Part:BBa_K5377300

thiM TPP riboswitch

Description

The thiM TPP riboswitch^[1] is a regulatory RNA element that specifically binds TPP (thiamine pyrophosphate) and controls the expression of genes involved in thiamine (vitamin B1) biosynthesis and transport, such as the thiM gene. This riboswitch is present in E. coli, functioning at either the transcriptional or translational level.^[2] Upon binding to TPP, the riboswitch undergoes a conformational change that represses gene expression by either terminating transcription or blocking translation initiation, depending on the organism and regulatory context.

Fig.1 The secondary structure of the FMN riboswitch. (From Wikipedia)

Usage and Biology

In the absence of TPP, the riboswitch adopts a structure that allows the transcription or translation of the thiM gene, promoting thiamine biosynthesis. When TPP is abundant, it binds to the riboswitch, triggering a structural rearrangement that halts the production of enzymes required for thiamine biosynthesis.^[3] This regulatory mechanism is highly conserved and plays a crucial role in maintaining proper cellular thiamine levels.

Fig.2 Schematic diagram of OFF/ON-state thiM TPP riboswitch.^[1]

By replacing the thiM gene downstream of the riboswitch sequence with the lacI gene, the riboswitch directly regulates the expression of the repressor protein instead of controlling TPP biosynthesis. Our team use this method to design the TPP self-induced production system. For more details, please refer to the BBa_K5377830page.

Characterization

Introduction

To validate the function of the riboswitch, we set up different TPP concentration gradients and added them manually.

Methods

We constructed the riboswitch 'OFF system' plasmid by using Gibson Assembly to insert the riboswitch fragment and sfgfp fragment into the pHG101 plasmid. This was then transformed into the E.coli MG1655 strain for fermentation. We took 200 μL of overnight culture solution, incubated it for 4 hours, and added different concentrations of TPP. Wild-type strains were used as negative controls, and strains with a dysfunctional riboswitch were used as positive controls. 4 hours later, we collected samples, centrifuged them at 13,000 rpm and 4℃ for 2 minutes, resuspended them in PBS buffer, and repeated this process three times. We then measured the optical density (OD) and fluorescence intensity. (excitation at 488 nm and emission at 509 nm)

Results

Fig.3 Downregulation of sfgfp expression by the thiM TPP riboswitch at different TPP concentrations.

We successfully validated the OFF function of the thiM TPP riboswitch. For both the positive and negative controls, there were no significant differences in the fluorescence intensity of the fermentation cultures when different concentrations of TPP were added. However, for the strain containing the complete riboswitch, the addition of TPP resulted in a significant decrease in the fluorescence intensity of the fermentation cultures. Moreover, within a certain concentration range, higher ligand concentrations led to more pronounced decreases in fluorescence intensity. For the thiM TPP riboswitch, the fluorescence intensity decreased at TPP concentrations of 0.5 mM, 1.0 mM, 1.5 mM, and 2.0 mM, with a maximum reduction of 54%. (The results are shown in Fig. 3.)

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]