Team:Hong Kong HKUST/riboregulator/results

Although there is a significant number of regulatory RNAs available in the registry, a comprehensive characterization information that the iGEM community can use to compare and contrast different regulatory RNAs (especially CR-TA riboregulators) is missing.Therefore we wanted to provide characterization information of regulatory RNAs so teams and labs will be confident in using these devices.

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 constructs like the diagram provided below: (provide diagram)

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 annealing. After annealing, these dsDNA was directly inserted into the vector containing BBa_I13401 ( GFP reporter for RHS of library test constructs). A promoter, BBa_J23102 was placed upstream by the scar formation of cis-repressing part therefore cis-repressing part can be constitutively transcribed.Trans-activating part was produced by the same method as cis-repressing part but an inducible promoter, P_bad, BBa_I0500 and double terminator, BBa_B0015 were used to replace BBa_J23102 and BBa_I13401 respectively. A low copy plasmid, pSB3K3 was used for GFP expression measurements. For the control of our characterisation, construct containing only RBS, 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 plasmid were used. We have submitted Cis-repressing and Trans-activating parts as well as some of intermediary parts.

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 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 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 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_bad (P_bad characterisation).

Furthermore the orthogonality of five different sets of riboregulators were tested in parallel to the repression and activation strength of riboregulators. The increase in fluorescence level was calculated using the fluorescence level obtained by plate reader at 0% and 2.5% of arabinose concentrations.