Revision as of 11:40, 16 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

This part was GFP(mut3b) followed by a variation of the WT SsrA tag sequence, which codes for the amino acid sequence AANDENYNYADAS. When the degradation tag fused to the C-terminal of proteins, it will make the protein susceptible to fast degradation through SspB-mediated binding to the ClpX protease as well as ClpA. DAS variants of SsrA tags require SspB for efficient degradation.

Note that tagged protein degradation rates are dependent on concentration of proteases (ClpX, ClpA) and binding mediators (SspB). Degradation rates are also heavily dependent on protein stability and depend on Km of binding to the protease, which is highly variable with each substrate. Be aware of ClpX, ClpA, and SspB knockouts when using SsrA tags; some expression strains have these mutations.

Result

Compared with GFP(mut3b), we found that the degradation tag has the ability to degrade the protein quickly.

Protocol

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 RNA

Sequence and Features