Team:UANL/Improve

IMPROVE

37°C inducible RNA Thermometer: An Improvement of BBa_K1140006

The conformation of RNA thermometers varies according to the temperature and their sequence. These structures can be associated to the ribosome binding site (RBS) and can be used for biotechnological purposes, such as controlling gene regulation to hide the RBS (off state) or leaving it exposed (on state), allowing the interaction of the RBS with the ribosome (Neupert, Karcher, & Bock, 2008). This strategy removes the need to add components in the environment that are usually of high cost or at certain concentrations that are toxic to the cells.

The primary objective was to perform an in silico redesign and in vivo characterization, to reduce the range of operation of previously reported thermometer [<partinfo>K1140006</partinfo>] (Rodríguez Ceja, 2015), obtaining a more precise range and allowing greater control of the system.


Experiments and Results

[<partinfo>K1140006</partinfo>] was redesigned in the area of the U6 thermometer(Figure 1), in order to reduce the operation temperature (28-37° C). The new piece, [<partinfo>K3317067</partinfo>], showed as RNA Thermometer Pro in the features, consists of 121 nucleotides, with a Hairpin formation in the position 91 to 101, before the RBS (AGGAGA), allowing greater control of the system.

Figure 1. Comparison of nucleotide sequences, corresponding to [<partinfo>K1140006</partinfo>] (Rodríguez Ceja, 2015) represented in pink, and the redesigned thermometer in blue. The modification sites are indicated with underlined and bold letters, while the site in gray is the RBS.

Because of the similarities of the images generated by RNA fold 2.3 (Figures 2 and 3), it was evaluated that the ΔG was lower in the redesigned thermometer, meaning that it requires more energy to denature the hairpin structure.

Figure 2. Simulation result at 36° C in the RNA fold 2.3 bioinformatics program. The structure on the left belongs to the [<partinfo>K1140006</partinfo>] thermometer (Rodríguez Ceja, 2015), and on the right, the Pro thermometer [<partinfo>K3317067</partinfo>]. The arrows and the thin contour rectangle indicate secondary structures that alter the stability of the RNA. The RBS sequence (is enclosed in thick contour rectangles) is not free, so the translation won't be allowed.

Figure 3. Simulation result at 37° C in the RNA fold 2.3 bioinformatics program. The structure on the left belongs to the [<partinfo>K1140006</partinfo>] thermometer (Rodríguez Ceja, 2015), and on the right, the Pro thermometer [<partinfo>K3317067</partinfo>]. The arrows and the thin contour rectangle indicate secondary structures that alter the stability of the RNA. The RBS is free allowing translation and is indicated by thick contour rectangles.

To compare both RNA thermometers, in vivo tests were performed.(Figure 4.) Bacteria were grown at 25, 30, 36, 37 and 40° C, growing overnight at 900 rpm, inoculating 5 µL of bacteria with [<partinfo>K3317067</partinfo>] (in microcentrifuge tubes with the cap pierced with a hot needle to allow aeration) 600 µL de LB broth with kanamycin. The same way with the RNA Thermometer from [<partinfo>K1140006</partinfo>] to make a comparison. The essays were made by triplicate.

Figure 4.Photographs of the bacteria pellets at the bottom of the tubes, growth at the temperatures indicated on every group. It can be seen by naked eye the different level of expression between the [<partinfo>K1140006</partinfo>] and [<partinfo>K3317067</partinfo>]

Every sample were homogenized and 200 µL were measured with a fluorometer at 587/610 nm (excitation/emission for mCherry(Tsien, Shaner, & Steinbach, 2005)) and 660 nm to obtain optical density and normalize results, which showed that the new thermometer controlled the expression of the reporter in a more precise range (Figure 4 and 5) like the predicted theoretically (Figures 2 and 3).

Analysis and Conclusions

Figure 5.Graph showing the dynamic range between the thermometers


As we can observe in the data obtained, our improved thermometer has an increment in the dynamic range of 9.59, compared to the original thermometer. This means that we were able to optimize the relation induction-leak, which is a desired attribute for a thermometer. In particular, this is a powerful tool when the desired gene to express represents a potential risk for the host, and the minimal expression causes toxicity or inhibits its proliferation.

Bibliography

  • Neupert, J., Karcher, D., & Bock, R. (2008). Design of simple synthetic RNA thermometers for temperature-controlled gene expression in Escherichia coli, 36(19), 1–9. https://doi.org/10.1093/nar/gkn545
  • Tsien, R. Y., Shaner, N. C., & Steinbach, P. A. (2005). A guide to choosing fluorescent proteins. Nature Methods, 2(12), 905–909. https://doi.org/10.1038/nmeth819
  • Rodríguez Ceja, J.G. (2015). Optimización de la Represión Génica Mediante el Uso Combinado de un Termómetro de ARN y el Factor Transcripcional TetR.