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

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<h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:&quot;calibri&quot;,&quot;sans-serif&quot;;="" 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"=""></span><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:&quot;calibri&quot;,&quot;sans-serif&quot;;="" 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"="" style=""><font size="5">RNA levels of lacZ and lacY</font><br /></span></h2>
 
<h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:&quot;calibri&quot;,&quot;sans-serif&quot;;="" 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"=""></span><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:&quot;calibri&quot;,&quot;sans-serif&quot;;="" 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"="" style=""><font size="5">RNA levels of lacZ and lacY</font><br /></span></h2>
  
<div class="paragraph" style="text-align:left;"><font size="3">To determine the levels of the <em style=""><span style="">lacZ </span></em>and <em style=""><span style="">lacY</span></em> transcripts, total RNA extraction was extracted, RNA was converted into cDNA by reverse transcription and the levels of the two transcripts were determined by real time PCR.<em style=""><span style=""></span></em><br /><br />  <strong>RNA extraction --&gt;&nbsp;Reverse transcription --&gt;&nbsp;Real time PCR</strong><br />&nbsp;<br /><br />  <strong>RNA extraction:</strong> (Link to the protocol)<br />  To extract the total RNA from the engineered <em style="">E. coli</em><br /><br />  <em style=""><span style="">&nbsp;</span></em><br />  <strong>Reverse transcription:</strong> (Link to the protocol)<br /> To convert the total RNA into cDNA for the subsequent real time PCR. Real time PCR can only quantify the levels of cDNA but not RNA.<br /><br />  &nbsp;<br />  <strong>Real time PCR:</strong> (Link to the protocol)<br />  <span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:="" &quot;calibri&quot;,&quot;sans-serif&quot;;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"="" style="">To amplify our desired cDNA (</span><em style=""><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:&quot;calibri&quot;,&quot;sans-serif&quot;;="" 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-hk;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"="" style="">lacZ</span></em><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:&quot;calibri&quot;,&quot;sans-serif&quot;;="" 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"="" style=""> and lacY) if the RNA extract contains lacZlacY RNA. It will be difficult to detect the small amount of RNA in cells if the RNA, which is then converted to cDNA), is not amplified.</span></font></div>
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<div class="paragraph" style="text-align:left;"><font size="3">To determine the levels of the <em style=""><span style="">lacZ </span></em>and <em style=""><span style="">lacY</span></em> transcripts, total RNA extraction was extracted, RNA was converted into cDNA by reverse transcription and the levels of the two transcripts were determined by real time PCR.<em style=""><span style=""></span></em><br /><br />  <strong>RNA extraction --&gt;&nbsp;Reverse transcription --&gt;&nbsp;Real time PCR</strong><br />&nbsp;<br /><br />  <strong>RNA extraction:</strong> <a href="https://2015.igem.org/Team:CityU_HK/Protocol#cloning" style="color: #000000"><b><u>(Link to the protocol)</u></b></a><br />  To extract the total RNA from the engineered <em style="">E. coli</em><br /><br />  <em style=""><span style="">&nbsp;</span></em><br />  <strong>Reverse transcription:</strong> <a href="https://2015.igem.org/Team:CityU_HK/Protocol#cloning" style="color: #000000"><b><u>(Link to the protocol)</u></b></a><br /> To convert the total RNA into cDNA for the subsequent real time PCR. Real time PCR can only quantify the levels of cDNA but not RNA.<br /><br />  &nbsp;<br />  <strong>Real time PCR:</strong> <a href="https://2015.igem.org/Team:CityU_HK/Protocol#cloning" style="color: #000000"><b><u>(Link to the protocol)</u></b></a><br />  <span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:="" &quot;calibri&quot;,&quot;sans-serif&quot;;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"="" style="">To amplify our desired cDNA (</span><em style=""><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:&quot;calibri&quot;,&quot;sans-serif&quot;;="" 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-hk;mso-fareast-language:="" zh-tw;mso-bidi-language:ar-sa"="" style="">lacZ</span></em><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:&quot;calibri&quot;,&quot;sans-serif&quot;;="" 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"="" style=""> and lacY) if the RNA extract contains lacZlacY RNA. It will be difficult to detect the small amount of RNA in cells if the RNA, which is then converted to cDNA), is not amplified.</span></font></div>
  
