns:v="urn:schemas-microsoft-com:vml" | + | <html> |
| − | xmlns:o="urn:schemas-microsoft-com:office:office"
| + | |
| − | xmlns:w="urn:schemas-microsoft-com:office:word"
| + | |
| − | xmlns:m="http://schemas.microsoft.com/office/2004/12/omml"
| + | |
| − | xmlns="http://www.w3.org/TR/REC-html40">
| + | |
| | | | |
| | <head> | | <head> |
| | <meta http-equiv=Content-Type content="text/html; charset=windows-1252"> | | <meta http-equiv=Content-Type content="text/html; charset=windows-1252"> |
| − | <meta name=ProgId content=Word.Document>
| + | <meta name=Generator content="Microsoft Word 15 (filtered)"> |
| − | <meta name=Generator content="Microsoft Word 15"> | + | |
| − | <meta name=Originator content="Microsoft Word 15">
| + | |
| − | <link rel=File-List href="Protocols_files/filelist.xml">
| + | |
| − | <link rel=Edit-Time-Data href="Protocols_files/editdata.mso">
| + | |
| − | <!--[if !mso]>
| + | |
| − | <style>
| + | |
| − | v\:* {behavior:url(#default#VML);}
| + | |
| − | o\:* {behavior:url(#default#VML);}
| + | |
| − | w\:* {behavior:url(#default#VML);}
| + | |
| − | .shape {behavior:url(#default#VML);}
| + | |
| − | </style>
| + | |
| − | <![endif]--><!--[if gte mso 9]><xml>
| + | |
| − | <o:DocumentProperties>
| + | |
| − | <o:Author>Ethan Steinberg</o:Author>
| + | |
| − | <o:Template>Normal</o:Template>
| + | |
| − | <o:LastAuthor>Kevin Yang</o:LastAuthor>
| + | |
| − | <o:Revision>2</o:Revision>
| + | |
| − | <o:TotalTime>3</o:TotalTime>
| + | |
| − | <o:Created>2015-09-18T05:05:00Z</o:Created>
| + | |
| − | <o:LastSaved>2015-09-18T05:05:00Z</o:LastSaved>
| + | |
| − | <o:Pages>1</o:Pages>
| + | |
| − | <o:Words>1066</o:Words>
| + | |
| − | <o:Characters>6082</o:Characters>
| + | |
| − | <o:Lines>50</o:Lines>
| + | |
| − | <o:Paragraphs>14</o:Paragraphs>
| + | |
| − | <o:CharactersWithSpaces>7134</o:CharactersWithSpaces>
| + | |
| − | <o:Version>15.00</o:Version>
| + | |
| − | </o:DocumentProperties>
| + | |
| − | <o:OfficeDocumentSettings>
| + | |
| − | <o:AllowPNG/>
| + | |
| − | </o:OfficeDocumentSettings>
| + | |
| − | </xml><![endif]-->
| + | |
| − | <link rel=themeData href="Protocols_files/themedata.thmx">
| + | |
| − | <link rel=colorSchemeMapping href="Protocols_files/colorschememapping.xml">
| + | |
| − | <!--[if gte mso 9]><xml>
| + | |
| − | <w:WordDocument>
| + | |
| − | <w:SpellingState>Clean</w:SpellingState>
| + | |
| − | <w:GrammarState>Clean</w:GrammarState>
| + | |
| − | <w:TrackMoves>false</w:TrackMoves>
| + | |
| − | <w:TrackFormatting/>
| + | |
| − | <w:PunctuationKerning/>
| + | |
| − | <w:ValidateAgainstSchemas/>
| + | |
| − | <w:SaveIfXMLInvalid>false</w:SaveIfXMLInvalid>
| + | |
| − | <w:IgnoreMixedContent>false</w:IgnoreMixedContent>
| + | |
| − | <w:AlwaysShowPlaceholderText>false</w:AlwaysShowPlaceholderText>
| + | |
| − | <w:DoNotPromoteQF/>
| + | |
| − | <w:LidThemeOther>EN-US</w:LidThemeOther>
| + | |
| − | <w:LidThemeAsian>JA</w:LidThemeAsian>
| + | |
| − | <w:LidThemeComplexScript>X-NONE</w:LidThemeComplexScript>
| + | |
| − | <w:Compatibility>
| + | |
| − | <w:BreakWrappedTables/>
| + | |
| − | <w:SnapToGridInCell/>
| + | |
| − | <w:WrapTextWithPunct/>
| + | |
| − | <w:UseAsianBreakRules/>
| + | |
| − | <w:UseWord2010TableStyleRules/>
| + | |
| − | <w:DontGrowAutofit/>
| + | |
| − | <w:SplitPgBreakAndParaMark/>
| + | |
| − | <w:EnableOpenTypeKerning/>
| + | |
| − | <w:DontFlipMirrorIndents/>
| + | |
| − | <w:OverrideTableStyleHps/>
| + | |
| − | <w:UseFELayout/>
| + | |
| − | </w:Compatibility>
| + | |
| − | <w:DoNotOptimizeForBrowser/>
| + | |
| − | <m:mathPr>
| + | |
| − | <m:mathFont m:val="Cambria Math"/>
| + | |
| − | <m:brkBin m:val="before"/>
| + | |
| − | <m:brkBinSub m:val="--"/>
| + | |
| − | <m:smallFrac m:val="off"/>
| + | |
| − | <m:dispDef/>
| + | |
| − | <m:lMargin m:val="0"/>
| + | |
| − | <m:rMargin m:val="0"/>
| + | |
| − | <m:defJc m:val="centerGroup"/>
| + | |
| − | <m:wrapIndent m:val="1440"/>
| + | |
| − | <m:intLim m:val="subSup"/>
| + | |
| − | <m:naryLim m:val="undOvr"/>
| + | |
| − | </m:mathPr></w:WordDocument>
| + | |
| − | </xml><![endif]--><!--[if gte mso 9]><xml>
| + | |
| − | <w:LatentStyles DefLockedState="false" DefUnhideWhenUsed="false"
| + | |
| − | DefSemiHidden="false" DefQFormat="false" DefPriority="99"
| + | |
| − | LatentStyleCount="371">
| + | |
| − | <w:LsdException Locked="false" Priority="0" QFormat="true" Name="Normal"/>
| + | |
| − | <w:LsdException Locked="false" Priority="9" QFormat="true" Name="heading 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="9" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" QFormat="true" Name="heading 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="9" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" QFormat="true" Name="heading 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="9" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" QFormat="true" Name="heading 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="9" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" QFormat="true" Name="heading 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="9" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" QFormat="true" Name="heading 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="9" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" QFormat="true" Name="heading 7"/>
| + | |
| − | <w:LsdException Locked="false" Priority="9" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" QFormat="true" Name="heading 8"/>
| + | |
| − | <w:LsdException Locked="false" Priority="9" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" QFormat="true" Name="heading 9"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="index 1"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="index 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="index 3"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="index 4"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="index 5"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="index 6"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="index 7"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="index 8"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="index 9"/>
| + | |
| − | <w:LsdException Locked="false" Priority="39" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" Name="toc 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="39" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" Name="toc 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="39" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" Name="toc 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="39" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" Name="toc 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="39" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" Name="toc 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="39" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" Name="toc 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="39" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" Name="toc 7"/>
| + | |
| − | <w:LsdException Locked="false" Priority="39" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" Name="toc 8"/>
| + | |
| − | <w:LsdException Locked="false" Priority="39" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" Name="toc 9"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Normal Indent"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="footnote text"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="annotation text"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="header"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="footer"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="index heading"/>
| + | |
