RiboTALs induce repression of constitutive promotersReturn to Top

We subjected our constructs to a no induction condition with no arabinose or theophylline added, which would result in maximal GFP expression for each promoter. We also subjected our constructs to the induction condition of 1% arabinose and 10mM Theophylline, which would result in maximal production RiboTALe transcript and TAL repressor proteins. Thus, difference in fluorescence between no induction and induction conditions would be due to the RiboTALe repression activity. We measured the fluorescence of our constructs in E. Coli strain MG1655Z1 over a course of 9-10 hours using the Tecan Infinite 200Pro microplate reader. Please refer to the Protocols page for details on our culture preparation and Tecan testing parameters. In the graphs shown here, error bars indicate the range over which the average fluorescence values were calculated.

The data show clear repression of the reporter under conditions of maximal induction of the RiboTALe transcription factor, indicating that gene expression is repressible by the synthetic transcription factor. In other words, the once constitutive promoters are now repressor-controlled by the RiboTALe device. The data also show that the promoters maintain, qualitatively, their relative strengths. The value listed under each promoter is it's relative promoter strength as listed in the Registry of Standard Biological Parts. The fold-decrease in fluorescence achieved by maximum induction of the RiboTALe was 56.8, 21.7, 3.99, and 1.38 for J23100, J23101, J23105, and J23109, respectively.

Repression is modulated by theophylline and promoter strengthReturn to Top

After ascertaining that the new promoter constructs were in fact repressible with addition of the RiboTALe inducers, the sensitivity to different levels of theophylline induction was investigated for each promoter construct. The promoters were subjected to theophylline induction levels ranging from 0 mM to 10 mM. Below are the results of these experiments, measured at the RiboTALe transcript induction level of 0.1% arabinose.

These data show greater repression at greater levels of theophylline induction, for all promoter constructs. The data indicate that the repression activity of the RiboTALe device is sensitive to the level of theophylline added. A RiboTALe transcription factor may be used to reliably modulate the expression of genes of interest by means of the riboswitch ligand.

Control of RiboTALe transcript levels reliably alters system responseReturn to Top

The sensitivity of the system to levels of RiboTALe transcript was investigated by subjecting the testing constructs to arabinose induction levels ranging from 0.01% to 0.50%, where arabinose is the inducer for the RiboTALe transcript. The constructs were also subjected to the previous range of theophylline induction levels to ascertain continued repression activity. The image below presents the results of this experiment for the J23101 promoter construct.

The data show a faster system response and greater repression at larger arabinose levels. Furthermore, theophylline sensitivity is maintained across different levels of RiboTALe transcript. The amount of RiboTALe transcript available is a controllable element of the repression system that affects gene expression in a consistent and predictable manner. The relative promoter strengths of the original constitutive promoter continues to be apparent, though each of the promoters has been shown to be repressor-controlled by the RiboTALe transcription factors.

3D RiboTALe Data PlotReturn to Top

Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view. The maximum Y value of the 2D plot can also be changed with the buttons in the upper left corner of the 2D plot.

(See the secondary data page for data on an additional construct involving J23106 and a smaller scale 3D plot for a better view of the J23105 and J23109 data.)