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

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<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"=""><font size="6">Characterization of the lactose-inducible promoter (BBa_K1695053)</font></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"=""><font size="6">Characterization of the lactose-inducible promoter (BBa_K1695053)</font></span><br /><span style=""></span></h2>
  
<div class="paragraph" style="text-align:left;"><br /><font color="#2a2a2a"><strong style="font-size: medium;"><em><span style="line-height: 107%;">&nbsp;</span></em></strong><span style="font-size: medium; line-height: 1.5em; text-align: justify; background-color: initial;"><font size ="3.5">The GFP reporter biobrick (BBa_K1695053) was constructed by linking
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<div class="paragraph" style="text-align:justify;"><br /><font color="#2a2a2a"><strong style="font-size: medium;"><em><span style="line-height: 107%;">&nbsp;</span></em></strong><span style="font-size: medium; line-height: 1.5em; text-align: justify; background-color: initial;"><font size ="3.5">The GFP reporter biobrick (BBa_K1695053) was constructed by linking
 
the above lacI-regulated promoter </span><span style="font-size: medium; line-height: 1.5em; text-align: justify; background-color: initial;">(BBa_K1695000)</span>
 
the above lacI-regulated promoter </span><span style="font-size: medium; line-height: 1.5em; text-align: justify; background-color: initial;">(BBa_K1695000)</span>
 
<span style="font-size: medium; line-height: 1.5em; text-align: justify; background-color: initial;">to GFP (BBa_I13504) (Figure 1). The response of this GFP reporter
 
<span style="font-size: medium; line-height: 1.5em; text-align: justify; background-color: initial;">to GFP (BBa_I13504) (Figure 1). The response of this GFP reporter
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</div></div>
 
</div></div>
  
<div class="paragraph" style="text-align:left;"><font size="4"><strong style="color: rgb(42, 42, 42);"><span "mso-bidi-font-size:="" 13.5pt;font-family:&quot;arial&quot;,sans-serif;color:#333333;mso-fareast-language:zh-hk"=""><font size ="2">Figure 3.</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=""> Quantitative RT-PCR analysis of <em style="">lacY</em> and <em style="">lacZ</em> expression in <em style="">E. coli </em>cells.</span></strong><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">&nbsp;</font></span><br /><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 size ="3"><font color="#2a2a2a">Expression of the </font><em style="color: rgb(42, 42, 42);">lacZ</em><font color="#2a2a2a"> and </font><em style="color: rgb(42, 42, 42);">lacY</em><font color="#2a2a2a"> genes was measured in control DH5&alpha; cells </font><strong><span "mso-bidi-font-size:12.0pt;font-family:="" &quot;arial&quot;,sans-serif;mso-fareast-font-family:&quot;times="" roman&quot;;color:#0f1368;="" mso-font-kerning:0pt"="" style=""><font color="#24678d">(BLUE</font></span></strong><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="#24678d">) </font></strong><font color="#2a2a2a">and BBa_S04055 recombinant </font><em style="color: rgb(42, 42, 42);">DH5&alpha;</em><font color="#2a2a2a"> cells</font><font color="#da4444"><strong> (</strong></font></span><span "mso-bidi-font-size:="" 12.0pt;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:&quot;times="" roman&quot;;="" color:#ca2323;mso-font-kerning:0pt"=""><font color="#da4444"><strong>RED</strong></font></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="#da4444"><strong>)</strong></font><font color="#2a2a2a">.</font></span><span "font-size:13.5pt;font-family:&quot;arial&quot;,sans-serif;color:#333333;="" mso-fareast-language:zh-hk"="" style="color: rgb(42, 42, 42);"> </span><span "mso-bidi-font-size:="" 9.0pt;font-family:&quot;arial&quot;,sans-serif;color:#333333;mso-fareast-language:zh-hk"="" style=""><font color="#2a2a2a"><i>Cells were cultured overnight in LB medium + antibiotic and total RNA harvested for qRT-PCR using 16S rRNA for normalization.</i></font></span><u style=""><span "font-size:13.5pt;font-family:&quot;arial&quot;,sans-serif;="" color:#333333;mso-fareast-language:zh-hk"="" style=""></span></u><br /></font><span style=""></span><br /><span style=""></span></div>
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<div class="paragraph" style="text-align:justify;"> <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"><font size="2">Figure 3. Quantitative RT-PCR analysis of <em style="">lacY</em> and <em style="">lacZ</em> expression in <em style="">E. coli </em>cells.</font></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 size="2"><font color="#2a2a2a">&#160;Expression of the </font><em style="color: rgb(42, 42, 42);">lacZ</em><font color="#2a2a2a"> and </font><em style="color: rgb(42, 42, 42);">lacY</em><font color="#2a2a2a"> genes was measured in control DH5&#945; cells </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"=""><strong><font color="#24678d">(BLUE)</font></strong><font color="#2a2a2a"> and BBa_S04055 recombinant </font><em style="color: rgb(42, 42, 42);">DH5&#945;</em><font color="#2a2a2a"> cells</font><font color="#da4444"><strong> (RED)</strong></font><font color="#2a2a2a">.</font></span><span "font-size:13.5pt;font-family:&quot;arial&quot;,sans-serif;color:#333333;="" mso-fareast-language:zh-hk"="" style="color: rgb(42, 42, 42);"> </span><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"><i>Cells were cultured overnight in LB medium + antibiotic and total RNA harvested for qRT-PCR using 16S rRNA for normalization.</i></font></span><br /><br /></div>
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</div></div>
 
