Difference between revisions of "Team:Austin UTexas LASA/Contribution"

Line 1: Line 1:
 
{{:Team:Austin_UTexas_LASA/Templates/header}}
 
{{:Team:Austin_UTexas_LASA/Templates/header}}
 
<html>
 
<html>
 +
  <div class ="column-full-size">
 +
      <h1>Contribution</h1>
 +
  </div>
 +
  <div class="container">
 +
      <h3>Group: Austin_UTexas_LASA 2017</h3>
 +
      <p>Summary: We validated the promoter function by a fluorescence detection assay utilizing green fluorescent protein (GFP)-fused construct to BBa_I14034 promoter part.</p>
 +
      <br></br>
 +
      <p>
 +
        This construct is produced by combining BBa_114034 promoter part and a GFP coding sequence. This plasmid contains ColE origin and CamR resistance.
 +
        <br></br>
  
−
 
+
      Three negative control constructs containing different promoter sequences were used and compared in this assay: 1) T7 promoter (Invitrogen, USA), 2) pCP25 (BBa_K1509003) and 3) pPA3 promoter (BBa_K2458000). These three promoter do not contain GFP coding sequence.
−
 
+
      <br></br>
−
<div class ="column-full-size">
+
      For the fluorescence detection, we used a plate reader Infinite M200pro (TECAN Trading, Switzerland) which is equipped at the Ellington lab in UT Austin.
−
<h1>Contribution</h1>
+
      <br></br>
−
</div>
+
      Because the part’s description stated that part BBa_I14034 is designed as a constitutive promoter, we did not add Isopropyl b-D-1-thiogalactopyranoside (IPTG) as a inducer for gene expression.
−
<div class="container">
+
      <br></br>
−
<h3>Group: Austin_UTexas_LASA 2017</h3>
+
      The plate reader measured the absorbance and fluorescence of each cell containing the BBa_I14034::GFP construct and non-GFP construct. Value of fluorescence was divided by cell density (absorbance) and the final result was shown as a graph in Figure 1 (below). The graph in figure 2 (below) shows a close-view of the fluorescence measured of the GFP driven by the control promoters.
−
<p>Summary: We validated the promoter function by a fluorescence detection assay utilizing green fluorescent protein (GFP)-fused construct to BBa_I14034 promoter part.</p>
+
      <br></br>
−
<br></br>
+
      <img src="https://static.igem.org/mediawiki/2017/b/ba/T--Austin_UTexas_LASA--promotercontrols.png">
−
<p>
+
      <img src="https://static.igem.org/mediawiki/2017/1/10/T--Austin_UTexas_LASA--driv.png">
−
This construct is produced by combining BBa_114034 promoter part and a GFP coding sequence. This plasmid contains ColE origin and CamR resistance.
+
      <br></br>
−
<br></br>
+
      RFU (relative fluorescence units) = value of fluorescence/value of absorbance
−
Protocol
+
      <br></br>
−
<ol>
+
      The result demonstrates that BBa_I14034 promoter is working to expression GFP in bacterial cells. Level of GFP expression was around 9,500 RFU. Cells containing non-GFP controls (T7::empty construct, CP25::empty construct, and PA3 empty construct) did not show significant detections.  
−
<li>Liquid cultures</li>
+
      <br></br>
−
<li>Pick colonies from plate</li>
+
      <br></br>
−
<li>Multiple controls </li>
+
      <img src ="https://static.igem.org/mediawiki/2017/6/6f/T--Austin_UTexas_LASA--contrib.jpg" width="50%" height = "50%" align = "right" style="margin:5px 5px">  
−
<li>Overnight in the 37 incubator </li>
+
      <br></br>
−
<li>Liquid cultures of already grown liquid cultures</li>
+
      </p>
−
<li>100 uL of grown liquid cultures into 5 mL of media + CamR</li>
+
  </div>
−
<li>Incubate cultures for another 4-6 hours </li>
+
−
<li>Spin down cultures </li>
+
−
<li>3000 rpm for 5 minutes</li>
+
−
<li>Resuspend in 1x Buffer</li>
+
−
<li>4 mL of 1x PBS </li>
+
−
<li>Pipette or vortex to resuspend cells</li>
+
−
<li>Plate reading</li>
+
−
<li>Take 100 uL of the resuspended cells</li>
+
−
<li>Load into a 96 well transparent plate</li>
+
−
<li>Measure absorbance and fluorescence with GFP assay (excitation: 495 nm, emission: 509 nm)
+
−
</li>
+
−
</ol>
+
−
 
