Difference between revisions of "Part:BBa K2904030"

Revision as of 06:33, 18 October 2019

Modular Four U riboswitch

Design

Background of 2019 OUC-China's project——RiboLego

Due to context-dependent performance and limited dynamic range, the widespread application of riboswitches is currently restricted. By replacing its original ORF with a new one, the structure of an aptamer domain can be subtly disrupted, resulting in a loss of ligand response. So riboswitch is still not be considered as a ‘plug and play' device. To tackle these problems, our project focuses on a standardized design principle to be used for modular and tunable riboswitch. The modular riboswitch we defined consists of the original riboswitch, Stabilizer and Tuner. Stabilizer can protect the structure of riboswitch from damage while Tuner can reduce the expression probability of fusion protein and make improvement of riboswitch function.

The construction of this part

After successfully constructing modular kinetic riboswitches, we then want to demonstrate that our guideline can also apply to the thermodynamic riboswitch. 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.

Result

To validate this new thermodynamic switch, we engineered a device including the tetracycline promoter, the modular Four U riboswitch and sfGFP. After construction, the circuit was transformed into DH5αZ1. Given that the temperature threshold of Four U is 37℃, we set the culture temperature to 28℃, 37℃ and 42℃ in liquid LB medium respectively. We can observe a noticeable difference when cultured for 30 hours. Though centrifugation, the recombination strain at 37°C and 42°C shows green while the strain at 28°C shows no fluorescence.

The result of other team

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.

Sequence and Features