| − | <w:LsdException Locked="false" Priority="35" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" QFormat="true" Name="caption"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="table of figures"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="envelope address"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="envelope return"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="footnote reference"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="annotation reference"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="line number"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="page number"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="endnote reference"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="endnote text"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="table of authorities"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="macro"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="toa heading"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List Bullet"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List Number"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List 3"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List 4"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List 5"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List Bullet 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List Bullet 3"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List Bullet 4"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List Bullet 5"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List Number 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List Number 3"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List Number 4"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List Number 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="10" QFormat="true" Name="Title"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Closing"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Signature"/>
| + | |
| − | <w:LsdException Locked="false" Priority="1" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" Name="Default Paragraph Font"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Body Text"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Body Text Indent"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List Continue"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List Continue 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List Continue 3"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List Continue 4"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="List Continue 5"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Message Header"/>
| + | |
| − | <w:LsdException Locked="false" Priority="11" QFormat="true" Name="Subtitle"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Salutation"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Date"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Body Text First Indent"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Body Text First Indent 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Note Heading"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Body Text 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Body Text 3"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Body Text Indent 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Body Text Indent 3"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Block Text"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Hyperlink"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="FollowedHyperlink"/>
| + | |
| − | <w:LsdException Locked="false" Priority="22" QFormat="true" Name="Strong"/>
| + | |
| − | <w:LsdException Locked="false" Priority="20" QFormat="true" Name="Emphasis"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Document Map"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Plain Text"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="E-mail Signature"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="HTML Top of Form"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="HTML Bottom of Form"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Normal (Web)"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="HTML Acronym"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="HTML Address"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="HTML Cite"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="HTML Code"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="HTML Definition"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="HTML Keyboard"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="HTML Preformatted"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="HTML Sample"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="HTML Typewriter"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="HTML Variable"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Normal Table"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="annotation subject"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="No List"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Outline List 1"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Outline List 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Outline List 3"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Simple 1"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Simple 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Simple 3"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Classic 1"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Classic 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Classic 3"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Classic 4"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Colorful 1"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Colorful 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Colorful 3"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Columns 1"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Columns 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Columns 3"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Columns 4"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Columns 5"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Grid 1"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Grid 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Grid 3"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Grid 4"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Grid 5"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Grid 6"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Grid 7"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Grid 8"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table List 1"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table List 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table List 3"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table List 4"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table List 5"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table List 6"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table List 7"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table List 8"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table 3D effects 1"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table 3D effects 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table 3D effects 3"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Contemporary"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Elegant"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Professional"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Subtle 1"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Subtle 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Web 1"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Web 2"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Web 3"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Balloon Text"/>
