Difference between revisions of "Team:Hong Kong HKUST/Description"

(Prototype team page)
 
 
(20 intermediate revisions by 3 users not shown)
Line 2: Line 2:
 
<html>
 
<html>
  
 +
<body>
 +
<section class="section jumbotron">
 +
<div class="content_wrapper_neo">
 +
    <h1 class="title text-center">Improvements on BBa_K592023 and BBa_K592024<br><p style="font-size:35px;">(Blue Fluorescent Protein mTagBFP generators)</p></h1>
 +
  <blockquote><p>The 2011 Uppsala Sweden iGEM team had previously submitted characterization data for parts <a href= "http://parts.igem.org/Part:BBa_K592023">BBa_K592023</a> and <a href = "http://parts.igem.org/Part:BBa_K592024">BBa_K592024</a>. However, we discovered that they did not put a terminator after the mTagBFP CDS. We wondered if the observed fluorescence outputs will be different with versus without a terminator. Therefore, we decided to perform an experiment for comparison. The experimental results showed that the observed fluorescence levels were lowered without a terminator.
 +
<br><br>Given our results, we would like to caution future users in referencing the relative promoter strengths reported by Uppsala Sweden 2011, as their measured expression levels, generated by parts without a terminator, may not fully represent the true strength of the tested promoters.</p></blockquote>
 +
                </div>
 +
        </section>
 +
<section class="section">
 +
<div class="content_wrapper_neo">
  
 +
  <h2 class="text-muted"><em><b>THE INVESTIGATION</b></em></h2><br>
 +
<p>We hypothesized that the expression level of mTagBFP is affected by the presence or absence of a terminator.
 +
<br><br>An experimental construct was built where the mTagBFP generator (<a href = "http://parts.igem.org/Part:BBa_K592100">BBa_K592100</a>) was driven by a constitutive promoter (BBa_J23101) and a medium (BBa_B0032). It was compared against its counterpart which harbored a terminator, BBa_B1006 3’ to the CDS. Constructs without promoters served as controls for auto-fluorescence. We then performed the same experiment but used BBa_B0034 as the RBS instead. Both of the results showed that the observed blue fluorescence outputs were lowered when the transcription was not properly terminated.</p>
 +
<br><br><br>&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;
 +
<img class="img-responsive" src= "https://static.igem.org/mediawiki/2016/4/48/Team--Hong_Kong_HKUST--Parts_Improvement.png" style="width:85%; margin-left:auto; margin-right:auto;"><br><br>
 +
<Figp style="font-size:14px; padding: 0 0.9em;"><b>Figure 1. Comparison of the expression levels of a BFP generator with and without a terminator.</b> (a) Circuit diagram illustrates experimental setup of the experiment harboring BBa_B0032. (b) Experimental results of the experiment harboring BBa_B0032.(c) Circuit diagram illustrates experimental setup of the experiment harboring BBa_B0034. (d) Experimental results of the experiment harboring BBa_B0034. Error bars represent SEM of 3 independent experiments on 3 different days.</Figp><br><br><br>
 +
  <p>Our results indicated that the proper expression of mTagBFP requires a terminator, thus the previously submitted part BBa_K592023 (BBa_B0032-BBa_K592100) and BBa_K592024 (BBa_B0032-BBa_K592100) are translational units only and should not be considered as acceptable alternatives to mTagBFP generators, which have terminators.</p>
 +
                </div>
 +
        </section>
 +
<section class="section jumbotron">
 +
<div class="content_wrapper_neo">
 +
<p>In this experiment, we have improved BBa_K592023 (BBa_B0032-BBa_K592100) and BBa_K592024 (BBa_B0034-BBa_K592100) by adding a terminator BBa_B1006 3’ to these parts. Both of the improved parts, <a href = "http://parts.igem.org/Part:BBa_K1899001">BBa_K1899001</a> (BBa_K592023-BBa_B1006) and <a href = "http://parts.igem.org/Part:BBa_K1899002">BBa_K1899002</a> (BBa_K592024-BBa_B1006) were submitted to the Parts Registry this year. With the improved parts, we hope to provide future users with better reporters for their assays.  </p><br>
 +
  <p> *For a detailed doucmentation of this characterization and the materials or methods used, please refer to our <a href = "https://static.igem.org/mediawiki/2016/b/b0/Team--Hong_Kong_HKUST--Improvement.pdf"target="_blank">PDF version</a> of this investigation. </p>
 +
                </div>
 +
        </section>
  
−
<div class="column full_size judges-will-not-evaluate">
+
<script>
−
<h3>★  ALERT! </h3>
+
    $(document).ready(function(){
−
<p>This page is used by the judges to evaluate your team for the<a href="https://2016.igem.org/Judging/Medals"> improve a previous part or project gold medal criterion</a>. </p>
+
        $("div#ustMenuWrapper li#ustProjectTab").addClass("ustActiveTab");
−
<p> Delete this box in order to be evaluated for this medal. See more information at <a href="https://2016.igem.org/Judging/Pages_for_Awards/Instructions"> Instructions for Pages for awards</a>.</p>
+
    });
−
</div>
+
</script>
−
 
