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Team:UC Davis/Data
From 2013.igem.org
Proof of Concept: Our Testing Construct
To characterize the behavior of a RiboTALe device, we acquired cells containing the sequences for TAL repressors from the Tagkopoulos Lab at UC Davis, with which we have worked closely. We placed the TAL repressors downstream of theophylline-responsive riboswitches, the sequences of which were taken from the studies A high-throughput screen for synthetic riboswitches reveals mechanistic insights into their function [1], and Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species [2]. The riboswitch-TALe sequences were placed under the regulation of a pBAD promoter.
We inserted previously engineered TALe binding sites corresponding to the TAL repressors used in our characterization experiments upstream of a reporter, GFP. This target sequence was placed under the regulation of a pTET promoter.
We tested our construct by subjecting the pBAD promoter, the theophylline riboswitch, and the pTET promoter to a range of induction levels with arabinose, theophylline, and aTc, respectively. It was expected that at low levels of arabinose and theophylline, but at high levels of aTc, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter, the theophylline riboswitch, and the pTET promoter.
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 KD 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 KD 240 ± 40 nM. We subjected both RiboTALes to the induction condition of 10 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 | |
| 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.
Riboswitch activation level modulates RiboTALe activity
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*. Both riboswitches had similar reported fold activation ratios, but Clone E has leakier and displays a higher maximal activation level of gene expression than Clone 8.1* [1,2]. 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 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 | |
| 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 similar 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 riboswitch leakiness and maximal activation levels do in fact impact system behavior[...complete .]
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 3-D graphs.
3D RiboTALe data plots
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.
