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:="" 12.0pt;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:&quot;times="" roman&quot;;="" color:black;mso-font-kerning:0pt"="">Figure 4. 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 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:&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="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 4. 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>
  
  

Revision as of 07:53, 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 A420 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.