Theophylline Riboswitches

To characterise the theophylline riboswitches (type 1 and 2), we quantified their activation at different theophylline concentrations (0 mM, 0.5 mM, 1 mM, 1.5 mM and 2 mM) over a period of time using fluorometry. Competent E. coli (strain DH5α) cells were transformed with plasmid vectors containing the riboswitch and were cultured until the mid-log phase of growth. Thereafter, a different concentration of theophylline was added to each culture for induction. The activation of the riboswitch was detected as a fluorescent response as a result of increased translation of the fluorescent fusion protein CheZ-Venus, in the presence of the activator. A Jasco FP-6300 spectrofluorometer was used to excite the cultures at 514 nm and the intensity of the emission peak was detected at 528 nm. Twenty readings were taken every 15 minutes for each culture, for a total period of 135 minutes. A non-fluorescent construct containing a Promoter-Cre-recombinase-double terminator was used as a negative control. Conversely, a construct that comprised of a Promoter-RBS-CheZ-Venus-double terminator (BBa_K537013) was used as a positive control. Fluorescent data for the 3D plots (Fig 1-4) were normalised by subtracting the fluorescent values of the negative control form that of the riboswitches.

Figure 1: The fluorescence of the CheZ-Venus fusion protein under the regulation of the theophylline riboswitch 1 (BBa_537011). A) The activation of the riboswitch over time at different concentrations of theophylline. The fluorescence increased over time, with an increasing concentration of theophylline, indicating the theophylline concentration-dependent expression of the CheZ-Venus fluorescent fusion protein. The fluorescence peaks 105 minutes after the addition of 1 mM theophylline. B) The maximum fluorescence detected was compared to negative and positive controls at 1 mM theophylline after 105 minutes incubation, and to that of baseline levels (0 minutes with 1 mM theophylline). 1 mM of theophylline induced the expression of CheZ-Venus to increase 4-fold after 105 minutes (values used for the calculation had the corresponding negative control value subtracted first).

Figure 2: The fluorescence of the CheZ-Venus fusion protein under the regulation of the theophylline riboswitch 2 (BBa_K537012). A) The activation of the riboswitch over time at different concentrations of theophylline. The same trend of increased switch activation over time, with increasing theophylline concentrations was seen as with theophylline riboswitch 1. However, with the type 2 switch, the fluorescence peaks 135 minutes after the addition of 1.5 mM theophylline. B) The maximum fluorescence detected was compared to negative and positive controls at 1.5 mM theophylline after 135 minutes incubation, and to that of baseline levels (0 minutes with 1.5 mM theophylline). 1.5 mM of theophylline induced a 37-fold increase in CheZ-Venus expression after 135 minutes (values used for the calculation had the corresponding negative control value subtracted first).

Figure 3: The fluorescence of the Venus reporter protein under the regulation of the theophylline riboswitch 1 (BBa_K537009). A) The fluorescent peak occurred prematurely after 75 minutes of incubation with 0.5 mM theophylline, compared to riboswitch 1 that contained the gene for the CheZ-Venus fusion protein. The maximum fluorescent intensity observed was also lower by a factor of 2 RFU. B) The maximum fluorescence detected was compared to negative and positive controls at 0.5 mM theophylline after 75 minutes of incubation, and to that of baseline levels (0 minutes with 0.5 mM theophylline). A 4.5-fold increase in protein expression was observed as the maximum (values used for the calculation had the corresponding negative control value subtracted first). This fold change is comparable to that described in Figure 1.

Figure 4: The fluorescence of the Venus reporter protein under the regulation of the theophylline riboswitch 2 (BBa_K537010). A) The activation of the riboswitch was affected by the lack of the CheZ gene in the switch construct. The increase in fluorescence was minimal upon induction and incubation over time. Furthermore, the fluorescent peak occurred prematurely after 60 minutes of incubation with 0.5 mM theophylline, when compared to the theophylline riboswitch 2 that contained the gene for the CheZ-Venus fusion protein. B) The maximum fluorescence detected was compared to negative and positive controls at 0.5 mM theophylline after 60 minutes of incubation, and to that of baseline levels (0 minutes with 0.5 mM theophylline). A 2-fold increase in protein expression was observed as the maximum (values used for the calculation had the corresponding negative control value subtracted first). This is far less than the value described for the same switch in Figure 2.

Discussion and conclusion:

Both the theophylline riboswitches displayed activation in response to increasing theophylline concentrations and incubation times. Theophylline riboswitch 2 controlling CheZ-Venus expression (BBa_K537012) clearly showed a superior level of activation compared to that theophylline riboswitch 1 (BBa_537011). This was expected of theophylline riboswitch 2 as it was selected by FACS for its optimised expression platform that allows for minimal leakiness at basal levels, and a strong activation in the presence of theophylline (Lynch and Gallivan, 2009). The activation shown by both the riboswitches regulating CheZ-Venus expression should be able to restore the motility of E. coli cells deficient in CheZ, in the presence of theophylline. More importantly, the theophylline concentration-dependent increase in CheZ-Venus expression alludes to the ability of directed cell movement, that is moving up concentration gradients of theophylline (Topp and Gallivan, 2007). Interestingly, the data suggests that the presence of the CheZ gene enhances the activation of the switches when compared to the constructs with venus alone. This may be due to some sort of structural stability conferred by the presence of the CheZ sequence.

