Part:BBa_K1678007

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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 964
  Illegal AgeI site found at 1076
  
• 1000
  COMPATIBLE WITH RFC[1000]