| + | |
| − | <w:LsdException Locked="false" Priority="59" Name="Table Grid"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
| + | |
| − | Name="Table Theme"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" Name="Placeholder Text"/>
| + | |
| − | <w:LsdException Locked="false" Priority="1" QFormat="true" Name="No Spacing"/>
| + | |
| − | <w:LsdException Locked="false" Priority="60" Name="Light Shading"/>
| + | |
| − | <w:LsdException Locked="false" Priority="61" Name="Light List"/>
| + | |
| − | <w:LsdException Locked="false" Priority="62" Name="Light Grid"/>
| + | |
| − | <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="65" Name="Medium List 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="66" Name="Medium List 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="70" Name="Dark List"/>
| + | |
| − | <w:LsdException Locked="false" Priority="71" Name="Colorful Shading"/>
| + | |
| − | <w:LsdException Locked="false" Priority="72" Name="Colorful List"/>
| + | |
| − | <w:LsdException Locked="false" Priority="73" Name="Colorful Grid"/>
| + | |
| − | <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="61" Name="Light List Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" SemiHidden="true" Name="Revision"/>
| + | |
| − | <w:LsdException Locked="false" Priority="34" QFormat="true"
| + | |
| − | Name="List Paragraph"/>
| + | |
| − | <w:LsdException Locked="false" Priority="29" QFormat="true" Name="Quote"/>
| + | |
| − | <w:LsdException Locked="false" Priority="30" QFormat="true"
| + | |
| − | Name="Intense Quote"/>
| + | |
| − | <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="61" Name="Light List Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="61" Name="Light List Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="61" Name="Light List Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="61" Name="Light List Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="61" Name="Light List Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="19" QFormat="true"
| + | |
| − | Name="Subtle Emphasis"/>
| + | |
| − | <w:LsdException Locked="false" Priority="21" QFormat="true"
| + | |
| − | Name="Intense Emphasis"/>
| + | |
| − | <w:LsdException Locked="false" Priority="31" QFormat="true"
| + | |
| − | Name="Subtle Reference"/>
| + | |
| − | <w:LsdException Locked="false" Priority="32" QFormat="true"
| + | |
| − | Name="Intense Reference"/>
| + | |
| − | <w:LsdException Locked="false" Priority="33" QFormat="true" Name="Book Title"/>
| + | |
| − | <w:LsdException Locked="false" Priority="37" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" Name="Bibliography"/>
| + | |
| − | <w:LsdException Locked="false" Priority="39" SemiHidden="true"
| + | |
| − | UnhideWhenUsed="true" QFormat="true" Name="TOC Heading"/>
| + | |
| − | <w:LsdException Locked="false" Priority="49" Name="Grid Table 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark"/>
| + | |
| − | <w:LsdException Locked="false" Priority="51" Name="Grid Table 6 Colorful"/>
| + | |
| − | <w:LsdException Locked="false" Priority="52" Name="Grid Table 7 Colorful"/>
| + | |
| − | <w:LsdException Locked="false" Priority="46"
| + | |
| − | Name="Grid Table 1 Light Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="51"
| + | |
| − | Name="Grid Table 6 Colorful Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="52"
| + | |
| − | Name="Grid Table 7 Colorful Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="46"
| + | |
| − | Name="Grid Table 1 Light Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="51"
| + | |
| − | Name="Grid Table 6 Colorful Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="52"
| + | |
| − | Name="Grid Table 7 Colorful Accent 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="46"
| + | |
| − | Name="Grid Table 1 Light Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="51"
| + | |
| − | Name="Grid Table 6 Colorful Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="52"
| + | |
| − | Name="Grid Table 7 Colorful Accent 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="46"
| + | |
| − | Name="Grid Table 1 Light Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="51"
| + | |
| − | Name="Grid Table 6 Colorful Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="52"
| + | |
| − | Name="Grid Table 7 Colorful Accent 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="46"
| + | |
| − | Name="Grid Table 1 Light Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="51"
| + | |
| − | Name="Grid Table 6 Colorful Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="52"
| + | |
| − | Name="Grid Table 7 Colorful Accent 5"/>
| + | |
| − | <w:LsdException Locked="false" Priority="46"
| + | |
| − | Name="Grid Table 1 Light Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="51"
| + | |
| − | Name="Grid Table 6 Colorful Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="52"
| + | |
| − | Name="Grid Table 7 Colorful Accent 6"/>
| + | |
| − | <w:LsdException Locked="false" Priority="46" Name="List Table 1 Light"/>
| + | |
| − | <w:LsdException Locked="false" Priority="47" Name="List Table 2"/>
| + | |
| − | <w:LsdException Locked="false" Priority="48" Name="List Table 3"/>
| + | |
| − | <w:LsdException Locked="false" Priority="49" Name="List Table 4"/>
| + | |
| − | <w:LsdException Locked="false" Priority="50" Name="List Table 5 Dark"/>
| + | |
| − | <w:LsdException Locked="false" Priority="51" Name="List Table 6 Colorful"/>
| + | |
| − | <w:LsdException Locked="false" Priority="52" Name="List Table 7 Colorful"/>
| + | |
| − | <w:LsdException Locked="false" Priority="46"
| + | |
| − | Name="List Table 1 Light Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="47" Name="List Table 2 Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="48" Name="List Table 3 Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="49" Name="List Table 4 Accent 1"/>
| + | |
| − | <w:LsdException Locked="false" Priority="50" Name="List Table 5 Dark Accent 1"/>
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| | | | |
| − | <p class=MsoNormal align=center style='text-align:center;line-height:150%'><b | + | <p class=MsoNormal align=center style='text-align:center;line-height:150%'><b><span |
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| − | 150%;font-family:"Calibri",sans-serif;mso-ascii-theme-font:major-latin; | + | color:#C00000'>Methods</span></b></p> |
| − | mso-hansi-theme-font:major-latin;mso-bidi-font-family:Arial;color:#C00000;
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| − | mso-bidi-font-style:italic'>Experiments and Protocols</span></b><b
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| | | | |
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| | | | |
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| − | line-height:150%;font-family:"Comic Sans MS";mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Comic Sans MS";color:black'>Oscillator System</span></p> |
| − | color:black;mso-bidi-font-style:italic'>Oscillator System</span><span | + | |
| − | style='font-size:16.0pt;line-height:150%;font-family:"Comic Sans MS";
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| | | | |
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| − | line-height:150%;font-family:"Garamond",serif;mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Garamond",serif;color:black'>We plan oscillations |
| − | color:black'>We plan oscillations using western blots and densitometry analysis | + | using western blots and densitometry analysis to track the different |
| − | to track the different phosphorylation states of <span class=SpellE>KaiC</span>. | + | phosphorylation states of KaiC. In order to synchronize a population of cells, |
