Part:BBa_K4654020

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

With this part, we introduce you to a riboswitch-reporter-construct that mediates the expression of lacZ 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 lacZ gene. 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 lacZ as a reporter because it prodces a with the eye easy visible signal. You can learn more about this reporter in our basic part BBa_K4654003.

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

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

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

Figure 1: ONPG-Assay examining the Response of the Riboswitch-LacZ constructs to Different LiCl concentrations in KRX Bacteria. Reporter activity is measured in Miller Units. Compared to the results from the other assays, it becomes clear that expression patterns are very different between fluroescent/luminescent reporters and the enzymatic reporter. Given the challenges of the ONPG assay, we made the decision to shift our focus towards utilizing reporters that were easier to handle and more reliable. This shift was motivated by our commitment to maintaining very high standards for precision and accuracy in our experiments. Additionally, we sought reporters that were less toxic, mitigating potential safety concerns associated with certain chemical agents.

Here you can see the riboswitch-lacZ assay results summarized in bullet points:

- Genomic sequence synthesis and assembly were challenging, requiring fragmentation.
- X-Gal-based reporter assays initially provided binary results but confirmed the basic idea's feasibility
- Limitation of X-Gal in providing quantitative measurements led to more sophisticated ONPG-based cell-based experiments.
- High toxicity of chemicals necessitated fume hood use and precluded plate reader utilization.
- Difficulty in high-throughput measurements due to fume hood setup and bacterial growth.
- Skepticism regarding data precision due to slight OD420 decreases during gradient program testing. Experiment results led to rejection of lacZ as reporter for our project

In contrast to BBa_K4654018 and BBa_K4654017, BBa_K4654020 was not used in a cell-free environment.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 30
  Illegal EcoRI site found at 3251
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 30
  Illegal EcoRI site found at 3251
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 30
  Illegal EcoRI site found at 3251
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 30
  Illegal EcoRI site found at 3251
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 30
  Illegal EcoRI site found at 3251
  Illegal NgoMIV site found at 123
  
• 1000
  COMPATIBLE WITH RFC[1000]