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"=""> | + | <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"="">Figure1.</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> | <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> | ||
| − | <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 ( | + | <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 (Figure1). 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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| − | <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 | + | <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 2. 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 | + | <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 3. 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> |
Revision as of 13:25, 17 September 2015
Figure1. 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 (Figure1). 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 2. 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 3. 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.
|