Part:BBa_K2904020
Modular Btub riboswitch containing Tuner A
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
To validate our design guideline, we employed a repressing Btub riboswitch, which can regulate the expression of TonB-dependent vitamin B12 receptor BtuB by binding adenosylcobalamin in Escherichia coli.The first 150bp of BtuB was chosen as Stabilizer of Btub riboswitch because our docking matrix suggested that a normal riboswitch structure would be observed when using this length of Stabilizer. This part was modular Btub riboswitch containing the original riboswitch, Stabilizer and Tuner A.
Result
We did two kinds of experiments to help us confirm the function of modular Btub riboswitch containing Tuner A.
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.
Summary
This year, we achieved a rational design principle of modular riboswitch. Many Tuners was utilized for tunable and efficient gene regulation. To verify the functionality of different Tuners, we engineered different modular Btub riboswitches including Tuner A, Tuner E and Tuner S respectively. All circuits selected sfGFP as the target gene. Using different Tuners, muti-level regulation can be achieved.
[http://2019.igem.org/Team:OUC-China/Model The method we used to design different Tuners is on this page!]
The microplate reader was used to measure the fluorescence intensity of sfGFP. The figure below selects the data when steady state is reached(at least two consecutive subsequent data points do not increase fluorescence). The results demonstrate that these Tuners are capable of shifting and tuning the induction response of modular Btub riboswitches.
By all the experiments mentioned before, we proved that Tuners work as expectations successfully. They are expected to serve as a powerful and tunable tool of riboswitch for future iGEM teams based on their demand.
If you are interested in the other parts we designed, you can click modular riboswitches containing Tuner A,Tuner B,Tuner C,Tuner D,Tuner E,and Tuner S.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 156
- 1000COMPATIBLE WITH RFC[1000]
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