| − | In order to synchronize a population of cells, we used M9 minimal media with no | + | we used M9 minimal media with no carbon supplements in order to limit the ATP |
| − | carbon supplements in order to limit the ATP provided to the E.coli cells. This | + | provided to the E.coli cells. This shock was conducted for both one and six |
| − | shock was conducted for both one and six hours (See results). </span><span | + | hours (See results). </span></p> |
| − | style='font-size:14.0pt;line-height:150%;font-family:"Garamond",serif;
| + | |
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| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:14.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Garamond",serif;mso-fareast-font-family:"Times New Roman"; | + | line-height:150%;font-family:"Garamond",serif'> </span></p> |
| − | mso-bidi-font-family:"Times New Roman"'><o:p> </o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:14.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Garamond",serif;mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Garamond",serif;color:black'>In addition to |
| − | color:black'>In addition to examining oscillations of our <span class=SpellE>KaiABC</span> | + | examining oscillations of our KaiABC biobrick, we also aimed to optimize these |
| − | <span class=SpellE>biobrick</span>, we also aimed to optimize these
| + | oscillations by altering the concentrations of KaiA, which is driven under an |
| − | oscillations by altering the concentrations of <span class=SpellE>KaiA</span>, | + | L-rhamnose inducible promoter. The input to output ratio of KaiA was first |
| − | which is driven under an L-<span class=SpellE>rhamnose</span> inducible | + | investigated using a 10 fold gradient of L-rhamnose concentrations. We assayed |
| − | promoter. The input to output ratio of <span class=SpellE>KaiA</span> was first | + | the amount of KaiA produced using Bradford assay and western blots. We |
| − | investigated using a 10 fold gradient of L-<span class=SpellE>rhamnose</span> | + | estimated the correct concentrations of L-rhamnose as well as the time of |
| − | concentrations. We assayed the amount of <span class=SpellE>KaiA</span> | + | |
| − | produced using Bradford assay and western blots. We estimated the correct | + | |
| − | concentrations of L-<span class=SpellE>rhamnose</span> as well as the time of | + | |
| | induction data from the Paris-Bettencourt 2014 team. Induction was completed | | induction data from the Paris-Bettencourt 2014 team. Induction was completed |
| | for 10 hours. We additionally conducted a time-course of induction for 16 hours | | for 10 hours. We additionally conducted a time-course of induction for 16 hours |
| − | in order to determine the saturation point of <span class=SpellE>KaiA</span>, | + | in order to determine the saturation point of KaiA, and to examine the |
| − | and to examine the degradation rate of <span class=SpellE>KaiA</span> after | + | degradation rate of KaiA after removal of the inducer from the |
| − | removal of the inducer from the synchronization/shock in M9 minimal media. </span><span | + | synchronization/shock in M9 minimal media. </span></p> |
| − | style='font-size:14.0pt;line-height:150%;font-family:"Garamond",serif;
| + | |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:10.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Times",serif;mso-fareast-font-family:"Times New Roman"; | + | line-height:150%;font-family:"Times",serif'> </span></p> |
| − | mso-bidi-font-family:"Times New Roman"'><o:p> </o:p></span></p>
| + | |
| | | | |
| | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; | | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Comic Sans MS";mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Comic Sans MS";color:black'>Read-Out System</span></p> |
| − | color:black;mso-bidi-font-style:italic'>Read-Out System</span><span | + | |
| − | style='font-size:16.0pt;line-height:150%;font-family:"Comic Sans MS";
| + | |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:14.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Garamond",serif;mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Garamond",serif;color:black'>We plan to assay the |
| − | color:black'>We plan to assay the read-out system by putting together pMC002 or | + | read-out system by putting together pMC002 or pMC003 with the KaiC |
| − | pMC003 with the <span class=SpellE>KaiC</span> <span class=SpellE>phosphomimetics</span> | + | phosphomimetics pMC004-7. pMC004-7 exhibit different phosphorylation states of |
| − | pMC004-7. <span class=GramE>pMC004-7</span> exhibit different phosphorylation | + | KaiC, essentially portraying a snapshot of KaiC throughout its oscillations in |
| − | states of <span class=SpellE>KaiC</span>, essentially portraying a snapshot of <span | + | the KaiABC system. We will pair each of these phosphomimetics with pMC002, |
| − | class=SpellE>KaiC</span> throughout its oscillations in the <span class=SpellE>KaiABC</span>
| + | containing the master regulator RpaA, its activator SasA, and a response |
| − | system. We will pair each of these <span class=SpellE>phosphomimetics</span> | + | regulator RpaB (the function of this has not been clearly characterized yet). |
| − | with pMC002, containing the master regulator <span class=SpellE>RpaA</span>, | + | We will also pair each of the phosphomimetics with pMC003, exhibiting the same |
| − | its activator <span class=SpellE>SasA</span>, and a response regulator <span | + | factors RpaA, RpaB, and SasA with the addition of CikA, a negative regulator of |
| − | class=SpellE>RpaB</span> (the function of this has not been clearly
| + | RpaA. We will assay the activation and deactivation of RpaA by measuring the |
| − | characterized yet). We will also pair each of the <span class=SpellE>phosphomimetics</span> | + | fluorescence of GFP under a plate reader. Given that the LVA-Stop tagged GFP is |
| − | with pMC003, exhibiting the same factors <span class=SpellE>RpaA</span>, <span | + | directly downstream the circadian kaibc promoter, we expect differences in |
| − | class=SpellE>RpaB</span>, and <span class=SpellE>SasA</span> with the addition
| + | activation/deactivation of RpaA to directly impact the amount of GFP expressed. |
| − | of <span class=SpellE>CikA</span>, a negative regulator of <span class=SpellE>RpaA</span>. | + | As such, we expect different fluorescence readings for each of the |
| − | We will assay the activation and deactivation of <span class=SpellE>RpaA</span> | + | phosphomimetics. Furthemore, we hypothesize that pMC003 will yield a greater |
| − | by measuring the fluorescence of GFP under a plate reader. Given that the | + | contrast in fluorescence readings between each phosphomimetic, given that it is |
| − | LVA-Stop tagged GFP is directly downstream the circadian <span class=SpellE>kaibc</span> | + | a negative regulator of RpaA.</span><span style='font-size:16.0pt;line-height: |
| − | promoter, we expect differences in activation/deactivation of <span | + | 150%;font-family:"Arial",sans-serif;color:black'> </span></p> |
| − | class=SpellE>RpaA</span> to directly impact the amount of GFP expressed. As
| + | |
| − | such, we expect different fluorescence readings for each of the <span | + | |
| − | class=SpellE>phosphomimetics</span>. <span class=SpellE>Furthemore</span>, we
| + | |
| − | hypothesize that pMC003 will yield a greater contrast in fluorescence readings | + | |
| − | between each <span class=SpellE>phosphomimetic</span>, given that it is a | + | |
| − | negative regulator of <span class=SpellE>RpaA</span>.</span><span | + | |
| − | style='font-size:11.0pt;line-height:150%;font-family:"Arial",sans-serif; | + | |
