Part:BBa_K1132008:Design

R4 riboregulator switch with with pTET and pOmpC

Assembly Compatibility:

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

  Illegal BamHI site found at 401
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 213
  

Design Notes

The R4 riboregulator sequence is designed based on the publication of Callura JM (http://www.ncbi.nlm.nih.gov.gate1.inist.fr/pubmed/22454498). The principle is to introduce short regulatory sequences that create RNA secondary structures to better control protein expression. The basic design is reported in the following scheme (red hexagonal boxes, terminators; green oval box, rbs; blue and red rectangles, riboregulator sequences; arrows, promoters). Promoters P1 and P2 are controlling the expression of a single gene X (not present in the biobrick). Two terminators are placed between P1 and P2.

Transcription occurring at P2 promoter stops at terminators placed after gene X. However, the red sequence can fold into a RNA secondary structure, blocking the RBS, preventing ribosome binding. The gene X immediately placed after the RBS is transcribed but not translated (no protein X).

The P1 promoter controls the expression of a small riboregulatory sequence capable of interacting with the one blocking RBS, but stronger in term of interaction. If transcription occurs at both sites P1 and P2, a small RNA is produced that destabilizes the loop created on the RBS and therefore releasing the riboregulator and enabling translation.

The part is released without any gene and can therefore be used to better control any protein expression. The P1 promoter is pTET (BBa_R0040) and the P2 promoter is pOmpC (BBA_R0082). P2 can be changed with Bam HI and Cla I restriction sites.

Source

later