Fluorescence is modulated by theophylline concentrations

We subjected our testing construct to the induction condition of 10 mM 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 the 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 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 (X) fold.

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

Variable binding affinities of the TAL repressors allow for system 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 10 mM aTC, which would result in constitutive and maximal expression of GFP given no repression. At the same time, we subjected both RiboTALes to the separate 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
	0.1	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
	0.1	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 0.1% 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.

Altering the riboswitch Shine-Delgarno sequence modulates RiboTALe activity

The study A flow cytometry-based screen for synthetic riboswitches by Sean Lynch and Justin Gallivan (Nucleic Acids Res. 2009 January; 37(1): 184–192) presents a library of theophylline riboswitches with randomized 8 base pair sequences in the Shine-Delgarno region of the transcript that were screened for riboswitch behavior. We proceeded to investigate the difference in RiboTALe system response achievable 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 10 mM aTC, which would result in constitutive and maximal expression of GFP given no repression. At the same time, we subjected both RiboTALes to the separate 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
	0.1	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	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
	0.0	10.0	Full GFP expression due to a lack of RiboTALe transcript and thus TAL repressor
	0.1	10.0	Full GFP repression, due the nominal expression of the RiboTALe transcript and the TAL repressor
	1.0	10.0	Full GFP repression, due the maximal expression of the RiboTALe transcript and the TAL repressor

The image above displays the relative change in fluorescence for the two RiboTALe devices tested in this experiment at arabinose induction levels of 0.0%, 0.1%, and 1.0% percent, where arabinose induces expression of the RiboTALe transcript. Riboswitch Clone E and riboswitch Clone 8.1* display the same activation ratio according to the studies from which we acquired the sequences. But it is clear that the two RiboTALe devices, differing only in the riboswitch controlling the translation of the TAL repressor, exhibit behavior indicating that differences in the SD sequence of the riboswitch do in fact impact system behavior[...complete .]

KO3D

Project Background Learn about how we combine riboswitches and TAL's into robust orthogonal mechanisms for inducible repression.	Results Check out the results of our experiments.
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.