Difference between revisions of "Part:BBa K2904080"

Revision as of 10:34, 21 October 2019

aTc inducible sfGFP regulated by 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

This part was used to validate our design principle of modular riboswitch. We employed 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. We used sfGFPas the reporter gene to reflect output of our system. Besides, Tuner A was inserted between Stabilizer and sfGFP. The modular Btub riboswitch containing the original riboswitch, Stabilizer and Tuner A was under control of the tetracycline promoter, which was induced by aTc.

Result

We did three kinds of experiments to help us confirm the function of modular Btub riboswitch containing Tuner A.

The result by confocal microscopy

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 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.

Figure1: Fluorescence microscopy of the uninduced and induced the original Btub, Btub fusion and modular Btub riboswitch, with the latter demonstrating a greater induction difference.

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.

Figure2: The results of modular Btub riboswitch containing Tuner A by microplate reader.

The result by flow cytometricy

Besides microplate reader, we also used flow cytometricy to test our system. The figure below indicated the good capability of modular Btub riboswitch containing Tuner A.

Figure3: The results of modular Btub riboswitch containing Tuner A by microplate reader. The purple group represents uninduced modular Btub riboswitch while the blue one represents induced modular riboswitch.

Summary

This year, we achieved a rational design principle of modular riboswitch. Many Tuners was utilized for tunable and efficient gene regulation. To validate Tuners can change the response curve of riboswitch, we constructed modular Btub riboswitches including Tuner A, E and 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!]
First, microplate reader was used to measure the fluorescence intensity of sfGFP. Data is shown for each construct until 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.

Figure4: The results of modular Btub riboswitch containing different Tuners by microplate reader.

Then we also tested our modular Btub 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 Eand Tuner S.

Sequence and Features