 
<h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:&quot;calibri&quot;,&quot;sans-serif&quot;;="" 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="5">Characterization on LacZ</font></span></h2>
 
<h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:&quot;calibri&quot;,&quot;sans-serif&quot;;="" 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="5">Characterization on LacZ</font></span></h2>
  
<div class="paragraph" style="text-align:left;"><font size="3"><span style="">To determine the level of &beta;-galactosidase encoded by <em style="">lacZ</em>, ONPG assay was performed.</span><br /><br /><span style=""></span>  <span style=""><strong>ONPG assay:</strong></span><span style="">&nbsp;(link to the protocol)</span><br /><span style=""></span><br /><span style=""></span>  <span style="">Besides lactose, ONPG (ortho-Nitrophenyl-&beta;-galactoside<strong style="">)</strong> is also a substrate of &beta;-galactosidase. ONPG is digested into galactose and ortho-nitrophenol (ONP) which is yellow in colour. Chloroform was used to lyse the cells and the enzymes were released into the surrounding. By measuring the O.D. at 420 nm after lysing the cells, the level of the &beta;-galatosidase was determined.</span></font><br /><span style=""></span><br /><span style=""></span></div>
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<div class="paragraph" style="text-align:left;"><font size="3"><span style="">To determine the level of &beta;-galactosidase encoded by <em style="">lacZ</em>, ONPG assay was performed.</span><br /><br /><span style=""></span>  <span style=""><strong>ONPG assay:</strong></span><span style="">&nbsp;<a href="https://2015.igem.org/Team:CityU_HK/Protocol#characterization" style="color: #000000"><b><u>(Link to the protocol)</u></b></a></span><br /><span style=""></span><br /><span style=""></span>  <span style="">Besides lactose, ONPG (ortho-Nitrophenyl-&beta;-galactoside<strong style="">)</strong> is also a substrate of &beta;-galactosidase. ONPG is digested into galactose and ortho-nitrophenol (ONP) which is yellow in colour. Chloroform was used to lyse the cells and the enzymes were released into the surrounding. By measuring the O.D. at 420 nm after lysing the cells, the level of the &beta;-galatosidase was determined.</span></font><br /><span style=""></span><br /><span style=""></span></div>
  
 
<h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:&quot;calibri&quot;,&quot;sans-serif&quot;;="" 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="5">pL8-UV5 characterization</font></span></h2>
 
<h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:&quot;calibri&quot;,&quot;sans-serif&quot;;="" 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="5">pL8-UV5 characterization</font></span></h2>
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<h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:&quot;calibri&quot;,&quot;sans-serif&quot;;="" 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="5">Characterization of the lacY lacZ operon BBa_S04055 (from 2008 Caltech: Curing lactose intolerance)</font></span></h2>
 
<h2 class="wsite-content-title" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:11.0pt;font-family:&quot;calibri&quot;,&quot;sans-serif&quot;;="" 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="5">Characterization of the lacY lacZ operon BBa_S04055 (from 2008 Caltech: Curing lactose intolerance)</font></span></h2>
  
<div class="paragraph" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:="" 11.0pt;font-family:&quot;calibri&quot;,&quot;sans-serif&quot;;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"="" style=""><font size="3">Western blotting (link to the protocol) is used to detect the expression level of &beta;-galactosidase inside <em style="">E. coli</em> harboring BBa_S04055.</font></span></div></div>
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<div class="paragraph" style="text-align:left;"><span "font-size:12.0pt;mso-bidi-font-size:="" 11.0pt;font-family:&quot;calibri&quot;,&quot;sans-serif&quot;;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"="" style=""><font size="3">Western blotting <a href="https://2015.igem.org/Team:CityU_HK/Protocol#characterization" style="color: #000000"><b><u>(Link to the protocol)</u></b></a> is used to detect the expression level of &beta;-galactosidase inside <em style="">E. coli</em> harboring BBa_S04055.</font></span></div></div>
 
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Revision as of 11:33, 18 September 2015

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