Part:BBa_K2904030
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 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. We did three kinds of experiments to help us confirm the function of modular Btub riboswitch containing Tuner E. </p>
The result by confocal microscopy
First, we selected sfGFP as the output of our system. It was under control of the tetracycline promoter, which was induced by aTc. For the sake of functional test, other 2 circuits are set, Btub-sfGFP and Btub-Stabilizer-sfGFP, which also were under control of the same promoter. By Confocal Microscopy Leica TCS SP8, it’s obvious that no fluorescence could be observed when the Btub riboswitch had sfGFP introduced directly. By comparison with Btub fusion, the modular Btub riboswitch demonstrates a greater induction difference since Tuner has the ability to improve the function of riboswitch.
The result by microplate reader
The qualitative experiment is not enough to analyze the modular Btub riboswitch. So we tested our system by microplate reader, which is used to reflect the intensity of sfGFP changing over time. The following chart shows the dynamic curve measured every two hours. It can prove that the modular Btub riboswitch can restore the structure of riboswitch and control the downstream gene expression during the whole cultivation period.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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