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).

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.

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