Part:BBa_K4654019

T7-Promoter_Spacer1-Li-+II_Riboswitch-Spacer2-5'nhaA_mScarlet-I3

With this part, we introduce you to a riboswitch-reporter-construct that mediates the expression of mScarlet-I3 in response to lithium ions.
The part can be used in a S1 laboratory

The riboswitch is the lithium-sensitive Li⁺-II riboswitch which was described by White et. al(2021)¹. They created the sequence by constructing a consensus sequence from several riboswitches of a lithium sensitive riboswitch class¹. It is a translational riboswitch that forms a three-dimensional structure on the mRNA in the absence of lithium ions, inhibiting translation by masking the ribosome binding site in the spacer sequence 2. Once lithium binds to the structure, a part of it unfolds so that the RBS becomes accessible to the ribosome which leads to translation of the mScarlet-I3 gene.

Figure 1: This figure shows the schematic work mechanism of the riboswitch-mScarlet-I3-construct.

The spacer sequences upstream and downstream of the riboswitch mimick the riboswitches natural environment to ensure correct folding. For the same reason we added the 5' region of the nhaA gene between the riboswitch and the reporter sequence . They are naturally regulated by the riboswitch (BBa_K4654011).

We chose mScarlet-I3 as a reporter as an alternative to sfGFP (BBa_K4654001). You can learn more about mScarlet-I3 reporter in our basic part BBa_K4654001.

The whole construct is placed under the control of the T7 promoter to ensure efficient transcription.

We characterize it in vivo to make sure it works as expected. Figure 2 shows the results.

Figure 2: Response of riboswitch-mScarlet-I3-construct 1M to 0.2, 0.5, 1, 1.5, 50 mM LiCl. The figure shows RFU values divided by the respective OD600 values. The 1M construct was used. Measurement was done with Tecan Spark Plate reader, the settings for the plate reader are in the protocol "sfGFP / mScarlet-I3 Assay". Bacteria were grown overnight as pre cultures. The next day, main cultures were inoculated and grown until OD600 0.1, then 0.1% rhamnose and 0.2 - 50 mM LiCl was added to induce mScarlet-I3 expression.

Since the 1M construct cannot reliably discriminate between different LiCl concentrations, we decided not to consider mScarlet-I3 for our experiments with cell-free systems. We suspect that the mScarlet-I3 sequence interferes with the proper folding of the riboswitch.

In contrast to BBa_K4654018 and BBa_K4654017, BBa_K4654019 was not used in a cell-free environment.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 30
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 30
  Illegal BglII site found at 878
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 30
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 30
  Illegal NgoMIV site found at 123
  Illegal NgoMIV site found at 336
  
• 1000
  COMPATIBLE WITH RFC[1000]