Part:BBa_K3933005

Reporter system for Vitamin B12 using a riboswitch

This part is a reporter system for Cobalamin (vitamin B12), using a riboswitch which acts as a sequester of the RBS located downstream of its sequence. It was inspired by a part created by Paris Bettencourt 2015 and modified by OUC Paris 2019 : BBa_K2904100 .

This part is a composite part that can be divided into two units. The first unit is a Tet R protein (BBa_C0040) , which is regulated by a constitutive promoter (BBa_J23106) and a double terminator BBa_B0015 . However, the translation of the Tet R protein is also regulated by a Cobalamin Riboswitch (BBa_K3933003) , which sequesters the RBS (BBa_B0032) when bound to vitamin B12.

The second unit is the reporter system related to the sensing of Vitamin B12 realised in the first part. It is composed of a Tet Operator (BBa_R0040) , which hinders the expression of the coding sequence placed after when it is bound by Tet R. It is followed by a mRFP (BBa_E1010) translated thanks to a RBS (BBa_B0032) . This unit is also closed by the double terminator BBa_B0015 .

When the riboswitch isn't bound to Cobalamin, the RBS is not sequestered and Tet R is expressed, which hinders the expression of the RFP.

However, when Cobalamin (or vitamin B12) binds to the riboswitch, it sequesters the RBS, and the Tet R is not translated. With no Tet R binding the Tet Operator, RFP can be translated and quantified. Thus, the RFP can be detected when Cobalamin binds to the riboswitch, thanks to a double inhibition system.

Thanks to Biorender

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 1891
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1741
  Illegal AgeI site found at 1853
  
• 1000
  COMPATIBLE WITH RFC[1000]