Composite

Part:BBa_K3933007

Designed by: Marie Guillaume   Group: iGEM21_Ionis_Paris   (2021-10-19)


Sensing system of Vitamin B12 coupled with Ldh

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 Lactate Dehydrogenase (BBa_K3102006) 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 Lactate Dehydrogenase.

BBa K3933007 schema-part1.png

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.

BBa K3933007 schema-part2.png

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
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1720
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1450


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