Difference between revisions of "Part:BBa K2904040"

Revision as of 13:37, 17 October 2019

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 project, we found that Paris_Bettencourt has created a cobalamin biosensor to measure vitamin B12 but got a bad result. The cobalamin biosensor was based on a repressing 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 vitamin B12. They associated this sequence with mRFP1 after suppressing the start codon of the fluorescent protein.

With our principle, the cobalamin biosensor can be optimized. The modular cobalamin biosensor consisting of the repressing riboswitch, the first 81bp 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. However, the modular Btub riboswitch demonstrates a greater induction difference since Stabilizer can maintain the structure of riboswitch and Tuner has the ability to improve the function of one.

The result by microplate reader

The qualitative experiment is not enough to analyze the improved riboswitch. So we tested our system by microplate reader, which is used to reflect the intensity of sfGFP changing over time. 改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.

Figure1: The results of modular Btub riboswitch containing Tuner A by microplate reader.

Sequence and Features