Part:BBa_K4654018

T7-Promoter_Spacer1-Li-+II_Riboswitch-Spacer2-5'nhaA_nanoLuc

With this part, we introduce you to a riboswitch-reporter-construct that mediates the expression of NLuc 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 NLuc gene (Figure 1).

Figure 1: This figure shows the schematic work mechanism of the riboswitch-nLuc-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 nanoLuc as a reporter because luminescence reporters usually exhibit higher sensitivity than other reporters. nanoLuc is very well suited for this system because of the protein's stability, small size and high luminescence efficiency upon exposure to it's substrate ². You can learn more about this reporter in our basic part BBa_K4654002.

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

This part was designed to be used in a cell-free environment. However, we decided to first characterize it in vivo to make sure it works as expected. Figure 2 shows the results for a cell based characterization of the contructs response to 10 mM LiCL.

Figure 2: nanoLuc Expression of the Construct in E. coli KRX under Different Conditions. Expression was measured in light counts per second immediately after the addition of the reagent substrate and 30 minutes after addition. NC: no assay reagent added to bacteria culture. -Li: No LiCl added to the bacteria culture. +Li: Addition of 10 mM LiCl to bacteria culture. method=none indicates that the attenuation setting was turned off in the Tecan Spark plate reader that was used to measure the luminescence. Strain=N1 is the construct ID used for the construct in this experiment.


This experiment shows increased nanoLuc activity when the riboswitch is induced with LiCl. This leads to two conclusions:

1. When no lithium is present, the riboswitch folds into the correct three-dimensional structure, thereby blocking translation
2. Upon lithium binding, the riboswitch unfolds and enables translation of the NLuc gene

These results indicate that our part works as expected in an in vivo setting.
Figure 3 shows the results we received in the cell-free assay:

Figure 3: nanoLuc Expression of the construct in vitro. Expression was measured in light counts per second 10 minutes after the addition of the reagent substrate. The X-Axis shows different LiCl concentrations that were added to the samples to examine signal development. A Tecan Spark plate reader that was used to measure the luminescence.

These results show that in principle, the part also works in a cell-free reaction setup. However, the results indicate a loss of signal with increasing LiCl concentration which is the exact opposite of what we expected. One hypothesis for this phenomenon could be that the substrate of the nanoLuc is used up faster when more riboswitch is present because of the stronger translation induction at higher LiCl levels. Further testing would be necessary to examine this further.

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
  
• 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 278
  
• 1000
  COMPATIBLE WITH RFC[1000]