Difference between revisions of "Team:CityU HK/Results"

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<div class="paragraph" style="text-align:justify;"><font size="1"><strong><span "font-size:11.0pt;="" line-height:107%;font-family:&quot;calibri&quot;,sans-serif;mso-ascii-theme-font:minor-latin;="" mso-fareast-font-family:&#26032;&#32048;&#26126;&#39636;;mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:="" minor-latin;mso-bidi-font-family:&quot;times="" roman&quot;;mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"=""><font color="#2a2a2a">Figure 2. Expression analysis of L8-UV5 promoter-GFP biobrick (BBa_K1695053). </font></span></strong><span "font-size:11.0pt;line-height:107%;font-family:&quot;calibri&quot;,sans-serif;="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:&#26032;&#32048;&#26126;&#39636;;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:&quot;times="" roman&quot;;="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa;mso-bidi-font-weight:bold"=""><font color="#2a2a2a">&nbsp;</font><font color="#2a2a2a">(A) Activation of GFP expression in the L8-UV5 promoter construct is dependent on IPTG concentrations below 1 mM. (B) Fluorescence response of the L8-UV5 promoter-GFP gene construct (BBa_K1695053) was measured over a 4.5-h time course under a range of IPTG concentrations (from 0.01 mM to 10 mM). Results show that induction of the L8-UV5 promoter is independent of IPTG at concentrations above 1 mM, where LacI is saturated and promoter is fully turned on<em>.&nbsp;</em></font></span><br /></font><br /><font size="4"><br /><span "font-size:11.0pt;line-height:="" 107%;font-family:&quot;calibri&quot;,sans-serif;mso-ascii-theme-font:minor-latin;="" mso-fareast-font-family:&#26032;&#32048;&#26126;&#39636;;mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:="" minor-latin;mso-bidi-font-family:&quot;times="" roman&quot;;mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style=""><font color="#2a2a2a">Results in Figure 2 show that GFP expression in the L8-UV5 promoter-GFP construct is dependent on IPTG concentrations below 1 mM. At IPTG concentrations above 1 mM, expression of GFP has reached a maxima regardless of the sampling time points.</font></span></font><span "font-size:11.0pt;line-height:107%;font-family:&quot;calibri&quot;,sans-serif;="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:&#26032;&#32048;&#26126;&#39636;;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:&quot;times="" roman&quot;;="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa;mso-bidi-font-weight:bold"="" style=""><br /></span></div>
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<div class="paragraph" style="text-align:justify;"><font size="3"><strong><span "font-size:11.0pt;="" line-height:107%;font-family:&quot;calibri&quot;,sans-serif;mso-ascii-theme-font:minor-latin;="" mso-fareast-font-family:&#26032;&#32048;&#26126;&#39636;;mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:="" minor-latin;mso-bidi-font-family:&quot;times="" roman&quot;;mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"=""><font color="#2a2a2a">Figure 2. Expression analysis of L8-UV5 promoter-GFP biobrick (BBa_K1695053). </font></span></strong><span "font-size:11.0pt;line-height:107%;font-family:&quot;calibri&quot;,sans-serif;="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:&#26032;&#32048;&#26126;&#39636;;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:&quot;times="" roman&quot;;="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa;mso-bidi-font-weight:bold"=""><font color="#2a2a2a">&nbsp;</font><font color="#2a2a2a">(A) Activation of GFP expression in the L8-UV5 promoter construct is dependent on IPTG concentrations below 1 mM. (B) Fluorescence response of the L8-UV5 promoter-GFP gene construct (BBa_K1695053) was measured over a 4.5-h time course under a range of IPTG concentrations (from 0.01 mM to 10 mM). Results show that induction of the L8-UV5 promoter is independent of IPTG at concentrations above 1 mM, where LacI is saturated and promoter is fully turned on<em>.