Part:BBa_K1333309
J23119-FourU-RFP
This part is a combination of a constitutive promoter(BBa_J23119), RNA thermometer(BBa_K115002), and a RFP gene(BBa_E1010).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 656
Illegal AgeI site found at 768 - 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
How the FourU Element functions compared to other RNAT
The 2008 iGEM team, TUDelft, has modified three kinds of natural zipper-like RNATs: Rose element (for short: ROSE), FourU element (for short FourU) and PrfA element (for short PrfA), which have already been made into basic parts of biobricks (BBa_K115001, BBa_K115002 and BBa_K115003). We then construct three plasmids containing these three RNATs respectively, between a constitutive promoter (BBa_J23119) and a reporter protein-mRFP (BBa_E1010), transformed the three plasmids into top10, to test whether these modified RNATs work well. We used the existing biobrick (BBa_J23100) in which only the FourU element is replaced by RBS compared to our experimental group, serving as the positive control. According to experiment results done by TUDelft, the temperature threshold of the part we choose is 37°C for FourU and PrfA, and 42 °C for Rose. So we set the culture temperature to 28°C, 37°C and 42°C, and if the RNAT work well, we could observe bacteria containing plasmid with FourU and PrfA cultured at 37°C and 42°C and the one with Rose cultured at 42°C changes to red and no change in the bacteria cultured in 28°C, that means these three RNAT change their conformations to the open state so that the ribosome could access the SD sequence.
According to the results, only the bacteria containing the plasmid with FourU element cultured at 37°C and 42°C become red. So the expression of mRFP could be regulated by FourU element tightly responding to temperature changes.
Further verification the working effect of FourU element as temperature-controlled switch
For further verifying the working effect of FourU element, we tested the fluorescence intensity of RFP protein to measure the expression level of the reporter protein regulated by FourU element as temperature-controlled switch. So we shake the bacteria containing plasmid with FourU element and the positive and negative control groups in the LB with chloramphenicol and set two culture temperature: 30°C and 37°C. After cultured for 24h, we test the fluorescent of RFP protein at excitation wavelength-584 nm and emission wavelength-607 nm.
And then, we extend the culture time to 30h and choose 3 time point (6h, 24h, 30h) to test fluorescence intensity of RFP protein expressed by each group, to test the level of leaked expression of RFP protein controlled by FourU element.
Figure 3 shows that, after cultured for 30h, the fluoresce intensity of RFP protein regulated by FourU at 30℃can still maintain a low level compared to the positive control cultured at the same temperature.
According to the results, FourU element works very well and the leak of its control is little.
Test the response of FourU element to temperature changes from 30°C to 37 °C
After further verifying the working effect of FourU element, we tested the response of FourU element to temperature changes from 30°C to 37 °C, for simulating the 3rd phase of our temperature control process. We cultured both the positive control and experimental bacteria at 30°C for 30h, and change the culture temperature to 37°C, and then test the fluoresce intensity of RFP protein after 1h, 2h and 3h. The result are as follows.
According to the results,after the bacteria shifted from 30°C to 37°C for 3h, the expression of RFP protein raise dramatically, that means FourU element shows a rapid response to temperature changes from 30°C to 37 °C.
For more information please visit our wiki
http://2014.igem.org/Team:SYSU-China/content.html#Project/Result/RNAT
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