Difference between revisions of "Team:CityU HK/Results"
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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:="" 13.5pt;font-family:"arial",sans-serif;color:#333333;mso-fareast-language:zh-hk"="">Figure_.</span></strong><strong style="color: rgb(42, 42, 42);"><span "mso-bidi-font-size:="" 12.0pt;font-family:"arial",sans-serif;mso-fareast-font-family:"times="" roman";="" color:black;mso-font-kerning:0pt"="" style=""> Quantitative RT-PCR analysis of <em style="">lacZ</em> and <em style="">lacY</em> transcripts in E. coli <em style="">DH5α</em> host cells.</span></strong><span "mso-bidi-font-size:12.0pt;font-family:"arial",sans-serif;="" mso-fareast-font-family:"times="" roman";color:black;mso-font-kerning:0pt"=""><font color="#2a2a2a"> </font></span><br /><span "mso-bidi-font-size:12.0pt;font-family:"arial",sans-serif;="" mso-fareast-font-family:"times="" roman";color:black;mso-font-kerning:0pt"=""><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 determined in control </font><em style="color: rgb(42, 42, 42);">DH5α</em><font color="#2a2a2a"> cells</font><font color="#24678d"> <strong>(</strong></font></span><strong><span "mso-bidi-font-size:12.0pt;font-family:="" "arial",sans-serif;mso-fareast-font-family:"times="" roman";color:#0f1368;="" mso-font-kerning:0pt"="" style=""><font color="#24678d">BLUE</font></span></strong><span "mso-bidi-font-size:="" 12.0pt;font-family:"arial",sans-serif;mso-fareast-font-family:"times="" roman";="" 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);">DH5α</em><font color="#2a2a2a"> cells</font><font color="#da4444"><strong> (</strong></font></span><span "mso-bidi-font-size:="" 12.0pt;font-family:"arial",sans-serif;mso-fareast-font-family:"times="" roman";="" color:#ca2323;mso-font-kerning:0pt"=""><font color="#da4444"><strong>RED</strong></font></span><span "mso-bidi-font-size:12.0pt;font-family:"arial",sans-serif;mso-fareast-font-family:="" "times="" roman";color:black;mso-font-kerning:0pt"=""><font color="#da4444"><strong>)</strong></font><font color="#2a2a2a">.</font></span><span "font-size:13.5pt;font-family:"arial",sans-serif;color:#333333;="" mso-fareast-language:zh-hk"="" style="color: rgb(42, 42, 42);"> </span><span "mso-bidi-font-size:="" 12.0pt;font-family:"arial",sans-serif;color:#333333;mso-fareast-language:zh-hk"="" style=""><font color="#2a2a2a">The cells were cultured overnight in LB medium and harvested for total RNA extraction.</font></span><u style=""><span "font-size:13.5pt;font-family:"arial",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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| + | <h2 class="wsite-content-title" style="text-align:left;"><strong style=""><u style=""><span "font-size:18.0pt;="" font-family:"arial",sans-serif;color:#333333"="" style=""><font size="6">Quantitative real-time PCR</font></span></u></strong><br /><span style=""></span></h2> | ||
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| + | <div class="paragraph" style="text-align:justify;"><font size="3"><span "mso-bidi-font-size:13.5pt;="" font-family:"arial",sans-serif;color:black;mso-themecolor:text1"="">Quantitative real-time PCR analyses showed that both </span><em><span "mso-bidi-font-size:13.5pt;font-family:"arial",sans-serif;="" color:black;mso-themecolor:text1;mso-fareast-language:zh-hk"="" style="">lacY </span></em><span "mso-bidi-font-size:13.5pt;font-family:"arial",sans-serif;="" color:black;mso-themecolor:text1;mso-fareast-language:zh-hk;mso-bidi-font-style:="" italic"="">and<em style=""> lacZ </em></span><span "mso-bidi-font-size:13.5pt;="" font-family:"arial",sans-serif;color:black;mso-themecolor:text1"="">transcripts were expressed at high levels in the recombinant <em style="">E. coli</em> cells as compared to control (Figure_). The <em style="">lacY</em> gene expression was 9-fold higher in recombinant cells than the control cells. For the <em style="">lacZ</em> gene</span><em><span "mso-bidi-font-size:13.5pt;="" font-family:"arial",sans-serif;color:black;mso-themecolor:text1;mso-fareast-language:="" zh-hk"="" style=""> </span></em><span "mso-bidi-font-size:13.5pt;="" font-family:"arial",sans-serif;color:black;mso-themecolor:text1;mso-fareast-language:="" zh-hk;mso-bidi-font-style:italic"="">mRNA expression, the recombinant cells </span><span "mso-bidi-font-size:13.5pt;font-family:"arial",sans-serif;="" color:black;mso-themecolor:text1"="">was 2-fold higher than the control cells. </span><span "mso-bidi-font-size:13.5pt;font-family:"arial",sans-serif;="" color:black;mso-themecolor:text1;mso-fareast-language:zh-hk"="" style=""></span><br /><span style=""></span><br /><span style=""></span> <span "font-size:12.0pt;mso-bidi-font-size:13.5pt;font-family:="" "arial",sans-serif;mso-fareast-font-family:新細明體;mso-fareast-theme-font:minor-fareast;="" color:#333333;mso-ansi-language:en-us;mso-fareast-language:zh-hk;mso-bidi-language:="" ar-sa"=""><br /> </span> <span "mso-bidi-font-size:13.5pt;font-family:"arial",sans-serif;color:#333333;="" mso-fareast-language:zh-hk"="">(Any replications on the experiments? N= times of replication)</span></font><br /><span style=""></span><br /><span style=""></span></div> | ||
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| + | <h2 class="wsite-content-title" style="text-align:right;"><strong style=""><u style=""><span "font-size:18.0pt;font-family:"arial",sans-serif;mso-fareast-font-family:="" 新細明體;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">Western Blot</font><font size="5"> </font></span></u></strong></h2> | ||
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| + | <div class="paragraph" style="text-align:justify;"><font size="3">Western blot analysis demonstrated that β-galactosidase protein (indicated by the arrow) was expressed higher in recombinant BBa_S04055 <em>E. coli</em> than the control cells. The result matches that of the qRT-PCR assay, suggesting β-galactosidase was overexpressed in the BBa_S04055 recombinant cells.</font></div> | ||
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| + | <div class="paragraph" style="text-align:left;"><font size="2"><font color="#2a2a2a"><strong><span "mso-bidi-font-size:="" 12.0pt;font-family:"arial",sans-serif;mso-fareast-font-family:"times="" roman";="" color:black;mso-font-kerning:0pt"="">Figure _. Western Blot analysis of the expression of β-galactosidase.</span></strong><span "font-size:13.5pt;="" font-family:"arial",sans-serif;color:#333333;mso-fareast-language:zh-hk"=""> </span></font><br /><span "mso-bidi-font-size:12.0pt;font-family:"arial",sans-serif;="" mso-fareast-font-family:"times="" roman";color:black;mso-font-kerning:0pt"=""><font color="#2a2a2a">Protein expression of β-galactosidase in the control cells (Lane 1) and BBa_S04055 transformant (Lane2).</font></span></font><br /><span style=""></span></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:"arial",sans-serif;mso-fareast-font-family:"times="" roman";="" color:black;mso-font-kerning:0pt"="">Figure _. Expression of </span></strong><strong style="color: rgb(42, 42, 42);"><span "mso-bidi-font-size:="" 12.0pt;font-family:"arial",sans-serif;color:#333333;mso-fareast-language:zh-hk"="" style="">β</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:"arial",sans-serif;mso-fareast-font-family:"times="" roman";="" 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:"arial",sans-serif;="" color:#333333;mso-fareast-language:zh-hk"=""><font color="#2a2a2a">Control</font> </span><strong><font color="#3387a2"><span "mso-bidi-font-size:12.0pt;="" font-family:"arial",sans-serif;mso-fareast-font-family:"times="" roman";="" color:black;mso-font-kerning:0pt"="" style="">(</span><span "mso-bidi-font-size:="" 12.0pt;font-family:"arial",sans-serif;mso-fareast-font-family:"times="" roman";="" color:#4f81bd;mso-themecolor:accent1;mso-font-kerning:0pt"="" style="">BLUE</span></font><span "mso-bidi-font-size:12.0pt;font-family:"arial",sans-serif;="" mso-fareast-font-family:"times="" roman";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:"arial",sans-serif;="" color:#333333;mso-fareast-language:zh-hk"="" style="">E. coli</span></em><span "mso-bidi-font-size:12.0pt;font-family:"arial",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:"arial",sans-serif;mso-fareast-font-family:="" "times="" roman";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:="" "arial",sans-serif;mso-fareast-font-family:"times="" roman";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:"arial",sans-serif;mso-fareast-font-family:="" "times="" roman";color:black;mso-font-kerning:0pt"=""><strong><font color="#da4444">)</font></strong> </span><span "mso-bidi-font-size:12.0pt;="" font-family:"arial",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:"arial",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:"arial",sans-serif;color:#333333;mso-fareast-language:zh-hk"="" style="color: rgb(42, 42, 42);">=0.3 were harvested and lysed to release intracellular β</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:"arial",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:"arial",sans-serif;color:black;mso-font-kerning:0pt;="" mso-fareast-language:zh-hk"="" style="color: rgb(42, 42, 42);"> was measured by Ab420 at 15 minute interval.