Team:NCTU Formosa/RNA data

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.

Circuit A: BBa_J23101 is a constitutive promoter, and the 37℃induced RBS we use is BBa_K115002. The mGFP is the gene coding for “Green Fluorescence Protein”.

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 shows the highest fluorescent protein expression at the 37°C and 42°C.

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

Figure 3: Testing RNA Thermometer
The flow cytometer’s data shows the highest fluorescent protein expression at the 37°C and 42°C. That proves the 37°C induced RBS (RNA thermometer) has an appropriate function.
The time-course expression results were measured with time to test RNA thermometer for 12 sets (4 samples by three repeats) of GFP expression devices. The circuit is inserted in backbone pSB3K3, and the host cell is EPI300. Each measuring was detected every 15 minutes. And all of the data represented the average of three independent measurements. Error bars indicated standard deviations. X-axis indicated the time units, and Y-axis indicated the fluorescence units with different scales (MEFL stands for molecules of equivalent fluorescence). Furthermore, the fluorescent signals changed with time per cell were measured by using a flow cytometer.


Circuit B: Ptet BBa_R0040 is a constitutive repressible promoter, and the RBS we use is BBa_B0034. The mGFP is the gene coding for “Green Fluorescence Protein”.

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 shows 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 4: The reporter circuit:
The result shows 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.
The time-course expression results were measured with time to test RNA thermometer for 12 sets (4 samples by three repeats) of GFP expression devices. The circuit is inserted in backbone pSB3K3, and the host cell is EPI300. Each measuring was detected every 15 minutes. And all of the data represented the average of three independent measurements. Error bars indicated standard deviations. X-axis indicated the time units, and Y-axis indicated the fluorescence units with different scales (MEFL stands for molecules of equivalent fluorescence). Furthermore, the fluorescent signals changed with time per cell were measured by using a flow cytometer.


Circuit A+B: BBa_J23101 is a constitutive promoter, and the 37℃induced RBS we use is BBa_K115002.Ptet BBa_R0040 is a constitutive repressible promoter, and the RBS we use is BBa_B0034. The mGFP is the gene coding for “Green Fluorescence Protein”. The terminators are all BBa_J61048.

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 (figure 5.), 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.

Figure 5: A low temperature release system with RNA thermometer
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.
The time-course expression results were measured with time to test RNA thermometer for 12 sets (4 samples by three repeats) of GFP expression devices. The circuit is inserted in backbone pSB3K3, and the host cell is EPI300. Each measuring was detected every 15 minutes. And all of the data represented the average of three independent measurements. Error bars indicated standard deviations. X-axis indicated the time units, and Y-axis indicated the fluorescence units with different scales (MEFL stands for molecules of equivalent fluorescence). Furthermore, the fluorescent signals changed with time per cell were measured by using a flow cytometer.

Comment

Our experimental results indicate that high temperature decreased the translation rate of the target protein, and this temperature-dependent genetic circuit can control the expression level of the target protein by the host cell's incubation temperature. However, the translational activity of the BBa_K115002 at different temperatures cannot be quantified directly from experimental data. To overcome this problem, we provided a dynamic model which can quantitatively assess the translation strength of the BBa_K115002 at temperatures 27°C, 32°C, 37°C and 42°C.