Latest revision as of 03:23, 22 October 2019

Vitamin B12 biosensor, composed by a cobalamin riboswitch in front of an mRFP1

This part can be used as a Vitamin B12 biosensor, we tested it for the AdoCobalamin form of B12 (AKA Coenzyme B12), but it might be used also to detect CyanoCobalamin, HydroxyCobalamin, MethylCobalamin according to the following publication : "A riboswitch sensor to determine vitamin B12 in fermented foods" Xuan Zhu 2015. We didn't tried this because we were out of time, anyway this would have to be characterized.

2019 OUC-China's characterization and improvement

Background

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.

Background of this part

2015Paris_Bettencourt iGEM team wanted to develop a reliable assay to measure vitamin B12, so they chose a riboswitch-based biosensor. 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 whose upstream is cobalamin riboswitch under control of the lac promoter. 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. They constructed this part consisting of the cobalamin riboswitch, the truncated cbiB gene, mRFP1 without start codon and rrnB T1 teminator.

Result

We transformed this circuit into DH5αZ1. To test the functionality of our construct, we measured the fluorescence level emitted by recombinant strain in the presence of different concentration of adocobalamin by microplate reader. To our surprise, the fluorescence intensity is very low at any concentration of adocobalamin.

Figure1: The results by microplate reader, which indicates that the cobalamin biosensor can't express mRFP1 at all. It's obvious that there is no fluorescence in E.coli.

Improvement

This year, our team’s vision is a standardized and easy adaptable design principle to be used for riboswitch of different purposes. 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. The result below by microplate reader shows that our improved cobalamin riboswitch works very well!

Figure2: The response curve of our improved cobalamin biosensor to Vb12. As we expected, an obvious decrease of mRFP1 expression was observed with the increased concentration of vb12. It demonstrates that the modular riboswitch functions properly.

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 can express mRFP1 normally since Stabilizer can maintain the structure of riboswitch and Tuner has the ability to improve the function of one.

Figure1: The results by confocal microscopy, which indicates that our principle can improve cobalamin biosensor successfully. It's obvious that the modular cobalamin riboswitch can express mRFP1.

Sequence and Features