Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id quod mazim placerat facer possim assum. Typi non habent claritatem insitam; est usus legentis in iis qui facit eorum claritatem. Investigationes demonstraverunt lectores legere me lius quod ii legunt saepius. Claritas est etiam processus dynamicus, qui sequitur mutationem consuetudium lectorum. Mirum est notare quam littera gothica, quam nunc putamus parum claram, anteposuerit litterarum formas humanitatis per seacula quarta decima et quinta decima. Eodem modo typi, qui nunc nobis videntur parum clari, fian

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 OD 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 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 (E.coli CheZ mutants), 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 (E.coli CheZ mutants) which were not transformed with any plasmid DNA. The brightfiled 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 mutants which were transformed with the StrongPromoter-ThRS1-venus-DoubleTerminator construct in pSB1A3 plasmid backbone. (Strong promoter – Bba_J23119 ; ThRS1- venus – Bba_K537003; double terminator – Bba_B0015). The brightfiled 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 mutants which were transformed with the StrongPromoter-ThRS2-venus-DoubleTerminator construct in pSB1A3 plasmid backbone. (Strong promoter – Bba_J23119 ; ThRS2- venus – Bba_K537004; double terminator – Bba_B0015). The brightfiled 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.

Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id quod mazim placerat facer possim assum. Typi non habent claritatem insitam; est usus legentis in iis qui facit eorum claritatem. Investigationes demonstraverunt lectores legere me lius quod ii legunt saepius. Claritas est etiam processus dynamicus, qui sequitur mutationem consuetudium lectorum. Mirum est notare quam littera gothica, quam nunc putamus parum claram, anteposuerit litterarum formas humanitatis per seacula quarta decima et quinta decima. Eodem modo typi, qui nunc nobis videntur parum clari, fian

Lorem ipsum dolor sit amet, consectetuer adipiscing elit, sed diam nonummy nibh euismod tincidunt ut laoreet dolore magna aliquam erat volutpat. Ut wisi enim ad minim veniam, quis nostrud exerci tation ullamcorper suscipit lobortis nisl ut aliquip ex ea commodo consequat. Duis autem vel eum iriure dolor in hendrerit in vulputate velit esse molestie consequat, vel illum dolore eu feugiat nulla facilisis at vero eros et accumsan et iusto odio dignissim qui blandit praesent luptatum zzril delenit augue duis dolore te feugait nulla facilisi. Nam liber tempor cum soluta nobis eleifend option congue nihil imperdiet doming id quod mazim placerat facer possim assum. Typi non habent claritatem insitam; est usus legentis in iis qui facit eorum claritatem. Investigationes demonstraverunt lectores legere me lius quod ii legunt saepius. Claritas est etiam processus dynamicus, qui sequitur mutationem consuetudium lectorum. Mirum est notare quam littera gothica, quam nunc putamus parum claram, anteposuerit litterarum formas humanitatis per seacula quarta decima et quinta decima. Eodem modo typi, qui nunc nobis videntur parum clari, fian