+
−
Three negative control constructs containing different promoter sequences were used and compared in this assay: 1) T7 promoter (Invitrogen, USA), 2) pCP25 (BBa_K1509003) and 3) pPA3 promoter (BBa_K2458000). These three promoter do not contain GFP coding sequence.
+
−
<br></br>
+
−
For the fluorescence detection, we used a plate reader Infinite M200pro (TECAN Trading, Switzerland) which is equipped at the Ellington lab in UT Austin.
+
−
<br></br>
+
−
Because the part’s description stated that part BBa_I14034 is designed as a constitutive promoter, we did not add Isopropyl b-D-1-thiogalactopyranoside (IPTG) as a inducer for gene expression.
+
−
<br></br>
+
−
The plate reader measured the absorbance and fluorescence of each cell containing the BBa_I14034::GFP construct and non-GFP construct. Value of fluorescence was divided by cell density (absorbance) and the final result was shown as a graph in Figure 1 (below). The graph in figure 2 (below) shows a close-view of the fluorescence measured of the GFP driven by the control promoters.
+
−
<br></br>
+
−
<img src="https://static.igem.org/mediawiki/2017/b/ba/T--Austin_UTexas_LASA--promotercontrols.png">
+
−
<img src="https://static.igem.org/mediawiki/2017/1/10/T--Austin_UTexas_LASA--driv.png">
+
−
 
+
−
<br></br>
+
−
RFU (relative fluorescence units) = value of fluorescence/value of absorbance
+
−
<br></br>
+
−
The result demonstrates that BBa_I14034 promoter is working to expression GFP in bacterial cells. Level of GFP expression was around 9,500 RFU. Cells containing non-GFP controls (T7::empty construct, CP25::empty construct, and PA3 empty construct) did not show significant detections.  
+
−
<br></br>
+
−
<br></br>
+
−
 
+
−
<img src ="https://static.igem.org/mediawiki/2017/6/6f/T--Austin_UTexas_LASA--contrib.jpg" width="50%" height = "50%" align = "right" style="margin:5px 5px">  
+
−
<br></br>
+
−
</p>
+
−
</div>
+
−
 
+
 
</html>
 
</html>
 
{{:Team:Austin_UTexas_LASA/Templates/footer}}
 
{{:Team:Austin_UTexas_LASA/Templates/footer}}

Revision as of 03:36, 2 November 2017

Contribution

Group: Austin_UTexas_LASA 2017

Summary: We validated the promoter function by a fluorescence detection assay utilizing green fluorescent protein (GFP)-fused construct to BBa_I14034 promoter part.



This construct is produced by combining BBa_114034 promoter part and a GFP coding sequence. This plasmid contains ColE origin and CamR resistance.

Three negative control constructs containing different promoter sequences were used and compared in this assay: 1) T7 promoter (Invitrogen, USA), 2) pCP25 (BBa_K1509003) and 3) pPA3 promoter (BBa_K2458000). These three promoter do not contain GFP coding sequence.

For the fluorescence detection, we used a plate reader Infinite M200pro (TECAN Trading, Switzerland) which is equipped at the Ellington lab in UT Austin.

Because the part’s description stated that part BBa_I14034 is designed as a constitutive promoter, we did not add Isopropyl b-D-1-thiogalactopyranoside (IPTG) as a inducer for gene expression.

The plate reader measured the absorbance and fluorescence of each cell containing the BBa_I14034::GFP construct and non-GFP construct. Value of fluorescence was divided by cell density (absorbance) and the final result was shown as a graph in Figure 1 (below). The graph in figure 2 (below) shows a close-view of the fluorescence measured of the GFP driven by the control promoters.



RFU (relative fluorescence units) = value of fluorescence/value of absorbance

The result demonstrates that BBa_I14034 promoter is working to expression GFP in bacterial cells. Level of GFP expression was around 9,500 RFU. Cells containing non-GFP controls (T7::empty construct, CP25::empty construct, and PA3 empty construct) did not show significant detections.





Our Sponsors
Follow Us
Contact Us
LASA iGEM
Liberal Arts & Science Academy
7309 Lazy Creek Dr, Austin, TX 78724