&nbsp;</em></font></span><br /></font><br /><font size="4"><br /><span "font-size:11.0pt;line-height:="" 107%;font-family:&quot;calibri&quot;,sans-serif;mso-ascii-theme-font:minor-latin;="" mso-fareast-font-family:&#26032;&#32048;&#26126;&#39636;;mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:="" minor-latin;mso-bidi-font-family:&quot;times="" roman&quot;;mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style=""><font color="#2a2a2a">Results in Figure 2 show that GFP expression in the L8-UV5 promoter-GFP construct is dependent on IPTG concentrations below 1 mM. At IPTG concentrations above 1 mM, expression of GFP has reached a maxima regardless of the sampling time points.</font></span></font><span "font-size:11.0pt;line-height:107%;font-family:&quot;calibri&quot;,sans-serif;="" mso-ascii-theme-font:minor-latin;mso-fareast-font-family:&#26032;&#32048;&#26126;&#39636;;mso-fareast-theme-font:="" minor-fareast;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:&quot;times="" roman&quot;;="" mso-bidi-theme-font:minor-bidi;mso-ansi-language:en-us;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa;mso-bidi-font-weight:bold"="" style=""><br /></span></div>
  
 
<h2 class="wsite-content-title" style="text-align:left;"><span "font-size:14.0pt;="" line-height:107%;font-family:&quot;calibri&quot;,sans-serif;mso-ascii-theme-font:minor-latin;="" mso-fareast-font-family:&#26032;&#32048;&#26126;&#39636;;mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:="" minor-latin;mso-bidi-font-family:&quot;times="" roman&quot;;mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"=""><u><font size="6">Improved/ expanded characterization of LacY-LacZ biobrick (BBa_S04055)</font></u></span><br /><span style=""></span></h2>
 
<h2 class="wsite-content-title" style="text-align:left;"><span "font-size:14.0pt;="" line-height:107%;font-family:&quot;calibri&quot;,sans-serif;mso-ascii-theme-font:minor-latin;="" mso-fareast-font-family:&#26032;&#32048;&#26126;&#39636;;mso-fareast-theme-font:minor-fareast;mso-hansi-theme-font:="" minor-latin;mso-bidi-font-family:&quot;times="" roman&quot;;mso-bidi-theme-font:minor-bidi;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"=""><u><font size="6">Improved/ expanded characterization of LacY-LacZ biobrick (BBa_S04055)</font></u></span><br /><span style=""></span></h2>
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<h2 class="wsite-content-title" style="text-align:right;"><strong style=""><u style=""><span "font-size:18.0pt;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:="" &#26032;&#32048;&#26126;&#39636;;mso-fareast-theme-font:minor-fareast;color:#333333;mso-ansi-language:en-us;="" mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style=""><font size="6">B. Western Blot Analysis</font><font size="5">&nbsp;</font></span></u></strong></h2>
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<h2 class="wsite-content-title" style="text-align:left;"><strong style=""><u style=""><span "font-size:18.0pt;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:="" &#26032;&#32048;&#26126;&#39636;;mso-fareast-theme-font:minor-fareast;color:#333333;mso-ansi-language:en-us;="" mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style=""><font size="6">B. Western Blot Analysis</font><font size="5">&nbsp;</font></span></u></strong></h2>
  
 
<div class="paragraph" style="text-align:justify;"><font size="3">Western blot analysis showed that the β-galactosidase protein (LacZ) (indicated by the arrow) is expressed at a significantly higher level in recombinant <em style="">E. coli</em> cells (BBa_S04055) relative to the control (Figure 4) and is in agreement with the <em style="">lacZ</em> mRNA results described in Figure 3.</font></div>
 
