Part:BBa_K2904100

Improved vitamin B12 biosensor, composed by modular cobalamin riboswitch in front of an mRFP1

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. 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.

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. Compared with the original riboswitch, the improved riboswitch demonstrates a greater induction difference.

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.

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 1044
  Illegal AgeI site found at 1156
  
• 1000
  COMPATIBLE WITH RFC[1000]