Atrazine Riboswitch

Preliminary activation data was obtained for the atrazine riboswitch.Fluorometry was performed on E. coli (stain DH5α) cells transformed with a construct that contained a Promoter-atrazine riboswitch-mRFP-double terminator (BBa_K537014). Similar to the theophylline riboswitches, different concentrations (0 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM) of atrazine were added to the cultures for induction. The cultures were excited at 584 nm and detected at 607 nm using a Jasco FP-6300 spectrofluorometer. Readings were taken every 15 minutes for 120 minutes in total. The same Promoter-Cre-recombinase-double terminator construct was used as a negative comtrol. The 3D plot data was normalised the same way as for the theophylline riboswitches.

Figure 5: The fluorescence of the mRFP1 reporter protein under the regulation of the atrazine riboswitch(BBa_K537014).A) The activation of the atrazine riboswitch at different atrazine concentrations over time. There was an increase in fluorescence as the atrazine concentration and incubation time increased. The amount of fluorescence detected was very small, with a maximum of 0.1 RFU detected after 75 minutes of incubation with 2 mM atrazine. B) The maximum was compared to the negative control at 75 minutes, 2 mM atrazine, and to that of fluorescent levels detected at basal levels (0 minutes, 2 mM atrazine). A 1.7-fold increase in expression of mRFP1 was observed over the 75 minute period (values used for the calculation had the corresponding negative control value subtracted first).

References

Lynch, S. A. & Gallivan, J. P. 2009. A flow cytometry-based screen for synthetic riboswitches. Nucleic Acids Res, 37, 184-92.

Topp, S. & Gallivan, J. P. 2007. Guiding bacteria with small molecules and RNA. J Am Chem Soc, 129, 6807-11.

Epi-fluorescence Wide-Field Microscopy

Fluorescent imaging was performed for a qualitative assay of the engineered bacteria.

Bacterial culture and Manipulation:

4mL of LB broth with Ampicillin was inoculated from colonies off a master plate of positive clones and grown overnight at 37°C in a shaking incubator. 2mL of each bacterial culture was then used to inoculate 25mL of LB broth with Ampicillin and grown for ± 3 hours in a shaking incubator at 37°C until the cells reached an OD600 of 0.55 (mid-log phase). 2 x 5mL of each sample culture was aliquoted into separate 15mL falcon tubes. One tube was left untreated; 150µL of 15mM theophylline was added to the other (to make a final concentration of 1.5mM theophylline). These 5mL aliquots were placed in a shaking incubator at 37°C for 30 min each to allow for activation. 200µL of each treated and untreated sample was then used in a microscope plate for imaging.

Equipment:

Zeiss Axioscope - Andor EMCCD Camera - Till Photonics Polychrome V

Imaging:

Widefield fluorescence microscopy was used to assess the expression of the Venus protein. The bacterial samples were excited at 500nm using the polychrome as an excitation source. The fluorescence was then observed by capturing light in the emission spectrum of Venus (528nm). The figure below represents a schematic of the imaging setup. Corresponding bright-field images (using white light) were also captured for each experiment.

Below are the images that were obtained for:

• The parental strain (BW25113) of the E. coli CheZ-deletion mutant, which served as the negative control

• ThRS1-venus

• ThRS2-venus

Please note: Bacteria which were transformed with constructs which contained the CheZ gene were not tested in this assay. This experiment served as a means of observing the activation of the theophylline riboswitches, hence only expression of the venus reporter protein was needed for detection of fluorescence. Activation of motility was not assessed in this case – it was examined in the motility assays on semi-solid agar plates. Differences in fluorescence intensities were not calculated from the images below since the fluorometry experiments served as the quantitative approach to measurement of riboswitch activation.

1. Negative Control:

Figure 1: The parental strain (BW25113) of the E. coli CheZ-deletion mutants were not transformed with any plasmid DNA. The brightfield images in the left column depict all bacterial cells. The venus images in the right column depict bacterial cells which emitted fluorescence. In both the absence and presence of theophylline, the parental bacteria did not fluoresce – which was the expected result.

2. Theophyllline Riboswitch (Type 1) fused to Venus:

Figure 2: E. coli CheZ-deletion mutants which were transformed with the Promoter-ThRS1-venus-DoubleTerminator (BBa_K537009) construct in pSB1A3 plasmid backbone. The brightfield images in the left column depict all bacterial cells. The venus images in the right column depict bacterial cells which emitted fluorescence. In the absence of theophylline, almost no fluorescence occurred. Upon the addition of theophylline at a concentration of 1.5mM, many of the cells emitted fluorescence showing activation of the theophylline riboswitch (type 1).

