Part:BBa_K598001

Theophylline Responsive Riboswitch 1G1 with Engineered RBS+GFP generator

This is a GFP generator regulated by theophylline responsive riboswitch. mRNA with a theophylline riboswitch in it responds to theophylline concerntration, producing different fluorescence strength of GFP. Regulated by different promoters, it would be used to demonstrate theophylline responsive curve, providing data for modeling.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 71
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 730
  

Background

Riboswitches are structured non-coding RNA domains performing as genetic control elements, regulating gene expression through ligand-RNA interactions without assistance of proteins. Several natural riboswitches have been reported, sensing small molecules including vitamin B1 and B12, FMN, SAM, lysine, guanine, adenine, TPP and theophylline [1]. Various mechanisms are utilized to regulate gene expression, ranging from transcription initiation to translation attenuation.

Theophylline responsive riboswitches have been widely studied in recent years. Theophylline is small molecule which is passable through bacterial cytomembrane, but not an essential nutrient of bacteria. Although theophylline has moderate toxicity leading to growth inhibition in logarithmic phase with 15mM theophylline in LB medium, this theophylline responsive riboswitch shows an approving regulation curve under 10mM theophylline concentration with negligible growth inhibition in M9 liquid medium (Fig.1).

File:PekingR parts theo 1G1 2D.png

Figure.7 Predicted secondary structure of 1G1 in the absence and presence of theophylline. Theophylline aptamer is boxed. 1nt slipping mechanism is shown in yellow. UGGA is shown in red as RBS sequence. AUG start codon is in black. .