Part:BBa_K1602053

RRlocked_site

This part is half of a two-part killswitch-system for E.coli utillizing a riboregulator for posttransciptional regulation of hokD. It's design is very simmilar to RRlocked as it consists of the fused sequences of a constitutive promoter (BBa_J23100), a cis-repressing sequence (crRNA), hokD (BBa_K1497008) and a terminator (BBa_B0015). When transcribed the cis-repressing sequence forms a hairpin-secondary-structure masking the ribosome binding site (RBS) and therefore prevents translation of the following hokD-sequence. In addition to RRlocked the sequence of hokD is flanked bei two restriction sites: BamHI and HindIII. If the corresponding trans-activating RNA-sequence (taRNA) (RRKey-BBa_K1602049) is present the two sequences form a RNA-RNA-complex. This leads to a helix shift and the release of the RBS enabling the expression of hokD resulting in cell death.

Figure 1: Interaction of taRNA and crRNA leads to expression of hokD.

The sequence of the crRNA is based on a existing riboregulator sequence pair published by Isaacs et al[1]. The original sequence contained an EcoRI restriction site which was removed by basepair-exchange. We used the Riboswitch Designer to find out which basepair-exchange is best suited to remove the unwanted EcoRI restriction site while not affecting the folding- and interaction-capabilities of the sequence.

References

1. Isaacs, F.J., et al., Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol, 2004. 22(7): p. 841-7.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 91
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]