Difference between revisions of "Team:Ionis Paris/Measurement"
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| − | <p>However, we encountered a major issue during the realization of this side-projects: in order for the software to work, we needed to know the RPU (Relative Promoter Unit) of our promoters. This RPU method is fairly recent, and no RPU database currently exists. We therefore wanted to determine by ourselves the RPU for the two promoters used in our biosensor, (Pr and Pu), and maybe add our results as the beginning of a crowdsourced RPU database. In order to perform this experiment, we designed | + | <p>However, we encountered a major issue during the realization of this side-projects: in order for the software to work, we needed to know the RPU (Relative Promoter Unit) of our promoters. This RPU method is fairly recent, and no RPU database currently exists. We therefore wanted to determine by ourselves the RPU for the two promoters used in our biosensor, (Pr and Pu), and maybe add our results as the beginning of a crowdsourced RPU database. In order to perform this experiment, we designed a series of BioBricks (see <a href="https://2016.igem.org/Team:Ionis_Paris/Parts">Parts</a>) where our promoters were expressing GFP. As the Pr promoter is constitutive, its GFP BioBrick was pretty simple. However, as Pu requires the XylR/Toluene complex to be activated, we modified our biosensor so it produces GFP instead of luciferase. The cells were then induced with toluene prior manipulation, in order to trigger GFP expression.</p> |
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| − | + | <p>However, the manipulation did not work as expected, as the standard used for RPU measurement gave no significant data. Though we observed fluorescence for both our BioBricks, we couldn't calculate RPUs due to that lack of standard data. The fluorescence measurement will however serve as characterization data.</p> | |
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Revision as of 16:34, 17 October 2016
Cello is a newly created software, allowing plasmid optimization through powerful bioinformatics tools. It was described in 2016 in “Genetic Circuit Design Automation” (Nielsen et al.), and is freely available here
The CelloCad takes a genetic construction as an input, and gives as an output the best possible plasmid for this genetic construction. We heard about Cello through one of our advisors, and were immediately appealed by the characterization possibilities of such a software. We wanted to design an enhanced version of our biosensor plasmid, and then compare its properties (detection range, response rapidity…) to the one we designed ourselves. However, we encountered a major issue during the realization of this side-projects: in order for the software to work, we needed to know the RPU (Relative Promoter Unit) of our promoters. This RPU method is fairly recent, and no RPU database currently exists. We therefore wanted to determine by ourselves the RPU for the two promoters used in our biosensor, (Pr and Pu), and maybe add our results as the beginning of a crowdsourced RPU database. In order to perform this experiment, we designed a series of BioBricks (see Parts) where our promoters were expressing GFP. As the Pr promoter is constitutive, its GFP BioBrick was pretty simple. However, as Pu requires the XylR/Toluene complex to be activated, we modified our biosensor so it produces GFP instead of luciferase. The cells were then induced with toluene prior manipulation, in order to trigger GFP expression. However, the manipulation did not work as expected, as the standard used for RPU measurement gave no significant data. Though we observed fluorescence for both our BioBricks, we couldn't calculate RPUs due to that lack of standard data. The fluorescence measurement will however serve as characterization data.CelloCAD Plasmid Optimization
Our team wanted to work with the Cello software in order to try to optimize our plasmid. Though we may not have the most complex genetic circuit, we were interested in the possible outcomes of such an experiment.