| − | color:black'> </span><span style='font-size:10.0pt;line-height:150%;font-family: | + | |
| − | "Times",serif;mso-bidi-font-family:"Times New Roman";mso-no-proof:yes'><o:p></o:p></span></p>
| + | |
| | | | |
| | <p class=MsoNormal align=center style='text-align:center;line-height:150%'><span | | <p class=MsoNormal align=center style='text-align:center;line-height:150%'><span |
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| − | filled="f" stroked="f">
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| − | <v:f eqn="sum @8 21600 0"/>
| + | |
| − | <v:f eqn="prod @7 21600 pixelHeight"/>
| + | |
| − | <v:f eqn="sum @10 21600 0"/>
| + | |
| − | </v:formulas>
| + | |
| − | <v:path o:extrusionok="f" gradientshapeok="t" o:connecttype="rect"/>
| + | |
| − | <o:lock v:ext="edit" aspectratio="t"/>
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| + | |
| − | style='width:534pt;height:181.5pt;visibility:visible;mso-wrap-style:square'>
| + | |
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| + | |
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| + | |
| − | alt="https://lh4.googleusercontent.com/pWda2PyQ3OVkertP1WQ3IpbHbnRP2oYVYTWA9rbP6devD_5FxIpJpZVUHp6FyFAtENPb5vqx58NKiR6baFdfQn9WyoOgfShLyk99QO6w4r0zQiCTjOtAeyltVK2WBOf5pok=s1600" | + | |
| − | v:shapes="Picture_x0020_1"><![endif]></span><span style='font-size:10.0pt;
| + | |
| − | line-height:150%;font-family:"Times",serif;mso-fareast-font-family:"Times New Roman";
| + | |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; | | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Comic Sans MS";mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Comic Sans MS";color:black'> </span></p> |
| − | color:black;mso-bidi-font-weight:bold'>Specific Assays:</span><span | + | |
| − | style='font-size:10.0pt;line-height:150%;font-family:"Times",serif;mso-fareast-font-family:
| + | |
| − | "Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:10.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Times",serif;mso-fareast-font-family:"Times New Roman"; | + | line-height:150%;font-family:"Comic Sans MS";color:black'> </span></p> |
| − | mso-bidi-font-family:"Times New Roman"'><o:p> </o:p></span></p>
| + | |
| | | | |
| | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; | | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Comic Sans MS";mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Comic Sans MS";color:black'> </span></p> |
| − | color:black;mso-bidi-font-style:italic'>Gibson Assembly </span><span | + | |
| − | style='font-size:16.0pt;line-height:150%;font-family:"Comic Sans MS";
| + | |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:14.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Garamond",serif;mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Comic Sans MS";color:black'> </span></p> |
| − | color:black'>Gibson assembly was conducted using NEBs 2x <span class=SpellE>HiFi</span> | + | |
| − | Assembly Master Mix. Volume of Gibson was halved in order to save reagent. 50
| + | |
| − | ng of the longest fragment in each reaction was added, and other fragments were
| + | |
| − | added in ratio relative to the longest fragment. 5uL of the master mix was
| + | |
| − | used, and H2O was added to bring whole volume of reaction to 10 <span
| + | |
| − | class=SpellE>uL</span>. Reaction was incubated at 50C for 1 hour. </span><span
| + | |
| − | style='font-size:14.0pt;line-height:150%;font-family:"Garamond",serif;
| + | |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:10.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Times",serif;mso-fareast-font-family:"Times New Roman"; | + | line-height:150%;font-family:"Comic Sans MS";color:black'>Specific Assays:</span></p> |
| − | mso-bidi-font-family:"Times New Roman"'><o:p> </o:p></span></p>
| + | |
| | | | |
| | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; | | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Comic Sans MS";mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Times",serif'> </span></p> |
| − | color:black;mso-bidi-font-style:italic'>PCR </span><span style='font-size:16.0pt;
| + | |
| − | line-height:150%;font-family:"Comic Sans MS";mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span class=SpellE><span | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | style='font-size:14.0pt;line-height:150%;font-family:"Garamond",serif; | + | line-height:150%;font-family:"Comic Sans MS";color:black'>Gibson Assembly </span></p> |
| − | mso-bidi-font-family:Arial;color:black'>Phusion</span></span><span
| + | |
| − | style='font-size:14.0pt;line-height:150%;font-family:"Garamond",serif;
| + | |
| − | mso-bidi-font-family:Arial;color:black'> DNAP from <span class=SpellE>Thermo</span>
| + | |
| − | Fisher with HF buffer used for reactions. Each reaction was supplemented with
| + | |
| − | DMSO. Gradient PCRs, <span class=SpellE>miniprep</span> PCRs, and colony PCRs
| + | |
| − | were conducted with 10uL as the final reaction volume. Samples that would be
| + | |
| − | gel extracted were conducted with 50 <span class=SpellE>uL</span> as the final
| + | |
| − | reaction volume. 10 <span class=SpellE>uM</span> primers were added. 30 cycles
| + | |
| − | of PCR were conducted. See Notebook for details on cycle temperatures. </span><span
| + | |
| − | style='font-size:14.0pt;line-height:150%;font-family:"Garamond",serif;
| + | |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:10.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Times",serif;mso-fareast-font-family:"Times New Roman"; | + | line-height:150%;font-family:"Garamond",serif;color:black'>Gibson assembly was |
| − | mso-bidi-font-family:"Times New Roman"'><o:p> </o:p></span></p>
| + | conducted using NEBs 2x HiFi Assembly Master Mix. Volume of Gibson was halved |
| | + | in order to save reagent. 50 ng of the longest fragment in each reaction was |
| | + | added, and other fragments were added in ratio relative to the longest fragment. |
| | + | 5uL of the master mix was used, and H2O was added to bring whole volume of |
| | + | reaction to 10 uL. Reaction was incubated at 50C for 1 hour. </span></p> |
| | | | |
| | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; | | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Comic Sans MS";mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Times",serif'> </span></p> |
| − | color:black;mso-bidi-font-style:italic'>Gel Electrophoresis</span><span
| + | |
| − | style='font-size:16.0pt;line-height:150%;font-family:"Comic Sans MS";
| + | |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:14.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Garamond",serif;mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Comic Sans MS";color:black'>PCR </span></p> |
| − | color:black'>Gel set up in TAE buffer. 1% agarose gel used for standard | + | |
| − | electrophoresis. 0.8% gel used for smaller fragments. 0.7 <span class=SpellE>uL</span>
| + | |
| − | of EtBr added for imaging. Gels run for 30 mins at 120V. Invitrogen 1 kB ladder
| + | |
| − | used for standard protocols. </span><span style='font-size:14.0pt;line-height:
| + | |
| − | 150%;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:10.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Times",serif;mso-fareast-font-family:"Times New Roman"; | + | line-height:150%;font-family:"Garamond",serif;color:black'>Phusion DNAP from |
| − | mso-bidi-font-family:"Times New Roman"'><o:p> </o:p></span></p>
| + | Thermo Fisher with HF buffer used for reactions. Each reaction was supplemented |
| | + | with DMSO. Gradient PCRs, miniprep PCRs, and colony PCRs were conducted with |
| | + | 10uL as the final reaction volume. Samples that would be gel extracted were |
| | + | conducted with 50 uL as the final reaction volume. 10 uM primers were added. 30 |