</div></div>
  
<div class="paragraph" style="text-align:left;"><font size="4"><font color="#2a2a2a"><strong><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 size ="2">Figure 4. Western Blot analysis of &beta;-galactosidase (LacZ) protein.</span></strong><span "font-size:13.5pt;="" font-family:&quot;arial&quot;,sans-serif;color:#333333;mso-fareast-language:zh-hk"="">&nbsp;</span></font><br /><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 size = "3"><font color="#2a2a2a"><i>Expression of the &beta;-galactosidase protein (~ 135 kDa band) in control (Lane 1) and recombinant (BBa_S04055) (Lane2) E. coli cells.</i></font></span></font><br /><span style=""></span></div>
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<div class="paragraph" style="text-align:justify;"> <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"><font size="2">Figure 4. Western Blot analysis of &#946;-galactosidase (LacZ) protein.</font></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 size="2"><font color="#2a2a2a">&#160; Expression of the &#946;-galactosidase protein (~ 135 kDa band) in control (Lane 1) and recombinant (BBa_S04055) (Lane2) <em style=””>E. coli</em> cells. </font></font></span><br /><br /><br /></div>
  
  
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</div></div>
 
</div></div>
  
<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"=""><font size ="2">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);">OD<font size="2">600</font></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;"> <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"><font size="2">Figure 5. Expression of &#946;-galactosidase as measured by the ONPG assay.</font></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 size="2"><font color="#2a2a2a">&#160; Control </font><strong><font color="#24678d">(BLUE)</font></strong><font color="#2a2a2a"> <em style=””>E. coli</em> cells and  BBa_S04055 recombinant cells </font><strong><font color="#da4444">(RED)</font></strong><font color="#2a2a2a"> at OD<sub>600</sub>=0.3 were harvested and lysed to release intracellular &#946;-galactosidase, which was then measured for its activity by the ONPG assay. The conversion was measured by A<sub>420</sub> at 15 minute interval.</font></font></font></span><br /><br /></div>
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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;color:#333333;="" mso-fareast-language:zh-hk"="" style=""><font size="6">C. Measurement of β-galactosidase enzyme activity using ONPG Assay</font></span></u></strong><br /><span style=""></span></h2>
 
<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;color:#333333;="" mso-fareast-language:zh-hk"="" style=""><font size="6">C. Measurement of β-galactosidase enzyme activity using ONPG Assay</font></span></u></strong><br /><span style=""></span></h2>
  
<div class="paragraph" style="text-align:left;"><font size="3"><span "mso-bidi-font-size:13.5pt;font-family:&quot;arial&quot;,sans-serif;="" color:black;mso-themecolor:text1;mso-fareast-language:zh-hk"="">The level of &beta;–galactosidase activity was measured in recombinant (BBa_S04055) and control E. coli cells using the ONPG colorimetric assay. The results in Figure 5 show that the concentration of the cleavage product (A<sub>420</sub>) increased linearly within 60 minutes in the recombinant cells (BBa_S04055) while no change in absorbance was observed in control cells which indicated that the &beta;-galactosidase enzyme activity is expressed in the recombinant cells and is in agreement with the results previously reported by the 2008 Caltech iGEM team.</span><br /></font><span style=""></span><br /><span style=""></span></div>
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<div class="paragraph"><font size="3"><span "mso-bidi-font-size:13.5pt;font-family:&quot;arial&quot;,sans-serif;="" color:black;mso-themecolor:text1;mso-fareast-language:zh-hk"="">The level of &beta;–galactosidase activity was measured in recombinant (BBa_S04055) and control E. coli cells using the ONPG colorimetric assay. The results in Figure 5 show that the concentration of the cleavage product (A<sub>420</sub>) increased linearly within 60 minutes in the recombinant cells (BBa_S04055) while no change in absorbance was observed in control cells which indicated that the &beta;-galactosidase enzyme activity is expressed in the recombinant cells and is in agreement with the results previously reported by the 2008 Caltech iGEM team.</span><br /></font><span style=""></span><br /><span style=""></span></div>
  
