Latest revision as of 20:10, 21 October 2019

Promoter+RNA thermometer (FourU) +mRFP

This is a device containing a promoter (BBa_J23119),a RNA thermometer (BBa_K115002) and a RFP coding sequence (BBa_E0010) on the pSB1C3, which can be easily transformed into E.coli. RNA Thermometers(RNAT) are temperature-sensing RNA sequences in 5’UTR of their mRNAs. At low temperature, RNAT folds into structure, blocking access of ribosome; At high temperature, RNAT switch from off to open conformation, increasing the efficiency of translation initiation. When the temperature rises above 37°C, RFP would be expressed in theory.

Experiments

TUDelft-2008 has modified 3 kinds of RNAT: ROSE(BBa_K115001), FourU(BBa_ K115002), PrfA(BBa_K115003). We constructed each RNAT under the control of 3 different constitutive promoters:BBa_J23101, BBa_J23106, BBa_J23119 and use RFP as a reporter. After construction, the 9 circuits were transformed into DH5α. According to the results of TUDelft-2008, the temperature threshold is 37℃ for FourU and PfrA, 42℃ for ROSE. Thus, we set the culture temperature to 28℃, 37℃ and 42℃ on solid LB medium while 28℃, 35℃, 37℃, 40℃, and 42℃ in liquid LB medium.

Agar plate test

For one temperature, we spotted one strain on 3 different plates, each plate contains 9 different strains. After 48 hours, as we can see in the photo: FourU worked best under all these promoters and show great switch activity when be used cooperatively with J23101 and J23119.

Liquid test

These are our K1679017 which were cultured at 28℃, 35℃, 37℃, 40℃, and 42℃ from right to left. It has significant differences between these temperature in liquid LB medium.

The 9 circuits were tested through plate reader as well. We set 5 temperature: 28℃, 35℃, 37℃, 40℃ and 42℃. After 41 h, when some replicates had changed color, we calculated the Fluorescence(excitation wavelength-584 nm and emission wavelength-607 nm) and OD(600).

After 41 h culturing in solid LB medium , when some replicates had changed color, we calculated the Fluorescence(excitation wavelength-584 nm and emission wavelength-607 nm) and OD(600).This part works well in liquid LB medium.

2019 OUC-China's characterization

Background

RNA Thermometers(RNAT) are temperature-sensing RNA sequences in 5’UTR of their mRNAs. At low temperature, RNAT folds into the structure, blocking access of ribosome; at high temperature, RNAT switch from off to open conformation, increasing the efficiency of translation initiation.

Unlike other thermosensitive regulators, especially those dependent on protein conformation change, RNA sequences of RNAT is short, conforming convenience. In addition, based on conformation changes, RNAT responses to temperature change in a more immediate manner, which can also be classified as the thermodynamic riboswitch.

2015 OUC-China iGEM team has found that FourU under the control of promoter J23119 worked best, whose temperature threshold is 37℃. They constructed this device containing a promoter,FourU and a RFP coding sequence. When the temperature rises above 37°C, RFP would be expressed in theory.

Result

The result by agar plate test

We transformed the circuit in the pSB1C3 into DH5αZ1, and then observed the colour of the colony in solid LB medium under 28℃, 37℃ and 42℃ after 48 hours of culture. According to the results, the bacteria containing the plasmid with FourU element cultured at 37°C and 42°C become red while no change in the bacteria cultured in 28°C. So the expression of mRFP could be regulated by Four U element tightly responding to temperature changes.

Figure1: The colour of the colony in different temperature shows the regulatory ability of Four U.

The result by liquid test

For further verifying the working effect of FourU element, we shake the bacteria containing plasmid with FourU element in the liquid LB medium and set three culture temperatures: 28°C, 30°C and 37°C. After cultured for 30h, we can observe a noticeable difference. After centrifugation, the recombination strain at 37°C and 42°C shows red while the strain at 28°C shows no fluorescence.

Figure2: The results of liquid test show the working effect of Four U in different temperature.

The above qualitative experiments are not enough to verify the effect of Four U, so we tested the fluorescence intensity by microplate reader to measure the expression level of the reporter protein legulated by FourU element as temperature-controlled switch. We still set three culture temperatures: 28°C, 30°C and 37°C. After overnight culture, we choose 8 time points (6h, 24h, 30h) to test fluorescence intensity of RFP protein at excitation wavelength-584 nm and emission wavelength-607 nm. The results demonstrate that FourU element is a useful thermodynamic riboswitch and works very well!

Figure3: The results show the working effect of Four U in different temperature by microplate reader.

Extend to our project

This year, OUC-China has proposed a guideline to construct modular riboswitch for the future iGEM teams. The modular riboswitch we designed consists of the original riboswitch, Stabilizer and Tuner. Stabilizer can protect the structure of riboswitch from damage while Tuner have the ability to separate Stabilizer and GOI. The design principle can tackle many problems about riboswitch including context-dependent performance and limited dynamic range.

Given that riboswitches can furthermore be classifified into thermodynamic and kinetic switches, we wonder that whether our design principle can apply to the RNAT successfully. So we designed a modular Four U riboswitch containing the original Four U riboswitch, Stabilizer and Tuner A. Using docking matrix, we selected the first 132bp of RFP as Stabilizer because RFP can express under the control of Four U. Finally, we constructed a device to regulate the expression of sfGFP with modular Four U riboswitch . The result demonstrate the universality of our guideline.

[http://2019.igem.org/Team:OUC-China/Design Click here, you can learn more about our project!]

Sequence and Features