| | + | cycles of PCR were conducted. See Notebook for details on cycle temperatures. </span></p> |
| | | | |
| | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; | | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Comic Sans MS";mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Times",serif'> </span></p> |
| − | color:black;mso-bidi-font-style:italic'>Gel Extraction </span><span
| + | |
| − | style='font-size:16.0pt;line-height:150%;font-family:"Comic Sans MS";
| + | |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span class=SpellE><span | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | style='font-size:14.0pt;line-height:150%;font-family:"Garamond",serif; | + | line-height:150%;font-family:"Comic Sans MS";color:black'>Gel Electrophoresis</span></p> |
| − | mso-bidi-font-family:Arial;color:black'>Machery</span></span><span
| + | |
| − | style='font-size:14.0pt;line-height:150%;font-family:"Garamond",serif;
| + | |
| − | mso-bidi-font-family:Arial;color:black'>-Nagel gel extraction kit used. See
| + | |
| − | notebook for complete protocol. Samples eluted in 15 <span class=SpellE>uL</span>
| + | |
| − | of elution buffer. </span><span style='font-size:14.0pt;line-height:150%;
| + | |
| − | font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:10.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Times",serif;mso-fareast-font-family:"Times New Roman"; | + | line-height:150%;font-family:"Garamond",serif;color:black'>Gel set up in TAE |
| − | mso-bidi-font-family:"Times New Roman"'><o:p> </o:p></span></p>
| + | buffer. 1% agarose gel used for standard electrophoresis. 0.8% gel used for |
| | + | smaller fragments. 0.7 uL of EtBr added for imaging. Gels run for 30 mins at |
| | + | 120V. Invitrogen 1 kB ladder used for standard protocols. </span></p> |
| | | | |
| | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; | | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Comic Sans MS";mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Times",serif'> </span></p> |
| − | color:black;mso-bidi-font-style:italic'>Transformation </span><span
| + | |
| − | style='font-size:16.0pt;line-height:150%;font-family:"Comic Sans MS";
| + | |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:14.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Garamond",serif;mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Comic Sans MS";color:black'>Gel Extraction </span></p> |
| − | color:black'>Competent cells made by team using RbCl2 and CaCl2 chemically | + | |
| − | competent protocols. </span><span style='font-size:14.0pt;line-height:150%;
| + | |
| − | font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:14.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Garamond",serif;mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Garamond",serif;color:black'>Machery-Nagel gel |
| − | color:black'>Efficiencies of competent cells were <u>4.00 x 10^5 <span | + | extraction kit used. See notebook for complete protocol. Samples eluted in 15 |
| − | class=SpellE>transformants</span>/<span class=GramE>ng <span style='text-decoration:
| + | uL of elution buffer. </span></p> |
| − | none;text-underline:none'> and</span></span></u> <u>1.15 x 10^6 <span
| + | |
| − | class=SpellE>transformants</span>/ng </u>respectively. Transformation was
| + | |
| − | conducted by incubating on ice for 30 mins and conducting a heat shock at 42 C
| + | |
| − | for 1 min. Colonies plated using glass beads method. Incubation at 37C
| + | |
| − | overnight. </span><span style='font-size:14.0pt;line-height:150%;font-family:
| + | |
| − | "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:10.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Times",serif;mso-fareast-font-family:"Times New Roman"; | + | line-height:150%;font-family:"Times",serif'> </span></p> |
| − | mso-bidi-font-family:"Times New Roman"'><o:p> </o:p></span></p>
| + | |
| | | | |
| | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; | | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Comic Sans MS";mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Comic Sans MS";color:black'>Transformation </span></p> |
| − | color:black;mso-bidi-font-style:italic'>Sequencing </span><span | + | |
| − | style='font-size:16.0pt;line-height:150%;font-family:"Comic Sans MS";
| + | |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:14.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Garamond",serif;mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Garamond",serif;color:black'>Competent cells made |
| − | color:black'>Sequencing conducted by the University of Chicago’s sequencing | + | by team using RbCl2 and CaCl2 chemically competent protocols. </span></p> |
| − | centers. </span><span style='font-size:14.0pt;line-height:150%;font-family:
| + | |
| − | "Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:10.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Times",serif;mso-fareast-font-family:"Times New Roman"; | + | line-height:150%;font-family:"Garamond",serif;color:black'>Efficiencies of |
| − | mso-bidi-font-family:"Times New Roman"'><o:p> </o:p></span></p>
| + | competent cells were <u>4.00 x 10^5 transformants/ng </u> and <u>1.15 |
| | + | x 10^6 transformants/ng </u>respectively. Transformation was conducted by |
| | + | incubating on ice for 30 mins and conducting a heat shock at 42 C for 1 min. |
| | + | Colonies plated using glass beads method. Incubation at 37C overnight. </span></p> |
| | | | |
| | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; | | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Comic Sans MS";mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Times",serif'> </span></p> |
| − | color:black;mso-bidi-font-style:italic'>Growing cultures</span><span
| + | |
| − | style='font-size:16.0pt;line-height:150%;font-family:"Comic Sans MS";
| + | |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:14.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Garamond",serif;mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Comic Sans MS";color:black'>Sequencing </span></p> |
| − | color:black'>Cultures grown for induction were incubated at 37C. OD600 for | + | |
| − | initial assays were measured to be around 1.5. In latter assays, cells were
| + | |
| − | diluted to be OD 600 ~0.6-0.7. Initial cultures were always grown in LB. </span><span
| + | |
| − | style='font-size:14.0pt;line-height:150%;font-family:"Garamond",serif;
| + | |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:10.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Times",serif;mso-fareast-font-family:"Times New Roman"; | + | line-height:150%;font-family:"Garamond",serif;color:black'>Sequencing conducted |
| − | mso-bidi-font-family:"Times New Roman"'><o:p> </o:p></span></p>
| + | by the University of Chicago Comprehensive Cancer Center’s sequencing centers. </span></p> |
| | | | |
| | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; | | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Comic Sans MS";mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Times",serif'> </span></p> |
| − | color:black;mso-bidi-font-style:italic'>Induction </span><span
| + | |
| − | style='font-size:16.0pt;line-height:150%;font-family:"Comic Sans MS";
| + | |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:14.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Garamond",serif;mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Comic Sans MS";color:black'>Growing cultures</span></p> |
| − | color:black'>Induction of <span class=SpellE>rhamnose</span> to drive <span | + | |
| − | class=SpellE>KaiA</span> expression conducted in M9 minimal media with
| + | |
| − | supplements: 0.4% glycerol and 0.1% <span class=SpellE>casamino</span> acids.
