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 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></blockquote>
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   <h2 class="text-muted"><em><b>THE INVESTIGATION</b></em></h2><br>
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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 (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>
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    <h1 class="text-muted"><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 <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.
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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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<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>
 
<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><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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<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>We hypothesized that the expression level of mTagBFP is affected by the presence or absence of a terminator. To check if that was true, we compared fluorescence outputs from BFP generators with terminator, versus one without 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>
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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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   <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. <br><br>
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We are not sure about the reason behind this phenomenon. It could be an issue related to energy expenditure: transcription run off might have used extra energy resources in the cell, which would normally be available to generate more mTagBFP production. Repeating the above experiment but substituting the strong terminator with terminators of weaker strengths might shed light on our guess.<br><br>
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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. Furthermore, we hope to raise the awareness on the standards of parts measurement through this investigation. We cautioned on the direct use of previous measurement data for promoters generated using BBa_K592023 or BBa_K592024, and we suggest future iGEM teams that undertake measurement experiments of any kind not to omit parts that might appear irrelevant in the measurement cassettes. </p><br>
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   <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>
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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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   <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>  
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Revision as of 07:44, 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 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.