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

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<div id="lactose" style="display:block;font-size:90%"></div>
 
<div id="lactose" style="display:block;font-size:90%"></div>
<h2  class="wsite-content-title" style="text-align:left;"><span "font-size:14.0pt;="" line-height:107%;font-family:&quot;calibri&quot;,sans-serif;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"=""><u><font size="6">Characterization of the lactose-inducible promoter (BBa_K1695053)</font></u></span><br /><span style=""></span></h2>
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<h2  class="wsite-content-title" style="text-align:left;"><span "font-size:14.0pt;="" line-height:107%;font-family:&quot;calibri&quot;,sans-serif;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="6">Characterization of the lactose-inducible promoter (BBa_K1695053)</font></span><br /><span style=""></span></h2>
  
 
<div class="paragraph" style="text-align:left;"><br /><font color="#2a2a2a"><strong style="font-size: medium;"><em><span style="line-height: 107%;">&nbsp;</span></em></strong><span style="font-size: medium; line-height: 1.5em; text-align: justify; background-color: initial;">The GFP reporter biobrick (BBa_K1695053) was constructed by linking
 
<div class="paragraph" style="text-align:left;"><br /><font color="#2a2a2a"><strong style="font-size: medium;"><em><span style="line-height: 107%;">&nbsp;</span></em></strong><span style="font-size: medium; line-height: 1.5em; text-align: justify; background-color: initial;">The GFP reporter biobrick (BBa_K1695053) was constructed by linking
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<div class="paragraph" style="text-align:justify;"><font size="3"><strong><span "font-size:11.0pt;="" line-height:107%;font-family:&quot;calibri&quot;,sans-serif;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 color="#2a2a2a">Figure 2. Expression analysis of L8-UV5 promoter-GFP biobrick (BBa_K1695053). </font></span></strong><span "font-size:11.0pt;line-height:107%;font-family:&quot;calibri&quot;,sans-serif;="" 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;mso-bidi-font-weight:bold"=""><font color="#2a2a2a">&nbsp;</font><font color="#2a2a2a">(A) Activation of GFP expression in the L8-UV5 promoter construct is dependent on IPTG concentrations below 1 mM. (B) Fluorescence response of the L8-UV5 promoter-GFP gene construct (BBa_K1695053) was measured over a 4.5-h time course under a range of IPTG concentrations (from 0.01 mM to 10 mM). Results show that induction of the L8-UV5 promoter is independent of IPTG at concentrations above 1 mM, where LacI is saturated and promoter is fully turned on<em>.&nbsp;</em></font></span><br /></font><br /><font size="4"><br /><span "font-size:11.0pt;line-height:="" 107%;font-family:&quot;calibri&quot;,sans-serif;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 color="#2a2a2a">Results in Figure 2 show that GFP expression in the L8-UV5 promoter-GFP construct is dependent on IPTG concentrations below 1 mM. At IPTG concentrations above 1 mM, expression of GFP has reached a maxima regardless of the sampling time points.</font></span></font><span "font-size:11.0pt;line-height:107%;font-family:&quot;calibri&quot;,sans-serif;="" 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;mso-bidi-font-weight:bold"="" style=""><br /><br /><br /></span></div>
 
<div class="paragraph" style="text-align:justify;"><font size="3"><strong><span "font-size:11.0pt;="" line-height:107%;font-family:&quot;calibri&quot;,sans-serif;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 color="#2a2a2a">Figure 2. Expression analysis of L8-UV5 promoter-GFP biobrick (BBa_K1695053). </font></span></strong><span "font-size:11.0pt;line-height:107%;font-family:&quot;calibri&quot;,sans-serif;="" 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;mso-bidi-font-weight:bold"=""><font color="#2a2a2a">&nbsp;</font><font color="#2a2a2a">(A) Activation of GFP expression in the L8-UV5 promoter construct is dependent on IPTG concentrations below 1 mM. (B) Fluorescence response of the L8-UV5 promoter-GFP gene construct (BBa_K1695053) was measured over a 4.5-h time course under a range of IPTG concentrations (from 0.01 mM to 10 mM). Results show that induction of the L8-UV5 promoter is independent of IPTG at concentrations above 1 mM, where LacI is saturated and promoter is fully turned on<em>.&nbsp;</em></font></span><br /></font><br /><font size="4"><br /><span "font-size:11.0pt;line-height:="" 107%;font-family:&quot;calibri&quot;,sans-serif;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 color="#2a2a2a">Results in Figure 2 show that GFP expression in the L8-UV5 promoter-GFP construct is dependent on IPTG concentrations below 1 mM. At IPTG concentrations above 1 mM, expression of GFP has reached a maxima regardless of the sampling time points.</font></span></font><span "font-size:11.0pt;line-height:107%;font-family:&quot;calibri&quot;,sans-serif;="" 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;mso-bidi-font-weight:bold"="" style=""><br /><br /><br /></span></div>
  
