Part:BBa_K2904004
Tuner E
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
We defined a Tuner element to include a repressing region, a RBS region and a coupled junction region. The repressing region is the reverse complement of a subsequence of the RBS region so that Tuner can form a hairpin with appropriate ∆G. The stop and start codon fused in the junction region. Ribosomes recruited by the upstream riboswitch can open up the hairpin of Tuner before dissociation at the stop codon in the junction region. Additional ribosomes can then assemble at the Tuner RBS and initiate translation at the first start codon of the introduced gene of interest. Therefore, Tuner can facilitate tuning of a riboswitch’s response and help GOI express normally.
The following diagram shows the structure of Tuner E and we marked each region clearly.
Result
RiboLego based on Adda
Using this part, we could engineer modular riboswitch. In order to validate the effect of Tuner E, we utilized 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. We used Tuner E to construct modular Adda riboswitch and sfGFPas the reporter gene to reflect output of our system.
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 Tuner E can enhance the function of riboswitch and help riboswitch control the downstream gene expression during the whole cultivation period.
RiboLego based on Btub
By employing the Adda riboswitch, we found it’s possible to use our design principle to optimize the function of riboswitch. So we chose the Btub riboswitch, a repressing riboswitch which responds to adenosylcobalamin to verify the universal applicability of our guideline. After running our program, the first 150bp of BtuB, the original target gene of the Btub riboswitch was used to serve as Stabilizer. We introduced Tuner E to construct modular Btub riboswitch and sfGFP was reporter gene as the output of this system.
By microplate reader, we measured the intensity of sfGFP changing over time in our system. The following chart shows the dynamic curve measured every two hours. As we can see, Tuner E can enhance the function of 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 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 A,Tuner B,Tuner C,Tuner Dand 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]
| None |