Difference between revisions of "Team:Hong Kong HKUST/Parts Improvement"

 
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    <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>
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  <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 lower without a terminator.
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<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>
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   <h2 class="text-muted"><em><b>THE INVESTIGATION</b></em></h2><br>
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   <li id="default"><a href="https://2016.igem.org/Team:Hong_Kong_HKUST/Parts_Improvement"></a></li>
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<p>We hypothesized that the expression level of mTagBFP is affected by the presence or absence of a terminator.  
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<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 RBS (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 lower when the transcription was not properly terminated.</p>
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<br><br><br>&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;
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<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>
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<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>
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  <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>
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      <h1> Navigation</h1>
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      <p>Before boarding, go check out below some tricks that will be useful in the course of your journey!</p>
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<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>
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      <hr>
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  <p> *For a detailed documentation 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>
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      <h2>Trick 1 - The Mode Switcher</h2>
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      <p1>The mode switcher, in analogue to a traffic light, combines 3 different interfaces into one powerful device switchable to one another. <br><br> Red: <b>Default mode</b> <small>(Veteran, no support needed!)</small><br> Yellow: <b>Tour mode</b> <small>(Summary of each and every page in one single map)</small>  <br> Green: <b>Construct mode</b> <small>(A reference diagram of our genetic construction)</small></p1>
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<area shape="circle" coords="242 126 20" alt="Home" href="https://2016.igem.org/Team:Hong_Kong_HKUST/Danson" onmouseover="tourFunction_home()" onmouseout="tourFunction_home()">
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<area shape="circle" coords="333 126 20" alt="About" href="https://2016.igem.org/Team:Hong_Kong_HKUST/The_Team" onmouseover="tourFunction_about()" onmouseout="tourFunction_home()">
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      <p id="tourPar">Welcome to HKUST 2016 iGEM team - Troika!!  We made a simple tour to make navigating our website a breeze! Follow us!</p>
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<h1><b>Improvements on BBa_K592023 and BBa_K592024 <br><br>(Blue Fluorescent Protein mTagBFP generators)</b></h1>
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<p>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.
+
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<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>
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<figp><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>
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document.getElementById("tourPar").innerHTML = "Let's meet our members and see how division of labour works out in our team! Also, check out the attributed parties of our project if you may.";
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document.getElementById("tourPar").innerHTML = "Get to know our project here! We built a biological device termed as tri-stable switch that could perform three discrete but alternating, steady states driven by three different repressible promoters. What's more, you can find a schematic diagram of the genetic switch by clicking on the green light button on the top right corner for your reference!";
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document.getElementById("tourPar").innerHTML = "We list out here the medal requirements that we have fulfilled, with other additional achievements such as new parts that we submitted to the Registry.";
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document.getElementById("tourPar").innerHTML = "You can have a grasp of how we are to sustain our project in the future as well as how an add-on investigation on the transcriptional manipulation in collaboration with the Rice University was conducted.";
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document.getElementById("tourPar").innerHTML = "For the outreach part, our main focus is on designing an applicable product of the tri-stable switch that is intended for public convenient use. Other missions that we have accomplished so far include educating high school students, exploring intellectual property right policies, and assisting other iGEM teams in plan-making.";
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document.getElementById("tourPar").innerHTML = "To evaluate our works, we identified the parameters potentially affecting our design, predicted, and verified the consistency of our experimental results by bringing about a mathematical prediction model and a stability analysis.";
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document.getElementById("tourPar").innerHTML = "We have compiled our logbooks together with the protocols we employed in this page. Safety information is also provided.";
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document.getElementById("tourPar").innerHTML = "We joined this year's iGEM Interlab Measurement Study where we conducted a relative expression compression by green fluorescence measurement.";
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document.getElementById("tourPar").innerHTML = "This year, we collaborate with two institutions, NUS and IIT in respective areas.";
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Latest revision as of 19:22, 19 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 lower 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 RBS (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 lower 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 documentation of this characterization and the materials or methods used, please refer to our PDF version of this investigation.