| + | |
| − | Induction solutions made with varying concentrations of <span class=SpellE>rhamnose</span>
| + | |
| − | in order to characterize the input-output ratio of <span class=SpellE>rhamnose</span>
| + | |
| − | to <span class=SpellE>KaiA</span>. This was significant for us to be able to
| + | |
| − | vary the concentrations of <span class=SpellE>KaiA</span> and to develop more
| + | |
| − | robust oscillations. Induction conducted for 10 hours at 30C. Induction
| + | |
| − | protocol followed from Silver Lab (Chen, A. H., <span class=SpellE>Lubkowicz</span>,
| + | |
| − | D., <span class=SpellE><span class=GramE>Yeong</span></span>, V., Chang, R. L.,
| + | |
| − | & Silver, P. a. (2015a). <span class=SpellE>Transplantability</span> of a
| + | |
| − | circadian clock to a <span class=SpellE>noncircadian</span> organism. Science
| + | |
| − | Advance, 1(5), 1–6. </span><a href="http://doi.org/10.1126/sciadv.1500358"><span
| + | |
| − | style='font-size:14.0pt;line-height:150%;font-family:"Garamond",serif;
| + | |
| − | mso-bidi-font-family:Arial;color:#1155CC'>http://doi.org/10.1126/sciadv.1500358</span></a><span
| + | |
| − | class=GramE><span style='font-size:14.0pt;line-height:150%;font-family:"Garamond",serif;
| + | |
| − | mso-bidi-font-family:Arial;color:black'>) .</span></span><span
| + | |
| − | style='font-size:14.0pt;line-height:150%;font-family:"Garamond",serif;
| + | |
| − | mso-bidi-font-family:Arial;color:black'> </span><span style='font-size:14.0pt;
| + | |
| − | line-height:150%;font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:10.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Times",serif;mso-fareast-font-family:"Times New Roman"; | + | line-height:150%;font-family:"Garamond",serif;color:black'>Cultures grown for |
| − | mso-bidi-font-family:"Times New Roman"'><o:p> </o:p></span></p>
| + | induction were incubated at 37C. OD600 for initial assays were measured to be |
| | + | around 1.5. In latter assays, cells were diluted to be OD 600 ~0.6-0.7. Initial |
| | + | cultures were always grown in LB. </span></p> |
| | | | |
| | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; | | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Comic Sans MS";mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Times",serif'> </span></p> |
| − | color:black;mso-bidi-font-style:italic'>Synchronization </span><span
| + | |
| − | style='font-size:16.0pt;line-height:150%;font-family:"Comic Sans MS";
| + | |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:14.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Garamond",serif;mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Comic Sans MS";color:black'>Induction </span></p> |
| − | color:black'>Synchronization to synchronize the <span class=SpellE>KaiABC</span> | + | |
| − | clock was conducted by incubating induced cells in M9 minimal media only (no
| + | |
| − | supplements). Synchronization conducted for both 1 hour and 6 hours. Silver lab
| + | |
| − | reported 1 hour synchronization while Rust lab has seen 6 hours to be adequate
| + | |
| − | for cyanobacteria. </span><span style='font-size:14.0pt;line-height:150%;
| + | |
| − | font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:10.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Times",serif;mso-fareast-font-family:"Times New Roman"; | + | line-height:150%;font-family:"Garamond",serif;color:black'>Induction of |
| − | mso-bidi-font-family:"Times New Roman"'><o:p> </o:p></span></p>
| + | rhamnose to drive KaiA expression conducted in M9 minimal media with |
| | + | supplements: 0.4% glycerol and 0.1% casamino acids. Induction solutions made |
| | + | with varying concentrations of rhamnose in order to characterize the |
| | + | input-output ratio of rhamnose to KaiA. This was significant for us to be able |
| | + | to vary the concentrations of KaiA and to develop more robust oscillations. |
| | + | Induction conducted for 10 hours at 30C. Induction protocol followed from |
| | + | Silver Lab (Chen, A. H., Lubkowicz, D., Yeong, V., Chang, R. L., & Silver, |
| | + | P. a. (2015a). Transplantability of a circadian clock to a noncircadian |
| | + | organism. Science Advance, 1(5), 1–6. </span><a |
| | + | href="http://doi.org/10.1126/sciadv.1500358"><span style='font-size:16.0pt; |
| | + | line-height:150%;font-family:"Garamond",serif;color:#1155CC'>http://doi.org/10.1126/sciadv.1500358</span></a><span |
| | + | style='font-size:16.0pt;line-height:150%;font-family:"Garamond",serif; |
| | + | color:black'>) . </span></p> |
| | | | |
| | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; | | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Comic Sans MS";mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Times",serif'> </span></p> |
| − | color:black;mso-bidi-font-style:italic'>Oscillation Time Course </span><span
| + | |
| − | style='font-size:16.0pt;line-height:150%;font-family:"Comic Sans MS";
| + | |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:14.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Garamond",serif;mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Comic Sans MS";color:black'>Synchronization </span></p> |
| − | color:black'>Following synchronization, cells were reintroduced in M9 media | + | |
| − | with 1mM leucine and 0.5% succinate supplements to allow for slow growth. Oscillations
| + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | were tracked by freezing 2 mL cell samples every 6 hours in a -80C freezer. The
| + | line-height:150%;font-family:"Garamond",serif;color:black'>Synchronization to |
| − | time duration for tracking oscillations was set at 3 days. </span><span
| + | synchronize the KaiABC clock was conducted by incubating induced cells in M9 |
| − | style='font-size:14.0pt;line-height:150%;font-family:"Garamond",serif; | + | minimal media only (no supplements or inducer). Synchronization conducted for |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | both 1 hour and 6 hours. Silver lab reported 1 hour synchronization while Rust |
| | + | lab has seen 6 hours to be adequate for cyanobacteria. </span></p> |
| | + | |
| | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| | + | line-height:150%;font-family:"Times",serif'> </span></p> |
| | + | |
| | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| | + | line-height:150%;font-family:"Comic Sans MS";color:black'>Oscillation Time |
| | + | Course </span></p> |
| | + | |
| | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| | + | line-height:150%;font-family:"Garamond",serif;color:black'>Following |
| | + | synchronization, cells were reintroduced in M9 media with 1mM leucine and 0.5% |
| | + | succinate supplements to allow for slow growth. Oscillations were tracked by |
| | + | freezing 2 mL cell samples every 6 hours in a -80C freezer. The time duration |
| | + | for tracking oscillations was set at 3 days. No rhamnose was added back, due to |
| | + | Silver lab’s reports of desynchronization of oscillations attributed to reintroduction |
| | + | of inducer with their construct.</span></p> |
| | | | |
