Part:BBa_K2904040
Modular cobalamin riboswitch
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
Our team’s vision is a standardized and easy adaptable design principle to be used for riboswitch of different purposes. By referencing the previous iGEM projects, we found that Paris_Bettencourt has created a cobalamin biosensor to measure vitamin B12. The cobalamin biosensor is based on a riboswitch taken from a transcribed fragment upstream of a cobalamin biosynthesis gene, cbiB, which is found in Propionibacterium shermanii and has been demonstrated to be sensitive to B12. At first, they used EGFP as their reporter gene, however, even in the absence of cobalamin, they had no GFP expression at all. Then they substituted EGFP with mRFP1 and inserted the first 24 bases of cbiB between them, the result was bad, too.
With our principle, the cobalamin biosensor can be optimized. The modular cobalamin biosensor consisting of the repressing riboswitch, the first 144bp of the original target gene and Tuner A. To test the functionality of the improved construct, the modular riboswitch was under the lac promoter and controlled the expression of mRFP1.
Result
The result by confocal microscopy
By Confocal Microscopy Leica TCS SP8, it’s obvious that no fluorescence could be observed when the original cobalamin biosensor designed by Paris_Bettencourt had mRFP1 introduced directly. It means that Stabilizer they chose was too short that couldn’t protect the structure of riboswitch. The structure offered by RNAfold also verified our prediction. On the contrary, the modular cobalamin riboswitch can express mRFP1 successfuly since Stabilizer can maintain the structure of riboswitch and Tuner has the ability to improve the function of one.
The result by microplate reader
To test the functionality of the improved construct, we measured the fluorescence level emitted by our strain in the presence of increasing levels of adocobalamin by microplate reader. We can observe an obvious decrease in mRFP1 expression with the adocobalamin concentration increased, which demonstrates that the riboswitch was improved successfully!
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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