<h2 class="wsite-content-title" style="text-align:left;"><span "font-size:14.0pt;="" line-height:107%;font-family:&quot;calibri&quot;,sans-serif;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"=""><u><font size="6">Improved/ expanded characterization of LacY-LacZ biobrick (BBa_S04055)</font></u></span><br /><span style=""></span></h2>
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<h2 class="wsite-content-title" style="text-align:left;"><span "font-size:14.0pt;="" line-height:107%;font-family:&quot;calibri&quot;,sans-serif;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="6">Improved/ expanded characterization of LacY-LacZ biobrick (BBa_S04055)</font></span><br /><span style=""></span></h2>
  
  
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<h2 class="wsite-content-title" style="text-align:left;"><span "font-size:14.0pt;="" line-height:107%;font-family:&quot;calibri&quot;,sans-serif;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"=""><u><font size="6">Characterization of Lysis Gene Cassette</span><span "font-size:12.0pt;line-height:107%;font-family:&quot;arial&quot;,sans-serif;="" color:black;mso-themecolor:text1;mso-font-kerning:12.0pt"="" style=""> </span><span "font-size:14.0pt;line-height:107%;font-family:&quot;arial&quot;,sans-serif;="" color:black;mso-themecolor:text1;mso-font-kerning:12.0pt"="" style="">S<span style="">mut</span><span style="">&lambda;</span>-R<span style="">&lambda;</span>-R<span style="">z</span><span style="">&lambda;</span></span><span style="">&nbsp;(BBa_K1695038)</font></u></span><br /><span style=""></span></h2>
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<h2 class="wsite-content-title" style="text-align:left;"><span "font-size:14.0pt;="" line-height:107%;font-family:&quot;calibri&quot;,sans-serif;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="6">Characterization of Lysis Gene Cassette</span><span "font-size:12.0pt;line-height:107%;font-family:&quot;arial&quot;,sans-serif;="" color:black;mso-themecolor:text1;mso-font-kerning:12.0pt"="" style=""> </span><span "font-size:14.0pt;line-height:107%;font-family:&quot;arial&quot;,sans-serif;="" color:black;mso-themecolor:text1;mso-font-kerning:12.0pt"="" style="">S<span style="">mut</span><span style="">&lambda;</span>-R<span style="">&lambda;</span>-R<span style="">z</span><span style="">&lambda;</span></span><span style="">&nbsp;(BBa_K1695038)</font></span><br /><span style=""></span></h2>
  
 
<div class="paragraph" style="text-align:left;"><span "font-size:11.0pt;line-height:="" 107%;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:&#26032;&#32048;&#26126;&#39636;;mso-fareast-theme-font:="" minor-fareast;color:black;mso-themecolor:text1;mso-font-kerning:12.0pt;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style=""><font color="#2a2a2a"><span id="selectionBoundary_1442551834478_6921464148908854" class="rangySelectionBoundary" style="line-height: 0; display: none;">&#65279;</span><font size="3">Upon induction with 0.2 mM, 1 mM and 5 mM of IPTG, recombinant </font><em style="font-size: medium;">E. coli</em><font size="3"> harboring the lysis cassette S</font><span "position:="" relative;top:3.0pt;mso-text-raise:-3.0pt"=""><font size="2">mut</font></span><font size="3">&lambda;-R&lambda;-Rz&lambda; (BBa_K1695038) showed a sharp drop in absorbance (A</font><font size="1">600</font><font size="3">) after 15 minutes whereas cells without IPTG induction showed continued as indicated by the steady increase in OD (Figure 6A). The results show that 0.2 mM IPTG is sufficient to initiate cell lysis after 15 minutes, and the speed of cell lysis is not increased at higher concentrations of IPTG (Figure 6B). Overall, the use of (1) OD</font><font size="1">600 </font><font size="3">measurement to determine cell growth or cell lysis (panel A) and (2) plate count (cfu; panel B) showed that the Lysis Gene Cassette is induced by IPTG and resulted in the initiation of cell lysis after 15 minutes of IPTG induction. Cells were completely lysed by 20 minutes. It would be interesting in future studies to determine if the time of cell lysis could be regulated by IPTG at concentrations below 0.2 mM.</font><span id="selectionBoundary_1442551834478_7756834849715233" class="rangySelectionBoundary" style="line-height: 0; display: none;">&#65279;</span></font></span></div>
 
