Part:BBa_K2904005
Tuner S
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. This Tuner contains a ssrA degradation tag which help Stabilizer degrade quickly.
Result
Overview
Using Tuner S, we could engineer modular Btub riboswitch. After running our program, the first 150bp of BtuB, the original target gene of the Btub riboswitch was used to serve as Stabilizer. 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, the modular Btub riboswitch has a beautiful response curve, which demonstrates that Tuner S has the ability to improve the function of 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!]
The 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.
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 Aand Tuner E.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 80
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