Team:NCTU Formosa/RNA data

We constructed three circuits to make sure the function of RNA thermometer. At the end we will combine those parts in one complete circuit with Low-temperature release system.

Figure 3: Testing RNA Thermometer

First, we combined a common promoter with 37°C induced RBS (RNA thermometer) and mGFP gene. It is for testing the function of RNA thermometer. Therefore, we can get the RNA thermometer’s regulation on green fluorescent protein expression. The flow cytometer’s data had shown the highest fluorescent protein expression at the 37°C and 42°C.

That prove the 37°C induced RBS (RNA thermometer) has an appropriate function (figure 3).

Figure 4: The report circuit

By this circuit we can realize the base line of the protein expression with common RBS and Ptet promoter. Later, we will add more parts on this circuit to regularly express target protein, so we have to build up the base line of protein expression.

The result shown E.coli will generate better volume of protein at the 32°C and 37°C, it is a reasonable conclusion. That’s because 32-37°C is the most appropriate growth temperature for E.coli. Therefore, we get the information about the basic growth temperature and base line of protein expression in this diagram (figure 4).

Figure 5: A low temperature release system with RNA thermometer

In this circuit, we combined the 37°C induced RBS (RNA thermometer), tetR gene and Ptet promoter with a reporter protein (GFP). We want to construct a circuit that will be turn on at low temperature and that will be turn off at high temperature. That’s why we combined 37°C induced RBS and tetR gene in the upstream circuit. When E.coli is cultivated above 37°C, it will make tetR protein to inhibit Ptet promoter and reporter protein cannot express. At the same time, E.coli will focus on expanding population and produce enough material for target product. So when we shift E.coli to lower temperature, it will smoothly produce target protein with plenty of material and no promoter inhibitor.

As the experimental data shown, the lower temperature 27°C and 32°C can lead E.coli to produce the greatest amount of GFP. In contrast, the higher 37°C and 42°C cause E.coli to produce GFP in slight amount.