+
</body>
−
 
+
−
<div class="column full_size">
+
−
 
+
−
<p>Tell us about your project, describe what moves you and why this is something important for your team.</p>
+
−
 
+
−
 
+
−
<h5>What should this page contain?</h5>
+
−
<ul>
+
−
<li> A clear and concise description of your project.</li>
+
−
<li>A detailed explanation of why your team chose to work on this particular project.</li>
+
−
<li>References and sources to document your research.</li>
+
−
<li>Use illustrations and other visual resources to explain your project.</li>
+
−
</ul>
+
−
 
+
−
 
+
−
</div>
+
−
 
+
−
<div class="column full_size" >
+
−
 
+
−
<h5>Advice on writing your Project Description</h5>
+
−
 
+
−
<p>
+
−
We encourage you to put up a lot of information and content on your wiki, but we also encourage you to include summaries as much as possible. If you think of the sections in your project description as the sections in a publication, you should try to be consist, accurate and unambiguous in your achievements.
+
−
</p>
+
−
 
+
−
<p>
+
−
Judges like to read your wiki and know exactly what you have achieved. This is how you should think about these sections; from the point of view of the judge evaluating you at the end of the year.
+
−
</p>
+
−
 
+
−
</div>
+
−
 
+
−
 
+
−
<div class="column half_size" >
+
−
 
+
−
<h5>References</h5>
+
−
<p>iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you thought about your project and what works inspired you.</p>
+
−
 
+
−
</div>
+
−
 
+
−
 
+
−
<div class="column half_size" >
+
−
<h5>Inspiration</h5>
+
−
<p>See how other teams have described and presented their projects: </p>
+
−
 
+
−
<ul>
+
−
<li><a href="https://2014.igem.org/Team:Imperial/Project"> Imperial</a></li>
+
−
<li><a href="https://2014.igem.org/Team:UC_Davis/Project_Overview"> UC Davis</a></li>
+
−
<li><a href="https://2014.igem.org/Team:SYSU-Software/Overview">SYSU Software</a></li>
+
−
</ul>
+
−
</div>
+
−
 
+
−
 
+
−
 
+
 
</html>
 
</html>

Latest revision as of 16:21, 18 October 2016

Home

Improvements on BBa_K592023 and BBa_K592024

(Blue Fluorescent Protein mTagBFP generators)

The 2011 Uppsala Sweden iGEM team had previously submitted characterization data for parts BBa_K592023 and BBa_K592024. However, we discovered that they did not put a terminator after the mTagBFP CDS. We wondered if the observed fluorescence outputs will be different with versus without a terminator. Therefore, we decided to perform an experiment for comparison. The experimental results showed that the observed fluorescence levels were lowered without a terminator.

Given our results, we would like to caution future users in referencing the relative promoter strengths reported by Uppsala Sweden 2011, as their measured expression levels, generated by parts without a terminator, may not fully represent the true strength of the tested promoters.

THE INVESTIGATION


We hypothesized that the expression level of mTagBFP is affected by the presence or absence of a terminator.

An experimental construct was built where the mTagBFP generator (BBa_K592100) was driven by a constitutive promoter (BBa_J23101) and a medium (BBa_B0032). It was compared against its counterpart which harbored a terminator, BBa_B1006 3’ to the CDS. Constructs without promoters served as controls for auto-fluorescence. We then performed the same experiment but used BBa_B0034 as the RBS instead. Both of the results showed that the observed blue fluorescence outputs were lowered when the transcription was not properly terminated.




                

Figure 1. Comparison of the expression levels of a BFP generator with and without a terminator. (a) Circuit diagram illustrates experimental setup of the experiment harboring BBa_B0032. (b) Experimental results of the experiment harboring BBa_B0032.(c) Circuit diagram illustrates experimental setup of the experiment harboring BBa_B0034. (d) Experimental results of the experiment harboring BBa_B0034. Error bars represent SEM of 3 independent experiments on 3 different days.


Our results indicated that the proper expression of mTagBFP requires a terminator, thus the previously submitted part BBa_K592023 (BBa_B0032-BBa_K592100) and BBa_K592024 (BBa_B0032-BBa_K592100) are translational units only and should not be considered as acceptable alternatives to mTagBFP generators, which have terminators.

In this experiment, we have improved BBa_K592023 (BBa_B0032-BBa_K592100) and BBa_K592024 (BBa_B0034-BBa_K592100) by adding a terminator BBa_B1006 3’ to these parts. Both of the improved parts, BBa_K1899001 (BBa_K592023-BBa_B1006) and BBa_K1899002 (BBa_K592024-BBa_B1006) were submitted to the Parts Registry this year. With the improved parts, we hope to provide future users with better reporters for their assays.


*For a detailed doucmentation of this characterization and the materials or methods used, please refer to our PDF version of this investigation.