Fluorescence is modulated by theophylline concentrations

We subjected our testing construct to the induction condition of 100 ng/mL aTc, which would result in constitutive and maximal expression of GFP given no repression. We also subjected the construct to the induction condition of 0.1% arabinose, which would produce a nominal level of RiboTALe transcript. We varied only the concentration of theophylline, over a range of 0 mM to 10 mM. Thus, difference in fluorescence between induction conditions would be due only to the RiboTALe repression activity. We measured the fluorescence of our construct 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.

The image above illustrates that GFP fluorescence is inversely related to theophylline concentrations, indicating that the TAL repressor is in fact being translated at rates corresponding to the theophylline induction levels, and effectively binding to its target site. At maximal theophylline concentrations, the expression of GFP is reduced 2.6 fold.

Next, we investigated what difference in system response we could achieve by altering the binding affinity of the TAL repressor protein.

Binding affinities of the TAL repressors provide tunability

The TAL repressor protein expressed by our RiboTALe device in our initial experiment has a dissociation constant K_D 1.3 ± .03 nM. We compared the activity of this RiboTALe to one under the control of the same theophylline riboswitch, but that expressed a TAL repressor with K_D 240 ± 40 nM. We subjected both RiboTALes to the induction condition of 100 ng/mL aTc, which would result in constitutive and maximal expression of GFP given no repression. At the same time, we subjected both RiboTALes to the following conditions:

	Arabinose Concentration (%)	Theophylline Concentration (mM)	Expected Result
	0.0	0.0	Full GFP expression due to a lack of RiboTALe transcript and thus TAL repressor
	1.0	0.0	Full GFP expression due to a lack of riboswitch inducer, and thus translation of the TAL protein. Decreased GFP expression could be attributed to riboswitch leakiness
	1.0	10.0	Full GFP repression, due the nominal expression of the RiboTALe transcript and the TAL repressor

The image above displays the peak fluorescence of two RiboTALe constructs, one expressing TALe 1 and the other expressing TALe 8, under different induction conditions for arabinose and theophylline. Both RiboTALes exhibit the expected behavior pattern given the induction conditions, but at consistently different levels of fluorescence. We have attributed this to the difference in binding affinities of the two TAL repressors to their respective binding sites.This variable, if well characterized for different TAL repressors, will provide a powerful means to control the tunability of these devices.

It is similarly interesting to note that under conditions of 1% arabinose, but no theophylline, there was clearly some reduction in fluorescence. We concluded that the riboswitch we used in this experiment had some degree of leakiness. We next investigated the possibility of altering riboswitch leakiness as another means to increase the tunability of our RiboTALe devices.

Riboswitch leakiness modulates RiboTALe activity

We proceeded to investigate the differences achievable in RiboTALe system response by varying the riboswitch controlling the translation of the TAL repressor. To this end we tested two RiboTALe devices, both of which expressed TALe 8. One of these RiboTALes was under the control of theophylline riboswitch Clone E and the other was under the control of theophylline riboswitch Clone 8.1*. We subjected both RiboTALes to the induction condition of 100 ng/uL aTC, which would result in constitutive and maximal expression of GFP given no repression. At the same time, we subjected both RiboTALes to the following conditions:

	Arabinose Concentration (%)	Theophylline Concentration (mM)	Expected Result
	0.0	0.0	Full GFP expression due to a lack of RiboTALe transcript and thus TAL repressor
	1.0	0.0	Full GFP expression due to a lack of riboswitch inducer, and thus translation of the TAL protein. Decreased GFP expression could be attributed to riboswitch leakiness
	1.0	10.0	Full GFP repression, due the nominal expression of the RiboTALe transcript and the TAL repressor

The image above displays the fluorescence results for the two RiboTALe devices tested in this experiment. According to the literature both riboswitches have similar reported fold activation ratios[1,2]. But it is clear that the two RiboTALe devices, differing only in the riboswitch controlling the translation of the TAL repressor, exhibit consistently different behavior. The data show that at 1% arabinose (the inducer for the RiboTALe transcript), but in the absence of theophylline, the RiboTALe under control of riboswitch Clone E is active. Under identical induction conditions, the RiboTALe under riboswitch Clone 8.1* exhibits no repression activity. The fluorescence measured was in fact higher than the baseline, possibly due to the extra carbon source provided by the arabinose. From the data we conclude that Clone E is leakier and yet stronger than Clone 8.1*, generating a 3.68 fold reduction in fluorescence as opposed to a 2.42 fold reduction. These data indicate that differences riboswitch leakiness and strength do impact RiboTALe system behavior, and can be engineered into RiboTALe designs as sources of tunability.

With all of the data that we have generated through testing the different parts of our system, we decided to put it all together with some 3D graphs.

3D RiboTALe Data Plot

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.

Project Background Learn about how we combine riboswitches and TAL's into robust orthogonal mechanisms for inducible repression.	Results Check out the cool results of our experiments with RiboTALs.
Human Practices Take a look at how we designed a new database for better raw data characterization of Biobricks.	Judging Criteria Here's the criteria that we met for this year's team.