Part:BBa_J100065

Synthetic Riboswitch

This part is a synthetic riboswitch that can be used in E. coli. In the presence of theophylline, the expression of the gene of interest is induced, but in the absence of theophylline, very little transcription takes place. This part contains a modified T5 promoter, which, in the absence of cymR, acts as a strong constitutive promoter. The ribosomal binding site is contained in the riboswitch.

Because the riboswitch must be right beside the gene of interest (the start codon is folded in the riboswitch), we added a BsaI site. This allows the riboswitch to be attached to the gene of interest using Golden Gate Assembly (as long as the first nucleotide after the start codon of the gene of interest is a C, we designed the part to use with superfolder GFP).

This riboswitch is modified from riboswitch D as described in the paper by http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2988590/?tool=pubmed Topp ''et al''.

Compare this part with Riboswitch E J100066. Riboswitch D has a shorter RBS. See Figure 1.B in Topp "et al" for the comparison of gene expression.

References: Shana Topp, Colleen M. K. Reynoso, Jessica C. Seeliger, Ian S. Goldlust, Shawn K. Desai, Dorothée Murat, Aimee Shen, Aaron W. Puri, Arash Komeili, Carolyn R. Bertozzi, June R. Scott, and Justin P. Gallivan. Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species. Applied and Environmental Microbiology, 76:23, 7881-7884. December 2010.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 1
  Illegal suffix found in sequence at 193
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1
  Illegal SpeI site found at 194
  Illegal PstI site found at 208
  Illegal NotI site found at 7
  Illegal NotI site found at 201
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 1
  Illegal suffix found in sequence at 194
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 1
  Illegal XbaI site found at 16
  Illegal SpeI site found at 194
  Illegal PstI site found at 208
  Illegal AgeI site found at 122
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 187