<div class="paragraph" style="text-align:justify;"><font size="3">Western blot analysis showed that the β-galactosidase protein (LacZ) (indicated by the arrow) is expressed at a significantly higher level in recombinant <em style="">E. coli</em> cells (BBa_S04055) relative to the control (Figure 4) and is in agreement with the <em style="">lacZ</em> mRNA results described in Figure 3.</font></div>
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<div class="paragraph" style="text-align:left;"><font size="2"><strong style="color: rgb(42, 42, 42);"><span "mso-bidi-font-size:="" 12.0pt;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:&quot;times="" roman&quot;;="" color:black;mso-font-kerning:0pt"="">Figure 5. Expression of </span></strong><strong style="color: rgb(42, 42, 42);"><span "mso-bidi-font-size:="" 12.0pt;font-family:&quot;arial&quot;,sans-serif;color:#333333;mso-fareast-language:zh-hk"="" style="">&beta;</span></strong><strong style="color: rgb(42, 42, 42);"><span style="">-galactosidase</span></strong><strong style="color: rgb(42, 42, 42);"><span "mso-bidi-font-size:="" 12.0pt;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:&quot;times="" roman&quot;;="" color:black;mso-font-kerning:0pt"="" style=""> as measured by the ONPG assay.</span></strong><br /><span "mso-bidi-font-size:12.0pt;font-family:&quot;arial&quot;,sans-serif;="" color:#333333;mso-fareast-language:zh-hk"=""><font size="3"><font color="#2a2a2a">Control</font> </span><strong><font color="#3387a2"><span "mso-bidi-font-size:12.0pt;="" font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:&quot;times="" roman&quot;;="" color:black;mso-font-kerning:0pt"="" style="">(</span><span "mso-bidi-font-size:="" 12.0pt;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:&quot;times="" roman&quot;;="" color:#4f81bd;mso-themecolor:accent1;mso-font-kerning:0pt"="" style="">BLUE</span></font><span "mso-bidi-font-size:12.0pt;font-family:&quot;arial&quot;,sans-serif;="" mso-fareast-font-family:&quot;times="" roman&quot;;color:black;mso-font-kerning:0pt"=""><font color="#3387a2">)</font> </span></strong><em style="color: rgb(42, 42, 42);"><span "mso-bidi-font-size:12.0pt;font-family:&quot;arial&quot;,sans-serif;="" color:#333333;mso-fareast-language:zh-hk"="" style="">E. coli</span></em><span "mso-bidi-font-size:12.0pt;font-family:&quot;arial&quot;,sans-serif;color:#333333;="" mso-fareast-language:zh-hk"="" style="color: rgb(42, 42, 42);"> cells and </span><span "mso-bidi-font-size:12.0pt;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:="" &quot;times="" roman&quot;;color:black;mso-font-kerning:0pt"=""><font color="#2a2a2a">BBa_S04055 recombinant cells</font><strong><font color="#da4444"> (</font></strong></span><span "mso-bidi-font-size:12.0pt;font-family:="" &quot;arial&quot;,sans-serif;mso-fareast-font-family:&quot;times="" roman&quot;;color:#c0504d;="" mso-themecolor:accent2;mso-font-kerning:0pt"=""><strong><font color="#da4444">RED</font></strong></span><span "mso-bidi-font-size:12.0pt;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:="" &quot;times="" roman&quot;;color:black;mso-font-kerning:0pt"=""><strong><font color="#da4444">)</font></strong> </span><span "mso-bidi-font-size:12.0pt;="" font-family:&quot;arial&quot;,sans-serif;color:#333333;mso-fareast-language:zh-hk"="" style="color: rgb(42, 42, 42);">at </span><span "mso-bidi-font-size:12.0pt;font-family:&quot;arial&quot;,sans-serif;="" color:black;mso-themecolor:text1;mso-font-kerning:12.0pt;mso-fareast-language:="" zh-hk"="" style="color: rgb(42, 42, 42);">OD600</span><span "mso-bidi-font-size:12.0pt;="" font-family:&quot;arial&quot;,sans-serif;color:#333333;mso-fareast-language:zh-hk"="" style="color: rgb(42, 42, 42);">=0.3 were harvested and lysed to release intracellular &beta;</span><span style="color: rgb(42, 42, 42);">-galactosidase, which was then measured for its activity by the ONPG assay.