Part:BBa_K1913012

Introduction

Guanine riboswitch with an inverter and reporter gene. This Biobrick allows for inducible mRFP production based on the concentration of guanine. The riboswitch used for this biobrick is the xpt of Bacillus subtilis which normally stops expression after it binds to guanine. 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_K1913012 consists of a guanine riboswitch, the TetR based inverter system and mRFP as a reporter gene.

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.

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]

Characterization of BBa K1913012

Bba K1913012 has been constructed out of Bba K1913009 and Bba K1913010. The biobrick has grown overnight in LB medium with different concentrations of guanine. The concentration range of guanine was based on the concentrations normally used for testing bacteria that are used to determine the amount of guanine in human blood. The amount of mRFP was measured every five minutes and divided by the OD. 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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]