Regulatory

Part:BBa_K1602053

Designed by: Christian Sator,Stefan Zens, Max Zander, Benedikt Spannenkrebs, Sebastian Jaeger   Group: iGEM15_TU_Darmstadt   (2015-09-16)
Revision as of 03:39, 25 September 2015 by CSator (Talk | contribs)

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.

Functional Parameters

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]


[edit]
Categories
//awards/part_collection/2015
Parameters
None