Part:BBa_K4654017

T7-Promoter_Spacer1_Li+-II_Riboswitch_Spacer2_5'nhaA

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
  
• 1000
  COMPATIBLE WITH RFC[1000]

With this part, we introduce you to a riboswitch-reporter-construct that mediates the expression of the sfGFP gene 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 sfGFP gene.

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

The spacer sequences upstream and downstream of the riboswitch mimic 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 into the Spacer2 sequence. They are naturally regulated by the riboswitch (BBa_K4654011).

We chose sfGFP as a reporter because it doesn’t require a substrate which makes it cheaper compared to other reporters. sfGFP is well suited for this system because of the protein's fast maturing time of 13.6 minutes compared to the “normal” GFP ². You can learn more about this reporter in our basic part BBa_K4654000.

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

Usage and biology

Figure 1: Response of Riboswitch-sfGFP Constructs to 50 mM LiCl. The figure shows RFU values divided by the respective OD600 values. The 1G, 2G, 3G, 4G and 5G constructs were 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 50 mM LiCl was added to the “induced”-cultures to induce sfGFP expression.

1G (BBa_K4654017) can be identified as the most suitable construct, since the riboswitch can close and open depending on the LiCl concentration. The induced expression is significantly higher than the uninduced expression.

Figure 2: Response of Riboswitch-sfGFP Construct 1G to 0 and 1.5 mM LiCl.. The figure shows RFU values divided by the respective OD600 values. The 1G 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 - 1.5 mM LiCl was added to induce sfGFP expression.

The sfGFP expression of the cell culture with a 0 mM LiCl induction is strongly significantly different (**, p=0.001) from the sfGFP expression of the cell culture with a 1.5 mM LiCl induction (t=-4.031;alpha=0.05;df=16;crit.t=2.12,p=0.001). Therefore, the therapeutic range of LiCl treatment (< 1.5 mM LiCl) can be reliably covered.

Figure 3: Response of Riboswitch-sfGFP Construct 1G to 0.5 and 1.5 mM LiCl. The figure shows RFU values divided by the respective OD600 values. The 1G 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 0.5 - 1.5 mM LiCl was added to induce sfGFP expression.

The sfGFP expression of the cell culture with a 0.5 mM LiCl induction is significantly different (*, p=0.03) from the sfGFP expression of the cell culture with a 1.5 mM LiCl induction (t=-2.1039;alpha=0.05;df=68;crit.t=1.99,p=0.03). Therefore, a reliable distinction can be made between a non-toxic concentration (0.5 mM LiCl) and a toxic concentration (1.5 mM LiCl). This proves the suitability of the 1G construct for our test system.

This part was designed to be used in a cell-free environment. Figure 4 shows the results we received in the cell-free assay:

Figure 4: Construct 1G in Cell-Free Assay.Response of Riboswitch-sfGFP Construct 1G to 50 mM LiCl compared to an uninduced sample as negative control in a cell-free environment. The figure shows RFU values, the measurement was done with Tecan Spark Plate reader, the settings for the plate reader are in the protocol "sfGFP / mScarlet-I3 Assay". For cell-free protein expression we used the biotechrabbit RTS E. coli HY KIT. The experiments were conducted according to the manufacturer's instructions. 50mM LiCl was added to induce sfGFP expression.

This experiment shows increased sfGFP 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 sfGFP gene

These results indicate that our part works as expected in an in vitro setting regarding the detection of lithium. Further tests are required to examine the response to different LiCl concentrations.