Team:Hong Kong HKUST/riboregulator/results

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<div class='content_1'><h3>Overview</h3>
 
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<p>Although there is a significant number of regulatory RNAs available in the registry, a comprehensive characterization information that the iGEM community can
 
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use to compare and contrast different regulatory RNAs (especially CR-TA riboregulators) is missing.Therefore we wanted to  provide characterization information of
 
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regulatory RNAs so teams and labs will be confident in using these devices. </p>
 
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<div class='content_1'><h3>Construct</h3>
 
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<p>In order to provide reliable characterisation data which will help iGEM teams to identify which CR-TA riboregulator works best for their projects. We have built
 
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constructs like the diagram provided below:
 
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<p>Cis-repressing part which was generated by using the Lock and Key algorithm generator from TU Delft 2009 iGEM team and followed by de novo synthesis from oligo
 
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annealing. After annealing, these dsDNA was directly inserted into the vector containing <a href="http://parts.igem.org/Part:BBa_I13401">BBa_I13401</a> ( GFP reporter for RHS of library test constructs). A promoter,
 
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<a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102</a> was placed upstream by the scar formation of cis-repressing part therefore cis-repressing part can be
 
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constitutively transcribed.Trans-activating part was produced by the same method as cis-repressing part but an inducible promoter, P<sub>bad</sub>, <a href="http://parts.igem.org/Part:BBa_I13401">BBa_I0500 </a>and double terminator,
 
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<a href="http://parts.igem.org/Part:BBa_BBa_B0015">BBa_B0015 </a>were used to replace <a href="http://parts.igem.org/Part:BBa_I13401">BBa_J23102 </a>and
 
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<a href="http://parts.igem.org/Part:BBa_I13401">BBa_I13401 </a>respectively. A low copy plasmid, pSB3K3 was used for GFP expression measurements. For the control of our characterisation,  construct containing only RBS,
 
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instead of CR  and another construct missing TA were built. To obtain the fluorescence level, autofluorescence had to be subtracted so bacteria containing pSB3K3-E0240
 
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plasmid were used. We have submitted Cis-repressing and Trans-activating parts as well as some of intermediary parts. </p>
 
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<div class='content_1'><h3> Results</h3>
 
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The above bar graphs represents the level of fluorescence measured by ? and it was plotted as fluorescence/OD600 on y-axis. Culture containing bacteria was
 
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incubated for X hours. (From left to right) For controls of each set of an experiment, (1) a construct without trans-activating part (2) a construct without
 
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cis-repressing part but with a ribosomal binding site (3) a construct without both cis-repressing and trans-activating parts. For our experiment, a construct
 
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containing both cis-repressing and trans-activating parts was used.  arabinose concentration of 0%, 1% and 2.5% were used to induce the inducible promoter, P<sub>bad</sub>
 
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(<a href="http://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterization">P<sub>bad</sub> characterisation</a>). </p>
 
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<p>Furthermore the orthogonality of five different sets of riboregulators were tested in parallel  to the repression and activation strength of riboregulators.
 
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The increase in fluorescence level was calculated using the fluorescence level obtained by plate reader at 0% and 2.5% of arabinose concentrations.</p>
 
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<div class='content_1'><h3> Discussion</h3>
 
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Latest revision as of 22:58, 17 October 2014