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

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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 the lactose-inducible promoter (BBa_K1695053)</font></u></span><br /><span style=""></span></h2>
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<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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the above lacI-regulated promoter </span><span style="font-size: medium; line-height: 1.5em; text-align: justify; background-color: initial;">(BBa_K1695000)</span>
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<span style="font-size: medium; line-height: 1.5em; text-align: justify; background-color: initial;">to GFP (BBa_I13504) (Figure 1). The response of this GFP reporter
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construct to IPTG induction was then measured based on the GFP fluorescence
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signal (excitation wavelength: 485nm; emission wavelength: 520 nm). GFP
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fluorescence signal from cells carrying the PL8-UV5-GFP construct (BBa_K1695053)
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was monitored over a 4.5-hour time course under a range of IPTG concentrations
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ranging from 0.01 &ndash; 10 mM. Normalization of GFP fluorescence was performed
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based on the A</span><font size="1">600</font></font><span style="font-size: medium; line-height: 1.5em; text-align: justify; background-color: initial;"><font color="#2a2a2a"> values of the culture.</font></span><span style=""></span><br /><span style=""></span><br /><span style=""></span><span style=""></span></div>
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<div class="paragraph" style="text-align:center;"><font color="#2a2a2a" size="3"><strong>Figure 1. Construct of the reporter biobrick, BBa_K1695053</strong></font><br /><span style=""></span></div>
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<div class="paragraph" style="text-align:justify;"><font size="1"><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 /></span></div>
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<h2 class="wsite-content-title" style="text-align:left;">Improved/ expanded characterization of LacY-LacZ biobrick (BBa_S04055)</h2>
 
<h2 class="wsite-content-title" style="text-align:left;">Improved/ expanded characterization of LacY-LacZ biobrick (BBa_S04055)</h2>
  

Revision as of 09:23, 18 September 2015

Results - iGEM2015 wiki

Characterization of the lactose-inducible promoter (BBa_K1695053)


 The GFP reporter biobrick (BBa_K1695053) was constructed by linking the above lacI-regulated promoter (BBa_K1695000) to GFP (BBa_I13504) (Figure 1). The response of this GFP reporter construct to IPTG induction was then measured based on the GFP fluorescence signal (excitation wavelength: 485nm; emission wavelength: 520 nm). GFP fluorescence signal from cells carrying the PL8-UV5-GFP construct (BBa_K1695053) was monitored over a 4.5-hour time course under a range of IPTG concentrations ranging from 0.01 – 10 mM. Normalization of GFP fluorescence was performed based on the A600 values of the culture.

Figure 1. Construct of the reporter biobrick, BBa_K1695053
Figure 2. Expression analysis of L8-UV5 promoter-GFP biobrick (BBa_K1695053).  (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. 


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.

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.