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

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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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    <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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<br><br><br>&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;&emsp;
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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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<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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<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. <br><br>
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<li> A clear and concise description of your project.</li>
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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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<li>A detailed explanation of why your team chose to work on this particular project.</li>
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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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<li>References and sources to document your research.</li>
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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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<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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<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>
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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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Revision as of 17:04, 14 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.




                

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.


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.

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.




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.

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.

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. 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.


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