3. Theophyllline Riboswitch (Type 2) fused to Venus:

Figure 3: E.coli CheZ-deletion mutants which were transformed with the Promoter-ThRS2-venus-DoubleTerminator (BBa_K537010) construct in pSB1A3 plasmid backbone. The brightfield images in the left column depict all bacterial cells. The venus images in the right column depict bacterial cells which emitted fluorescence. In the absence of theophylline, some fluorescence was observed. This result showed the leakiness of the riboswitch. A substantial amount of venus translation is permitted because of the flexible conformation of this riboswitch. Upon the addition of theophylline at a concentration of 1.5mM, much more fluorescence was detected. More venus was expressed in these cells due to the activation of the riboswitch via theophylline.

Semi-solid agar plates containing different concentrations of theophylline (0mM, 0.5mM, 1.0mM and 1.5mM) were made. Promoter-theophylline riboswitch 1-CheZ-Venus-double terminator (T1-Ch-V)(BBa_K537011), Promoter-theophylline riboswitch 2-CheZ-Venus-double terminator (t2-Ch-V) (BBa_K537012), Promoter-theophylline riboswitch 1-Venus-double terminator (t1-V) (BBa_K537009) and Promoter-theophylline riboswitch 2-Venus-double terminator (t2-V) (BBa_B573013) were transformed into CheZ deletion mutants and grown to mid-log phase (OD600 = 0.55). 1.5μl of culture was plated into the centre of the plates containing the various concentrations of theophylline and incubated at 37°C for 24 hours. This experiment was performed in triplicate. The protocol for this experiment can be found here.

Figure 1: The distance travelled by CheZ deletion mutants (mm) over a 24 hour period at 37°C on semi-solid agar plates containing different theophylline concentrations (mM)

The t1-V and t2-V transformants did not travel from the point of inoculation for all concentrations of theophylline. The CheZ deletion mutants containing t1-Ch-V travelled 0mm at 0mM theophylline and reached a maximum travel distance of 5mm at 1.5mM theophylline. The t2-Ch-V containing CheZ deletion mutants travelled 2mm in the absence of theophylline and at a concentration of 1.5 mM theophylline, were able to travel 22mm from the point of inoculation

Figure 2 85mm semi-solid agar plates containing 1.0mM theophylline indicating the point of inoculation and distance travelled by the bacteria over a 24 hour period. A) Inoculation of 1.5μl of mid-log phase E. coli CheZ deletion mutants transformed with theophylline riboswitch 1-venus. No evidence of movement of bacteria from point of inoculation. B) Inoculation of 1.5μl of mid-log phase E. coli CheZ deletion mutants transformed with theophylline riboswitch 2-venus. There is clearly no evidence of movement of bacteria from the point of inoculation

Both the t1-V and t2-V constructs transformed into the CheZ deletion mutants are non-motile, each having travelled a distance of 0mm at all concentrations of theophylline (See A and B). This indicates that in the absence of the CheZ protein, the bacteria are non-motile (Topps and Gallivan, 2007).

In order to determine whether we had successfully engineered the E. coli CheZ deletion mutants to chemotactically migrate towards theophylline, we performed a capillary assay. Bacteria transformed with t1-V, t2-V, RBS-CheZ, t1-Ch-V and t2-Ch-V were grown to mid-log phase. Activation of chemotaxis was achieved by incubating the cultures with 1 mM theophylline prior to the assay. A 50μl drop of each culture was then placed onto separate sterile surfaces and two capillary tubes were placed into each drop. The first was a control, containing only PBS and 20 μM EDTA. The second tube contained PBS, 20 μM and 2mM theophylline. The two capillary tubes were suspended into each drop of culture for 30 minutes. A dilution series of the solutions in the tubes were plated and colonies counted after 12 hours. Further details of this protocol can be found here.

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Discussion and conclusion

Cells transformed with t1-V, t2-V and RBS-CheZ all displayed an index of approximately 100, indicating the same number of bacteria moved towards the control (0mM theophylline) and the experiment (2mM theophylline) capillary tube. This proves that none of these constructs induce chemotactic behaviour. In terms of both t1-V and t2-V, the lack of CheZ results in the inability of CheY dephosphorylation and the swimming motion observed in chemotaxis, which is a key driver in directed movement towards an attractant (Topps and Gallivan, 2007). Although the RBS-CheZ construct constitutively expresses CheZ, and bacterial swimming is achieved through CheY dephosphorylation, the expression levels remain constant regardless of the presence or absence of theophylline. Random movement will occur as a result.

The t1-Ch-V and t2-Ch-V transformants, with chemotaxis indices of 180 and 227 respectively, both indicated a significantly higher number of bacteria that travelled to the 2mM theophylline solution in the capillary tube compared to that of the control. The chemotaxis index of the t2-Ch-V construct is significantly higher (p=0.005) than that of t1-Ch-V, indicating that cells transformed with t2-Ch-V is more attracted to theophylline than t1-Ch-V. Should maximum chemotaxis towards theophylline be desired, t2-Ch-V would be the more suitable construct.

The graph indicates that the control constructs RBS-CheZ, t1-V and t2-V have similar chemotaxis indices. Both t1-Ch-V and t2-Ch-V have chemotaxis indices (180 and 227 respectively) significantly higher than those constructs where CheZ expression is not under the control of riboswitches