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Team:NCTU Formosa/RNA design
From 2011.igem.org
RNA Thermometer
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
At the left, the RNA forms stable base-pairs on the Shine-Dalgarno sequence (SD sequence), disabling the ribosome to bind. (SD sequence is the polypurine sequence AGGAGG centered about 10 bp before the AUG initiation codon on bacterial mRNA.) It is now in the off-state. The base-pairing of this RNA region will block the expression of the protein encoded behind it. In the other hand, at the right, certain external factor is added and 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.
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 inside 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 , 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, the temperature change 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.
We used the RNA thermometer to build up a low-temperature released system which was designed to control the expression of targeted protein. 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, 37°C and 40°C.
We also gather the data of green fluorescent protein’s expression via flow cytometer.