  
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<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"=""><font size="6">Characterization of Lysis Gene Cassette</span><span "font-size:12.0pt;line-height:107%;font-family:&quot;arial&quot;,sans-serif;="" color:black;mso-themecolor:text1;mso-font-kerning:12.0pt"="" style=""> </span><span "font-size:14.0pt;line-height:107%;font-family:&quot;arial&quot;,sans-serif;="" color:black;mso-themecolor:text1;mso-font-kerning:12.0pt"="" style="">S<span style="">mut</span><span style="">&lambda;</span>-R<span style="">&lambda;</span>-R<span style="">z</span><span style="">&lambda;</span></span><span style="">&nbsp;(BBa_K1695038)</font></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"=""><font size="6">Characterization of Lysis Gene Cassette</span><span "font-size:12.0pt;line-height:107%;font-family:&quot;arial&quot;,sans-serif;="" color:black;mso-themecolor:text1;mso-font-kerning:12.0pt"="" style=""> </span><span "font-size:14.0pt;line-height:107%;font-family:&quot;arial&quot;,sans-serif;="" color:black;mso-themecolor:text1;mso-font-kerning:12.0pt"="" style="">S<span style="">mut</span><span style="">&lambda;</span>-R<span style="">&lambda;</span>-R<span style="">z</span><span style="">&lambda;</span></span><span style="">&nbsp;(BBa_K1695038)</font></span><br /><span style=""></span></h2>
  
<div class="paragraph" style="text-align:left;"><span "font-size:11.0pt;line-height:="" 107%;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:&#26032;&#32048;&#26126;&#39636;;mso-fareast-theme-font:="" minor-fareast;color:black;mso-themecolor:text1;mso-font-kerning:12.0pt;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style=""><font color="#2a2a2a"><span id="selectionBoundary_1442551834478_6921464148908854" class="rangySelectionBoundary" style="line-height: 0; display: none;">&#65279;</span><font size="3.5">Upon induction with 0.2 mM, 1 mM and 5 mM of IPTG, recombinant </font><em style="font-size: medium;">E. coli</em><font size="3.5"> harboring the lysis cassette S</font><span "position:="" relative;top:3.0pt;mso-text-raise:-3.0pt"=""><font size="2">mut</font></span><font size="3.5">&lambda;-R&lambda;-Rz&lambda; (BBa_K1695038) showed a sharp drop in absorbance (A</font><font size="1">600</font><font size="3.5">) 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) OD</font><font size="1">600 </font><font size="3.5">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.</font><span id="selectionBoundary_1442551834478_7756834849715233" class="rangySelectionBoundary" style="line-height: 0; display: none;">&#65279;</span></font></span></div>
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<div class="paragraph" ><span "font-size:11.0pt;line-height:="" 107%;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:&#26032;&#32048;&#26126;&#39636;;mso-fareast-theme-font:="" minor-fareast;color:black;mso-themecolor:text1;mso-font-kerning:12.0pt;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style=""><font color="#2a2a2a"><span id="selectionBoundary_1442551834478_6921464148908854" class="rangySelectionBoundary" style="line-height: 0; display: none;">&#65279;</span><font size="3.5">Upon induction with 0.2 mM, 1 mM and 5 mM of IPTG, recombinant </font><em style="font-size: medium;">E. coli</em><font size="3.5"> harboring the lysis cassette S</font><span "position:="" relative;top:3.0pt;mso-text-raise:-3.0pt"=""><font size="2">mut</font></span><font size="3.5">&lambda;-R&lambda;-Rz&lambda; (BBa_K1695038) showed a sharp drop in absorbance (A</font><font size="1">600</font><font size="3.5">) 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) OD</font><font size="1">600 </font><font size="3.5">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.</font><span id="selectionBoundary_1442551834478_7756834849715233" class="rangySelectionBoundary" style="line-height: 0; display: none;">&#65279;</span></font></span></div>
  
 
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Latest revision as of 20:10, 18 September 2015

Results - iGEM2015 wiki

Characterization of Lysis Gene Cassette Smutλ-Rλ-Rzλ (BBa_K1695038)

Upon induction with 0.2 mM, 1 mM and 5 mM of IPTG, recombinant E. coli harboring the lysis cassette Smutλ-Rλ-Rzλ (BBa_K1695038) 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_K1695038) 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.