Part:BBa_K2904010
Modular Adda 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 activating Adda riboswitch, which can regulate the expression of adenosine deaminase by binding 2-aminopurine in Vibrio vulnificus.The first 150bp of adenosine deaminase was chosen as Stabilizer of Adda riboswitch because our docking matrix suggested that a normal riboswitch structure would be observed when using this length of Stabilizer. This part was modular Adda riboswitch containing the original riboswitch, Stabilizer and Tuner A.
Result
We did two kinds of experiments to help us confirm the function of modular Adda riboswitch containing Tuner A. What's more, sfGFP and EYFP was used to ensure that our modular riboswitch will work with different proteins.
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, Adda-sfGFP and Adda-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 adenine riboswitch had sfGFP introduced directly. The direct fusion of sfGFP to Stabilizer yielded very clear inclusion bodies, manifested as distinct spots present at one pole of the cell which are formed by misfolded insoluble proteins. By comparison, the modular Adda riboswitch yielded working protein since Tuner has the ability to insulation the target gene from Stabilizer.
The result by microplate reader
The qualitative experiment is not enough to analyze the modular Adda 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 Adda riboswitch can restore the structure of riboswitch and control the downstream gene expression during the whole cultivation period.
Then sfGFP was substituted by EYFP. To test its effect, we used microplate reader to measure the fluorescence intensity of EYFP. 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 modular Adda riboswitch can achieve rational regulation of EYFP by responsive to different concentrations of 2-AP.
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 Adda riboswitches including Tuner A to E 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!]
First, 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 Adda riboswitches.
Then we also tested our modular Adda riboswitches by flow cytometer. The figure below shows the measured expression distributions at the same induction for modular Adda riboswitch with different Tuners.
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 B,Tuner C,Tuner D,Tuner Eand Tuner S.
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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