</span></font><u style="font-size: x-small; color: rgb(42, 42, 42);"><span "font-size:13.5pt;font-family:"arial",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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| + | <h2 class="wsite-content-title" style="text-align:left;"><strong style=""><u style=""><span "font-size:18.0pt;font-family:"arial",sans-serif;color:#333333;="" mso-fareast-language:zh-hk"="" style=""><font size="6">ONPG Assay</font></span></u></strong><br /><span style=""></span></h2> | ||
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| + | <div class="paragraph" style="text-align:left;"><font size="3"><span "mso-bidi-font-size:13.5pt;font-family:"arial",sans-serif;="" color:black;mso-themecolor:text1;mso-fareast-language:zh-hk"="">The assay </span><span "mso-bidi-font-size:13.5pt;font-family:"arial",sans-serif;="" color:black;mso-themecolor:text1"="">showed that<span style=""> the cleavage product</span></span><em><span "mso-bidi-font-size:="" 13.5pt;font-family:"arial",sans-serif;color:black;mso-themecolor:text1;="" mso-fareast-language:zh-hk"="" style=""> </span></em><span "mso-bidi-font-size:="" 13.5pt;font-family:"arial",sans-serif;color:black;mso-themecolor:text1;="" mso-fareast-language:zh-hk;mso-bidi-font-style:italic"="">increased linearly within 60 minutes in </span><span "mso-bidi-font-size:13.5pt;="" font-family:"arial",sans-serif;color:black;mso-themecolor:text1;mso-fareast-language:="" zh-hk"="">BBa_S04055</span><span "mso-bidi-font-size:13.5pt;="" font-family:"arial",sans-serif;color:black;mso-themecolor:text1"=""> recombinant<span style=""> cells </span></span><span "mso-bidi-font-size:13.5pt;font-family:"arial",sans-serif;color:black;="" mso-themecolor:text1;mso-fareast-language:zh-hk"="">and </span><span "mso-bidi-font-size:13.5pt;font-family:"arial",sans-serif;color:black;="" mso-themecolor:text1"="">no change in absorbance in was observed in the</span><span "mso-bidi-font-size:13.5pt;font-family:"arial",sans-serif;color:black;="" mso-themecolor:text1;mso-fareast-language:zh-hk"=""> control cells, suggesting that LacZ protein was expressed in BBa_S04055 recombinant cells, which matches the result reported by the iGEM 2008 Caltech team. </span><span style=""></span><br /></font><span style=""></span><br /><span style=""></span></div> | ||
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Revision as of 13:20, 17 September 2015
Figure_. Quantitative RT-PCR analysis of lacZ and lacY transcripts in E. coli DH5α host cells.
Expression of the lacZ and lacY genes was determined in control DH5α cells (BLUE) and BBa_S04055 recombinant DH5α cells (RED). The cells were cultured overnight in LB medium and harvested for total RNA extraction. |
Quantitative real-time PCR
Quantitative real-time PCR analyses showed that both lacY and lacZ transcripts were expressed at high levels in the recombinant E. coli cells as compared to control (Figure_). The lacY gene expression was 9-fold higher in recombinant cells than the control cells. For the lacZ gene mRNA expression, the recombinant cells was 2-fold higher than the control cells.
(Any replications on the experiments? N= times of replication) |
Western BlotWestern blot analysis demonstrated that β-galactosidase protein (indicated by the arrow) was expressed higher in recombinant BBa_S04055 E. coli than the control cells. The result matches that of the qRT-PCR assay, suggesting β-galactosidase was overexpressed in the BBa_S04055 recombinant cells.
|
Figure _. Western Blot analysis of the expression of β-galactosidase.
Protein expression of β-galactosidase in the control cells (Lane 1) and BBa_S04055 transformant (Lane2). |
Figure _. Expression of β-galactosidase as measured by the ONPG assay.
Control (BLUE) E. coli cells and BBa_S04055 recombinant cells (RED) at OD600=0.3 were harvested and lysed to release intracellular β-galactosidase, which was then measured for its activity by the ONPG assay. The conversion was measured by Ab420 at 15 minute interval. |
ONPG Assay
The assay showed that the cleavage product increased linearly within 60 minutes in BBa_S04055 recombinant cells and no change in absorbance in was observed in the control cells, suggesting that LacZ protein was expressed in BBa_S04055 recombinant cells, which matches the result reported by the iGEM 2008 Caltech team.
|