Part:BBa_K1913011

Introduction

Vitamin B12 riboswitch with an inverter and reporter gene. This BioBrick allows for inducible mRFP production based on the concentration of vitamin B12. The riboswitch used for this BioBrick is the btuB of Escherichia coli which normally stops expression after it binds to vitamin B12. Thanks to the TetR QPI system, expression of mRFP will start after binding to the ligand. Riboswitches are pieces of mRNA that can regulate gene expression depending on if it is bound to a ligand. They consists of two parts. An aptamer which bounds to a specific ligand and an expression platform. This dual system makes riboswitches interesting tools for utilization within synthetic biology: aptamers and expression platform of different riboswitch sequences could potentially be combined to create new riboswitches. Another advantage of riboswitches is the fact that they regulate gene expression at the mRNA level, essentially skipping the first part of the central dogma. In the case of inducible promoters, DNA needs first to be transcribed into RNA before translation.

Design and construction

For an in-depth explanation about the design and considerations, please check: http://2016.igem.org/Team:Wageningen_UR/Description/Regulation As can be seen in figure 1, BBa_K1913011 consists of a vitamin B12 riboswitch, the TetR based inverter system and mRFP as a reporter gene.

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Figure 1. Schematic overview showing that the system has been constructed via a 3A-assembly out of two constructs: one containing the riboswitch and one containing the TetR QPI and a reporter gene.

Usage

BBa_K1913011 has in our case be used for the detection of vitamin B12 in beehives, but the btuB riboswitch can be used for multiple applications. For example, it could be used to detect traces of blood, as vitamin B12 is present in (human) blood.

Characterization of BBa K1913011

Bba_K1913011 has been constructed out of Bba_K1913008 and Bba_K1913010. The BioBrick has grown overnight in LB medium with different concentrations of guanine and vitamin B12. The concentration range of vitamin B12 was based on the concentrations normally used for testing bacteria that are used to determine the amount of vitamin B12 in human blood. The amount of mRFP was measured every five minutes and divided by the OD.

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Figure 2. BBa_K1913011 was grown overnight in LB medium with different concentrations of vitamin B12.

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 NgoMIV site found at 199
  Illegal AgeI site found at 1965
  Illegal AgeI site found at 2077
  
• 1000
  COMPATIBLE WITH RFC[1000]