</span><span "mso-bidi-font-size:12.0pt;font-family:&quot;arial&quot;,sans-serif;="" color:#333333;mso-fareast-language:zh-hk"="" style="color: rgb(42, 42, 42);"> The </span><span style="color: rgb(42, 42, 42);">conversion</span><span "mso-bidi-font-size:="" 12.0pt;font-family:&quot;arial&quot;,sans-serif;color:black;mso-font-kerning:0pt;="" mso-fareast-language:zh-hk"="" style="color: rgb(42, 42, 42);"> was measured by A<sub>420</sub> at 15 minute interval.</span></font><u style="font-size: x-small; color: rgb(42, 42, 42);"><span "font-size:13.5pt;font-family:&quot;arial&quot;,sans-serif;="" color:#333333;mso-fareast-language:zh-hk"="" style=""></span></u><br /><span style=""></span><br /><span style=""></span></div>
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<div class="paragraph" style="text-align:left;"><font size="4"><strong style="color: rgb(42, 42, 42);"><span "mso-bidi-font-size:="" 12.0pt;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:&quot;times="" roman&quot;;="" color:black;mso-font-kerning:0pt"="">Figure 5. Expression of </span></strong><strong style="color: rgb(42, 42, 42);"><span "mso-bidi-font-size:="" 12.0pt;font-family:&quot;arial&quot;,sans-serif;color:#333333;mso-fareast-language:zh-hk"="" style="">&beta;</span></strong><strong style="color: rgb(42, 42, 42);"><span style="">-galactosidase</span></strong><strong style="color: rgb(42, 42, 42);"><span "mso-bidi-font-size:="" 12.0pt;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:&quot;times="" roman&quot;;="" color:black;mso-font-kerning:0pt"="" style=""> as measured by the ONPG assay.</span></strong><br /><span "mso-bidi-font-size:12.0pt;font-family:&quot;arial&quot;,sans-serif;="" color:#333333;mso-fareast-language:zh-hk"=""><font size="3"><font color="#2a2a2a">Control</font> </span><strong><font color="#3387a2"><span "mso-bidi-font-size:12.0pt;="" font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:&quot;times="" roman&quot;;="" color:black;mso-font-kerning:0pt"="" style="">(</span><span "mso-bidi-font-size:="" 12.0pt;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:&quot;times="" roman&quot;;="" color:#4f81bd;mso-themecolor:accent1;mso-font-kerning:0pt"="" style="">BLUE</span></font><span "mso-bidi-font-size:12.0pt;font-family:&quot;arial&quot;,sans-serif;="" mso-fareast-font-family:&quot;times="" roman&quot;;color:black;mso-font-kerning:0pt"=""><font color="#3387a2">)</font> </span></strong><em style="color: rgb(42, 42, 42);"><span "mso-bidi-font-size:12.0pt;font-family:&quot;arial&quot;,sans-serif;="" color:#333333;mso-fareast-language:zh-hk"="" style="">E. coli</span></em><span "mso-bidi-font-size:12.0pt;font-family:&quot;arial&quot;,sans-serif;color:#333333;="" mso-fareast-language:zh-hk"="" style="color: rgb(42, 42, 42);"> cells and </span><span "mso-bidi-font-size:12.0pt;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:="" &quot;times="" roman&quot;;color:black;mso-font-kerning:0pt"=""><font color="#2a2a2a">BBa_S04055 recombinant cells</font><strong><font color="#da4444"> (</font></strong></span><span "mso-bidi-font-size:12.0pt;font-family:="" &quot;arial&quot;,sans-serif;mso-fareast-font-family:&quot;times="" roman&quot;;color:#c0504d;="" mso-themecolor:accent2;mso-font-kerning:0pt"=""><strong><font color="#da4444">RED</font></strong></span><span "mso-bidi-font-size:12.0pt;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:="" &quot;times="" roman&quot;;color:black;mso-font-kerning:0pt"=""><strong><font color="#da4444">)</font></strong> </span><span "mso-bidi-font-size:12.0pt;="" font-family:&quot;arial&quot;,sans-serif;color:#333333;mso-fareast-language:zh-hk"="" style="color: rgb(42, 42, 42);">at </span><span "mso-bidi-font-size:12.0pt;font-family:&quot;arial&quot;,sans-serif;="" color:black;mso-themecolor:text1;mso-font-kerning:12.0pt;mso-fareast-language:="" zh-hk"="" style="color: rgb(42, 42, 42);">OD600</span><span "mso-bidi-font-size:12.0pt;="" font-family:&quot;arial&quot;,sans-serif;color:#333333;mso-fareast-language:zh-hk"="" style="color: rgb(42, 42, 42);">=0.3 were harvested and lysed to release intracellular &beta;</span><span style="color: rgb(42, 42, 42);">-galactosidase, which was then measured for its activity by the ONPG assay.</span><span "mso-bidi-font-size:12.0pt;font-family:&quot;arial&quot;,sans-serif;="" color:#333333;mso-fareast-language:zh-hk"="" style="color: rgb(42, 42, 42);"> The </span><span style="color: rgb(42, 42, 42);">conversion</span><span "mso-bidi-font-size:="" 12.0pt;font-family:&quot;arial&quot;,sans-serif;color:black;mso-font-kerning:0pt;="" mso-fareast-language:zh-hk"="" style="color: rgb(42, 42, 42);"> was measured by A<sub>420</sub> at 15 minute interval.</span></font><u style="font-size: x-small; color: rgb(42, 42, 42);"><span "font-size:13.5pt;font-family:&quot;arial&quot;,sans-serif;="" color:#333333;mso-fareast-language:zh-hk"="" style=""></span></u><br /><span style=""></span><br /><span style=""></span></div>
  
  
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<div class="paragraph" style="text-align:justify;"><span "font-family:="" &quot;arial&quot;,sans-serif;color:black;mso-themecolor:text1;mso-font-kerning:12.0pt"=""></span><font size="2"><span "font-family:="" &quot;arial&quot;,sans-serif;color:black;mso-themecolor:text1;mso-font-kerning:12.0pt"="" style="color: rgb(42, 42, 42);"><span "font-family:&quot;arial&quot;,sans-serif;="" color:black;mso-themecolor:text1;mso-font-kerning:12.0pt"=""><strong>Figure 6. Cell lysis upon induction of lysis cassette by IPTG in&nbsp;<em>E. coli</em>&nbsp;cells.&nbsp;</strong></span>Recombinant<em> E. coli</em> carrying lysis cassette Smut&lambda;-R&lambda;-Rz&lambda;&nbsp;(BBa_1695038) was cultured in minimal medium (supplemented with 0.2% glucose and </span><span "font-family:="" &quot;arial&quot;,sans-serif;color:red;mso-font-kerning:12.0pt"="" style="color: rgb(42, 42, 42);">0.5(0.02%)&nbsp;</span><span "font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:="" text1;mso-font-kerning:12.0pt"="" style="color: rgb(42, 42, 42);">casamino acid). IPTG at various concentrations (0 mM, </span><span "font-size:80.0pt;font-family:symbol;="" mso-ascii-font-family:arial;mso-hansi-font-family:arial;mso-bidi-font-family:="" arial;color:#5b9bd5;mso-font-kerning:50.0pt;mso-char-type:symbol;mso-symbol-font-family:="" symbol"=""><span id="selectionBoundary_1442552977967_1015325584448874" class="rangySelectionBoundary" style="line-height: 0; display: none;">&#65279;</span><font color="#3a96b8">&middot;</font><span id="selectionBoundary_1442552977966_4542458744253963" class="rangySelectionBoundary" style="line-height: 0; display: none;">&#65279;</span></span><span "font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:="" text1;mso-font-kerning:12.0pt"="" style="color: rgb(42, 42, 42);">) (0.2 mM,</span><span "font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:="" text1;mso-font-kerning:50.0pt"="" style="color: rgb(42, 42, 42);"> </span><span "font-size:80.0pt;font-family:symbol;mso-ascii-font-family:arial;="" mso-hansi-font-family:arial;mso-bidi-font-family:arial;color:#ed7d31;="" mso-font-kerning:12.0pt;mso-char-type:symbol;mso-symbol-font-family:symbol"=""><span id="selectionBoundary_1442553009231_3344129309989512" class="rangySelectionBoundary" style="line-height: 0; display: none;">&#65279;</span><font color="#da8044">&middot;</font><span