Part:BBa_K2223004

B_12 Riboswitch

The riboswitch part contains a psbAI promoter which transcribes an mRNA strand consisting of a riboswitch and repressor gene, TetR. The riboswitch binds to vitamin B₁₂ and regulates the expression of the downstream repressor gene. In the presence of vitamin B₁₂, the riboswitch specifically anneals onto the cobalamin molecule inducing a conformational change within the mRNA sequestering the riboswitch binding site upstream of TetR. However, in the absence of vitamin B₁₂, TetR is continuously expressed.

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 NgoMIV site found at 392
  
• 1000
  COMPATIBLE WITH RFC[1000]

Intro

This riboswitch part contains a psbAI promoter, modified for expression in both S. elongatus and E. coli, which transcribes an mRNA strand consisting of a riboswitch and repressor gene, TetR. This riboswitch binds to vitamin B₁₂ and regulates the expression of the downstream repressor gene. In the presence of vitamin B₁₂, the riboswitch specifically anneals onto the cobalamin molecule inducing a conformational change within the mRNA sequestering the riboswitch binding site upstream of TetR, allowing expression of the downstream reporter gene. However, in the absence of vitamin B₁₂, TetR is constitutively expressed and the Tet repressor trans-regulates the Tet operator, TetO, which inhibits expression of the downstream reporter. Thus, in the presence of vitamin B₁₂ the cells will fluoresce.

Design

This riboswitch system was designed to detect intracellular vitamin B12 (adenosylcobalamin) in S. elongatus via fluorescence using the btuB gene from E. coli. It was designed to be used with a downstream mRFP reporter (BBa_K2223005), which contains the same a modified psbAI promoter as this part. The riboswitch and reporter produce two separate mRNA strands. The reporter has the TetO operator upstream of the mRFP reporter gene. This riboswitch system was adapted from BBa_K1913011, from the Wageningen 2016 iGEM, to work in both E. coli and S. elongatus with the modified psbAI promoter.

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Promoter

psbAI is a light induced promoter that is documented to be constitutively expressed in cyanoabacteria, such as S. elongatus. Previous characterization of the wild-type psbAI promoter can be found at the 2012 Valencia iGEM team parts page on the registry (BBa_K754000). In this riboswitch part system, this promoter was modified at the -10 element for expression in E. coli.

This modified promoter was shown to be fully functional in E. coli. Three E. coli samples containing a plasmid with both the riboswitch and reporter were grown in LB/ampicillin liquid cultures and their fluorescence was monitored via nanodrop throughout their growth. Because LB contains vitamin B12 (from yeast extract), the cultures were expected to all fluoresce at similar levels. Fluorescence indicates that the promoter is fully functional in E. coli, indicated by the mRFP reporter being expressed.

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Riboswitch response to vitamin B12: After confirming that the modified psbAI promoter functions in E. coli, this riboswitch was characterized for the detection of vitamin B12. Liquid cultures of E. coli were grown in M9 minimal media, which does not contain vitamin B12. These cultures were inoculated from an LB culture described in the promoter characterization. The cultures were supplemented with a gradient of Adenosylcobalamin (coenzyme B12) ranging from 0 ug/ml to 10 ug/ml. These cultures increased in fluorescence as the vitamin B12 concentration increased. We also monitored the growth of these cultures; the cultures grown in M9 minimal media grew much slower than the cultures grown in LB. This growth difference is because M9 minimal media has less nutrients available than LB and the adenosylcobalamin stock used was dissolved in methanol rather than water, which would inhibit cell viability. We chose to start with 2.5ug/ml of vitamin B12 because the UCSC 2017 igem team modeled their organism to produce about 2.5ug/ml of vitamin B12.