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

 
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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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<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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<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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<h1><b> Project Description </b></h1>
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<h4> Tri-stable switch is a biological device that could perform three discrete, but alternating, steady states driven by three different repressible promoters. The presence of a transient pulse of inducer allows effective state shifting, while signal interference is prevented. In order to enhance the specificities towards inducers, improvements were made based on the Brown’s tri-stable switch model in 2006. This year, three well-characterized repressible promoters are used: phlFp, tetp, and lacp. The whole construct is divided into three parts, and each contains one functional system with two protein coding sequences(CDS), creating an interconnected tri-stable toggle switch. Moreover, mathematical modelling is applied to predict and verify the consistency of the experimental results. At present, it is by far possible and practical to apply the switch in biosensing which could be achieved by developing a combinatorial circuit of promoters and CDS. With its advantageous characteristics, it is foreseeable that the switch could be applied in a wider spectrum of fields in the near future, for example, biocomputational system and diseases diagnostic.</h4>
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<p>Tell us about your project, describe what moves you and why this is something important for your team.</p>
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<h5>What should this page contain?</h5>
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<li> A clear and concise description of your project.</li>
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<li>A detailed explanation of why your team chose to work on this particular project.</li>
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<h5>Advice on writing your Project Description</h5>
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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.
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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.
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<h5>Inspiration</h5>
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<p>See how other teams have described and presented their projects: </p>
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<li><a href="https://2014.igem.org/Team:Imperial/Project"> Imperial</a></li>
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<li><a href="https://2014.igem.org/Team:UC_Davis/Project_Overview"> UC Davis</a></li>
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<li><a href="https://2014.igem.org/Team:SYSU-Software/Overview">SYSU Software</a></li>
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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.