id="selectionBoundary_1442553009231_27489691064693034" class="rangySelectionBoundary" style="line-height: 0; display: none;">&#65279;</span></span><span "font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:="" text1;mso-font-kerning:50.0pt"="" style="color: rgb(42, 42, 42);">), 1 mM,</span><span "font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:="" text1;mso-font-kerning:50.0pt"="" style="color: rgb(42, 42, 42);"> </span><span "font-size:80.0pt;font-family:symbol;mso-ascii-font-family:arial;="" mso-hansi-font-family:arial;mso-bidi-font-family:arial;color:#a5a5a5;="" mso-font-kerning:50.0pt;mso-char-type:symbol;mso-symbol-font-family:symbol"=""><font color="#818181">&middot;</font></span><span "font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:="" text1;mso-font-kerning:50.0pt"="" style="color: rgb(42, 42, 42);">) (5 mM,</span><span "font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:="" text1;mso-font-kerning:12.0pt"="" style="color: rgb(42, 42, 42);"> </span><span "font-size:80.0pt;font-family:symbol;mso-ascii-font-family:arial;="" mso-hansi-font-family:arial;mso-bidi-font-family:arial;color:#ffc000;="" mso-font-kerning:12.0pt;mso-char-type:symbol;mso-symbol-font-family:symbol"=""><font color="#e0bf5c">&middot;</font></span><span "font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:="" text1;mso-font-kerning:12.0pt"=""><font color="#2a2a2a">) was added to the bacterial culture at OD600 ~ 0.6. Samples were taken at 5-min &nbsp;and 10-min intervals from IPTG induced and uninduced cultures for </font><a title=""><font color="#2a2a2a">(A) OD600 measurements and (B) cell plating on LB solid medium (CFU count) to determine the percentage (%) of cell survival, respectively.</font></a></span><br /></font><br /></div></div>
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<div class="paragraph" style="text-align:justify;"><span "font-family:="" &quot;arial&quot;,sans-serif;color:black;mso-themecolor:text1;mso-font-kerning:12.0pt"=""></span><font size="4"><span "font-family:="" &quot;arial&quot;,sans-serif;color:black;mso-themecolor:text1;mso-font-kerning:12.0pt"="" style="color: rgb(42, 42, 42);"><span "font-family:&quot;arial&quot;,sans-serif;="" color:black;mso-themecolor:text1;mso-font-kerning:12.0pt"=""><strong>Figure 6. Cell lysis upon induction of lysis cassette by IPTG in&nbsp;<em>E. coli</em>&nbsp;cells.&nbsp;</strong></span>Recombinant<em> E. coli</em> carrying lysis cassette Smut&lambda;-R&lambda;-Rz&lambda;&nbsp;(BBa_1695038) was cultured in minimal medium (supplemented with 0.2% glucose and </span><span "font-family:="" &quot;arial&quot;,sans-serif;color:red;mso-font-kerning:12.0pt"="" style="color: rgb(42, 42, 42);">0.5(0.02%)&nbsp;</span><span "font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:="" text1;mso-font-kerning:12.0pt"="" style="color: rgb(42, 42, 42);">casamino acid). IPTG at various concentrations (0 mM, </span><span "font-size:80.0pt;font-family:symbol;="" mso-ascii-font-family:arial;mso-hansi-font-family:arial;mso-bidi-font-family:="" arial;color:#5b9bd5;mso-font-kerning:50.0pt;mso-char-type:symbol;mso-symbol-font-family:="" symbol"=""><span id="selectionBoundary_1442552977967_1015325584448874" class="rangySelectionBoundary" style="line-height: 0; display: none;">&#65279;</span><font color="#3a96b8">&middot;</font><span id="selectionBoundary_1442552977966_4542458744253963" class="rangySelectionBoundary" style="line-height: 0; display: none;">&#65279;</span></span><span "font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:="" text1;mso-font-kerning:12.0pt"="" style="color: rgb(42, 42, 42);">) (0.2 mM,</span><span "font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:="" text1;mso-font-kerning:50.0pt"="" style="color: rgb(42, 42, 42);"> </span><span "font-size:80.0pt;font-family:symbol;mso-ascii-font-family:arial;="" mso-hansi-font-family:arial;mso-bidi-font-family:arial;color:#ed7d31;="" mso-font-kerning:12.0pt;mso-char-type:symbol;mso-symbol-font-family:symbol"=""><span id="selectionBoundary_1442553009231_3344129309989512" class="rangySelectionBoundary" style="line-height: 0; display: none;">&#65279;</span><font color="#da8044">&middot;</font><span id="selectionBoundary_1442553009231_27489691064693034" class="rangySelectionBoundary" style="line-height: 0; display: none;">&#65279;</span></span><span "font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:="" text1;mso-font-kerning:50.0pt"="" style="color: rgb(42, 42, 42);">), 1 mM,</span><span "font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:="" text1;mso-font-kerning:50.0pt"="" style="color: rgb(42, 42, 42);"> </span><span "font-size:80.0pt;font-family:symbol;mso-ascii-font-family:arial;="" mso-hansi-font-family:arial;mso-bidi-font-family:arial;color:#a5a5a5;="" mso-font-kerning:50.0pt;mso-char-type:symbol;mso-symbol-font-family:symbol"=""><font color="#818181">&middot;</font></span><span "font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:="" text1;mso-font-kerning:50.0pt"="" style="color: rgb(42, 42, 42);">) (5 mM,</span><span "font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:="" text1;mso-font-kerning:12.0pt"="" style="color: rgb(42, 42, 42);"> </span><span "font-size:80.0pt;font-family:symbol;mso-ascii-font-family:arial;="" mso-hansi-font-family:arial;mso-bidi-font-family:arial;color:#ffc000;="" mso-font-kerning:12.0pt;mso-char-type:symbol;mso-symbol-font-family:symbol"=""><font color="#e0bf5c">&middot;</font></span><span "font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:="" text1;mso-font-kerning:12.0pt"=""><font color="#2a2a2a">) was added to the bacterial culture at OD600 ~ 0.6. Samples were taken at 5-min &nbsp;and 10-min intervals from IPTG induced and uninduced cultures for </font><a title=""><font color="#2a2a2a">(A) OD600 measurements and (B) cell plating on LB solid medium (CFU count) to determine the percentage (%) of cell survival, respectively.</font></a></span><br /></font><br /></div></div>
  
 
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Revision as of 11:01, 18 September 2015

Results - iGEM2015 wiki

Characterization of Lysis Gene Cassette Smutλ-Rλ-Rzλ (BBa_1695038)

Upon induction with 0.2 mM, 1 mM and 5 mM of IPTG, recombinant E. coli harboring the lysis cassette Smutλ-Rλ-Rzλ (BBa_1695038) showed a sharp drop in absorbance (A600) after 15 minutes whereas cells without IPTG induction showed continued as indicated by the steady increase in OD (Figure 6A). The results show that 0.2 mM IPTG is sufficient to initiate cell lysis after 15 minutes, and the speed of cell lysis is not increased at higher concentrations of IPTG (Figure 6B). Overall, the use of (1) OD600 measurement to determine cell growth or cell lysis (panel A) and (2) plate count (cfu; panel B) showed that the Lysis Gene Cassette is induced by IPTG and resulted in the initiation of cell lysis after 15 minutes of IPTG induction. Cells were completely lysed by 20 minutes. It would be interesting in future studies to determine if the time of cell lysis could be regulated by IPTG at concentrations below 0.2 mM.
Figure 6. Cell lysis upon induction of lysis cassette by IPTG in E. coli cells. Recombinant E. coli carrying lysis cassette Smutλ-Rλ-Rzλ (BBa_1695038) was cultured in minimal medium (supplemented with 0.2% glucose and 0.5(0.02%) casamino acid). IPTG at various concentrations (0 mM, ·) (0.2 mM, ·), 1 mM, ·) (5 mM, ·) was added to the bacterial culture at OD600 ~ 0.6. Samples were taken at 5-min  and 10-min intervals from IPTG induced and uninduced cultures for (A) OD600 measurements and (B) cell plating on LB solid medium (CFU count) to determine the percentage (%) of cell survival, respectively.