Team:NCTU Formosa/RNA design

RNA Thermometer system is about an RNA-based system that is able to have differential gene expression when temperature changes. The expression of heat-shock, cold-shock and some virulence genes are coordinated in response to temperature changes. Apart from protein-mediated transcriptional control mechanisms, translational control by RNA thermometers is a widely used regulatory strategy. RNA thermometers are thermo-sensors that regulate gene expression by temperature-induced changes in RNA conformation.

Figure 1: RNA thermometer

There are several systems suggested in literature that are based on RNA secondary structure. The idea in general is that if the temperature drops below a certain temperature, the RNA will form stable base-pairs on the Shine-Dalgarno sequence, disabling the ribosome to bind. The base-pairing of this RNA region will block the expression of the protein encoded behind it (figure 1). In this way gene expression can be regulated on the RNA level by temperature.

Figure 2: Post-transcriptional regulation acts at the RNA level.

At the left (figure 2), in the off-state, the RNA is folded into a hairpin structure that occludes the Shine Dalgarno region (ribosome binding site). In this situation the translation is blocked because the ribosome cannot bind to the RNA.

At the right (figure 2), an external factor causes a conformational change of the RNA, exposing the Shine Dalgarno region. Translation can now initiate, because the ribosome is able to bind to Shine Dalgarno region. The system is now in the on-state (figure 2).

We used the RNA thermometer to build up a low-temperature release system which was designed to control target protein expression. A specific ribosome binding site (RBS) BBa_K115002 with high translation activity at high temperature(> 37°C) and low translation activity at room temperature was used to design the temperature-dependent genetic circuit in E. coli, with a green fluorescent protein (GFP) used as the reporter protein. We analyzed fluorescence intensity during E. coli growth at log phase and stationary phase at temperatures 25°C, 30°C, 30°C and 40°C.

We also gather the data of green fluorescent protein’s expression via flow cytometer.