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

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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"="" style=""><font size="6">Quantitative real-time PCR</font></span></u></strong><br /><span style=""></span></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;color:#333333"="" style=""><font size="6">A. 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:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:text1"="">Quantitative real-time PCR analysis showed that both lacY and lacZ mRNA transcripts are expressed at high levels in recombinant E. coli cells (harboring the BBa_S04055 biobrick) as compared to control DH5α cells (Figure 1). Expression of lacY and lacZ is 9- fold and 2-fold higher, respectively, in recombinant cells with respect to the control cells.</span></font></div>
 
<div class="paragraph" style="text-align:justify;"><font size="3"><span "mso-bidi-font-size:13.5pt;="" font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:text1"="">Quantitative real-time PCR analysis showed that both lacY and lacZ mRNA transcripts are expressed at high levels in recombinant E. coli cells (harboring the BBa_S04055 biobrick) as compared to control DH5α cells (Figure 1). Expression of lacY and lacZ is 9- fold and 2-fold higher, respectively, in recombinant cells with respect to the control cells.</span></font></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:&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">Western Blot</font><font size="5">&nbsp;</font></span></u></strong></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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<div class="paragraph" style="text-align:justify;"><font size="3">Western blot analysis demonstrated that &beta;-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 &beta;-galactosidase was overexpressed in the BBa_S04055 recombinant cells.</font></div>
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<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 2) and is in agreement with the <em style="">lacZ</em> mRNA results described in Figure 1.</font></div>
  
  
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</div></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:&quot;arial&quot;,sans-serif;mso-fareast-font-family:&quot;times="" roman&quot;;="" color:black;mso-font-kerning:0pt"="">Figure 3. Western Blot analysis of the expression of &beta;-galactosidase.</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">Protein expression of &beta;-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"><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"="">Figure 2. 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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<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">ONPG Assay</font></span></u></strong><br /><span style=""></span></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;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>
  
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<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 assay </span><span "mso-bidi-font-size:13.5pt;font-family:&quot;arial&quot;,sans-serif;="" color:black;mso-themecolor:text1"="">showed that<span style="">&nbsp;the cleavage product</span></span><em><span "mso-bidi-font-size:="" 13.5pt;font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:text1;="" mso-fareast-language:zh-hk"="" style=""> </span></em><span "mso-bidi-font-size:="" 13.5pt;font-family:&quot;arial&quot;,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:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:text1;mso-fareast-language:="" zh-hk"="">BBa_S04055</span><span "mso-bidi-font-size:13.5pt;="" font-family:&quot;arial&quot;,sans-serif;color:black;mso-themecolor:text1"=""> recombinant<span style="">&nbsp;cells </span></span><span "mso-bidi-font-size:13.5pt;font-family:&quot;arial&quot;,sans-serif;color:black;="" mso-themecolor:text1;mso-fareast-language:zh-hk"="">and </span><span "mso-bidi-font-size:13.5pt;font-family:&quot;arial&quot;,sans-serif;color:black;="" mso-themecolor:text1"="">no change in absorbance in &nbsp;was observed in the</span><span "mso-bidi-font-size:13.5pt;font-family:&quot;arial&quot;,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.&nbsp;</span><span style=""></span><br /></font><span style=""></span><br /><span style=""></span></div>
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<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 3 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>
  
  

Revision as of 07:32, 18 September 2015

Results - iGEM2015 wiki

Improved/ expanded characterization of LacY-LacZ biobrick (BBa_S04055)

Figure 1. Quantitative RT-PCR analysis of lacY and lacZ expression in E. coli cells. 
Expression of the lacZ and lacY genes was measured in control DH5α cells (BLUE) and BBa_S04055 recombinant DH5α cells (RED). Cells were cultured overnight in LB medium + antibiotic and total RNA harvested for qRT-PCR using 16S rRNA for normalization.

A. Quantitative real-time PCR

Quantitative real-time PCR analysis showed that both lacY and lacZ mRNA transcripts are expressed at high levels in recombinant E. coli cells (harboring the BBa_S04055 biobrick) as compared to control DH5α cells (Figure 1). Expression of lacY and lacZ is 9- fold and 2-fold higher, respectively, in recombinant cells with respect to the control cells.

B. Western Blot Analysis 

Western blot analysis showed that the β-galactosidase protein (LacZ) (indicated by the arrow) is expressed at a significantly higher level in recombinant E. coli cells (BBa_S04055) relative to the control (Figure 2) and is in agreement with the lacZ mRNA results described in Figure 1.
Figure 2. Western Blot analysis of β-galactosidase (LacZ) protein. 
Expression of the β-galactosidase protein (~ 135 kDa band) in control (Lane 1) and recombinant (BBa_S04055) (Lane2) E. coli cells.
Figure 4. 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.

C. Measurement of β-galactosidase enzyme activity using ONPG Assay

The level of β–galactosidase activity was measured in recombinant (BBa_S04055) and control E. coli cells using the ONPG colorimetric assay. The results in Figure 3 show that the concentration of the cleavage product (A420) 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 β-galactosidase enzyme activity is expressed in the recombinant cells and is in agreement with the results previously reported by the 2008 Caltech iGEM team.