<div class="paragraph" style="text-align:left;"><span "font-size:11.0pt;line-height:="" 107%;font-family:&quot;arial&quot;,sans-serif;mso-fareast-font-family:&#26032;&#32048;&#26126;&#39636;;mso-fareast-theme-font:="" minor-fareast;color:black;mso-themecolor:text1;mso-font-kerning:12.0pt;="" mso-ansi-language:en-us;mso-fareast-language:zh-tw;mso-bidi-language:ar-sa"="" style=""><font color="#2a2a2a"><span id="selectionBoundary_1442551834478_6921464148908854" class="rangySelectionBoundary" style="line-height: 0; display: none;">&#65279;</span><font size="3">Upon induction with 0.2 mM, 1 mM and 5 mM of IPTG, recombinant </font><em style="font-size: medium;">E. coli</em><font size="3"> harboring the lysis cassette S</font><span "position:="" relative;top:3.0pt;mso-text-raise:-3.0pt"=""><font size="2">mut</font></span><font size="3">&lambda;-R&lambda;-Rz&lambda; (BBa_K1695038) showed a sharp drop in absorbance (A</font><font size="1">600</font><font size="3">) after 15 minutes whereas cells without IPTG induction showed continued as indicated by the steady increase in OD (Figure 6A). The results show that 0.2 mM IPTG is sufficient to initiate cell lysis after 15 minutes, and the speed of cell lysis is not increased at higher concentrations of IPTG (Figure 6B). Overall, the use of (1) OD</font><font size="1">600 </font><font size="3">measurement to determine cell growth or cell lysis (panel A) and (2) plate count (cfu; panel B) showed that the Lysis Gene Cassette is induced by IPTG and resulted in the initiation of cell lysis after 15 minutes of IPTG induction. Cells were completely lysed by 20 minutes. It would be interesting in future studies to determine if the time of cell lysis could be regulated by IPTG at concentrations below 0.2 mM.</font><span id="selectionBoundary_1442551834478_7756834849715233" class="rangySelectionBoundary" style="line-height: 0; display: none;">&#65279;</span></font></span></div>

Revision as of 11:56, 18 September 2015

Results - iGEM2015 wiki

Characterization of Lysis Gene Cassette Smutλ-Rλ-Rzλ (BBa_K1695038)

Upon induction with 0.2 mM, 1 mM and 5 mM of IPTG, recombinant E. coli harboring the lysis cassette Smutλ-Rλ-Rzλ (BBa_K1695038) showed a sharp drop in absorbance (A600) after 15 minutes whereas cells without IPTG induction showed continued as indicated by the steady increase in OD (Figure 6A). The results show that 0.2 mM IPTG is sufficient to initiate cell lysis after 15 minutes, and the speed of cell lysis is not increased at higher concentrations of IPTG (Figure 6B). Overall, the use of (1) OD600 measurement to determine cell growth or cell lysis (panel A) and (2) plate count (cfu; panel B) showed that the Lysis Gene Cassette is induced by IPTG and resulted in the initiation of cell lysis after 15 minutes of IPTG induction. Cells were completely lysed by 20 minutes. It would be interesting in future studies to determine if the time of cell lysis could be regulated by IPTG at concentrations below 0.2 mM.
Figure 6. Cell lysis upon induction of lysis cassette by IPTG in E. coli cells. Recombinant E. coli carrying lysis cassette Smutλ-Rλ-Rzλ (BBa_K1695038) was cultured in minimal medium (supplemented with 0.2% glucose and 0.5(0.02%) casamino acid). IPTG at various concentrations (0 mM, ·) (0.2 mM, ·), 1 mM, ·) (5 mM, ·) was added to the bacterial culture at OD600 ~ 0.6. Samples were taken at 5-min  and 10-min intervals from IPTG induced and uninduced cultures for (A) OD600 measurements and (B) cell plating on LB solid medium (CFU count) to determine the percentage (%) of cell survival, respectively.