| | <p class=MsoNormal style='margin-bottom:12.0pt;line-height:150%'><span | | <p class=MsoNormal style='margin-bottom:12.0pt;line-height:150%'><span |
| − | style='font-size:10.0pt;line-height:150%;font-family:"Times",serif;mso-fareast-font-family: | + | style='font-size:16.0pt;line-height:150%;font-family:"Times",serif'> </span></p> |
| − | "Times New Roman";mso-bidi-font-family:"Times New Roman"'><o:p> </o:p></span></p>
| + | |
| | | | |
| | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; | | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Comic Sans MS";mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Comic Sans MS";color:black'>Western Blot</span></p> |
| − | color:black;mso-bidi-font-style:italic'>Western Blot</span><span | + | |
| − | style='font-size:16.0pt;line-height:150%;font-family:"Comic Sans MS";
| + | |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:14.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Garamond",serif;mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Garamond",serif;color:black'>In order to track |
| − | color:black'>In order to track expression of <span class=SpellE>KaiA<span | + | expression of KaiA,B,C, and phosphorylation states of KaiC western blots were |
| − | class=GramE>,B,C</span></span>, and phosphorylation states of <span
| + | conducted. For simple expression assays, such as when characterizing the pRha |
| − | class=SpellE>KaiC</span> western blots were conducted. For simple expression
| + | promoter and quantifying KaiA expression, a 14-20% BioRad precast protein gel |
| − | assays, such as when characterizing the <span class=SpellE>pRha</span> promoter | + | was used during SDS page gel run. For tracking different phoshphorylation |
| − | and quantifying <span class=SpellE>KaiA</span> expression, a 14-20% <span | + | states of KaiC, a 4-7% BioRad precast protein gel was used. Bradford assays |
| − | class=SpellE>BioRad</span> precast protein gel was used during SDS page gel
| + | were conducted before SDS-page in order to standardize the amount of protein |
| − | run. For tracking different <span class=SpellE>phoshphorylation</span> states | + | added per sample. A positive control of cyanobacterial lysate, and a negative |
| − | of <span class=SpellE>KaiC</span>, a 4-7% <span class=SpellE>BioRad</span> | + | control of E.coli cells containing an empty Cam circular backbone were run. |
| − | precast protein gel was used. Bradford assays were conducted before SDS-page in | + | Purified protein samples provided by the Rust lab were run to quantify amount |
| − | order to standardize the amount of protein added per sample. A positive control | + | of KaiA, B, and C expressed by transformed E.coli cells. Transfer accomplished |
| − | of cyanobacterial lysate, and a negative control of E.coli cells containing an | + | using PVDF membranes. Membrane sandwich apparatus was prepared under Towbin |
| − | empty Cam circular backbone were run. Purified protein samples provided by the | + | |
| − | Rust lab were run to quantify amount of <span class=SpellE>KaiA</span>, B, and | + | |
| − | C expressed by transformed E.coli cells. Transfer accomplished using PVDF | + | |
| − | membranes. Membrane sandwich apparatus was prepared under <span class=SpellE>Towbin</span> | + | |
| | transfer buffer. Blocking to prevent non-specific binding of antibodies was | | transfer buffer. Blocking to prevent non-specific binding of antibodies was |
| | conducted using TBST+2% dry milk. This was followed by a stain of primary | | conducted using TBST+2% dry milk. This was followed by a stain of primary |
| Line 1,222: |
Line 325: |
| | membranes were washed using wash buffer then stained with anti-goat HRP | | membranes were washed using wash buffer then stained with anti-goat HRP |
| | secondary antibody allowing us to visualize the primary antibody stain. The | | secondary antibody allowing us to visualize the primary antibody stain. The |
| − | membranes were washed then imaged using <span class=SpellE>chemilumenescnece</span> | + | membranes were washed then imaged using chemilumenescnece imaging systems. </span></p> |
| − | imaging systems. </span><span style='font-size:14.0pt;line-height:150%; | + | |
| − | font-family:"Garamond",serif;mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:10.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Times",serif;mso-fareast-font-family:"Times New Roman"; | + | line-height:150%;font-family:"Times",serif'> </span></p> |
| − | mso-bidi-font-family:"Times New Roman"'><o:p> </o:p></span></p>
| + | |
| | | | |
| | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; | | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Comic Sans MS";mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Comic Sans MS";color:black'>Imaging and |
| − | color:black;mso-bidi-font-style:italic'>Imaging and Quantification</span><span | + | Quantification</span></p> |
| − | style='font-size:16.0pt;line-height:150%;font-family:"Comic Sans MS";
| + | |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><span style='font-size:14.0pt; | + | <p class=MsoNormal style='line-height:150%'><span style='font-size:16.0pt; |
| − | line-height:150%;font-family:"Garamond",serif;mso-bidi-font-family:Arial; | + | line-height:150%;font-family:"Garamond",serif;color:black'>Densitometry of |
| − | color:black'>Densitometry of images was conducted in ImageJ. <i> </i></span><span | + | images was conducted in ImageJ. For the induction time course and the oscillation |
| − | style='font-size:14.0pt;line-height:150%;font-family:"Garamond",serif;
| + | assays (oscillation data not up on wiki, but to be presented at the Jamboree), |
| − | mso-bidi-font-family:"Times New Roman"'><o:p></o:p></span></p>
| + | the nanogram amount of each protein was determined by calibrating to a |
| | + | logarithm curve of purified standards of known concentration ran on the same |
| | + | Western blot. This allowed for determination of accurate stoichiometric ratios |
| | + | between KaiA on the rhamnose inducible promoter and KaiB and KaiC, both on the |
| | + | constitutive promoter. Normalization for OD was performed for post-shock time |
| | + | points, including oscillation time points, to control for changes in cell |
| | + | density and protein degradation after induction of KaiA was turned off.</span></p> |
| | | | |
| | <p class=MsoNormal style='line-height:150%'><span style='font-size:10.0pt; | | <p class=MsoNormal style='line-height:150%'><span style='font-size:10.0pt; |
| − | line-height:150%;font-family:"Times",serif;mso-fareast-font-family:"Times New Roman"; | + | line-height:150%;font-family:"Times",serif'> </span></p> |
| − | mso-bidi-font-family:"Times New Roman"'><o:p> </o:p></span></p>
| + | |
| | | | |
| − | <p class=MsoNormal style='line-height:150%'><o:p> </o:p></p> | + | <p class=MsoNormal style='line-height:150%'> </p> |
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| | </div> | | </div> |
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| | </html> | | </html> |
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| | {{UChicago/Footer}} | | {{UChicago/Footer}} |
