Difference between revisions of "Part:BBa K1602050"
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<partinfo>BBa_K1602050 short</partinfo>
 
<partinfo>BBa_K1602050 short</partinfo>
  
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This part is half of a two-part killswitch-system for <i>E.coli</i> utillizing a riboregulator for posttransciptional regulation of hokD. It consists of the fused sequences of a constitutive promoter (<html><a href="/Part:BBa_J23100">BBa_J23100</a></html>), a cis-repressing sequence (crRNA), hokD (<html><a href="/Part:BBa_K1497008">BBa_K1497008</a></html>) and a terminator (<html><a href="/Part:BBa_B0015">BBa_B0015</a></html>). 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. If the corresponding trans-activating RNA-sequence (taRNA) (<html><a href="/Part:BBa_K1602049">RRKey-BBa_K1602049</a></html>) 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.
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This part is half of a two-part killswitch-system for <i>E.coli</i> utillizing a riboregulator for posttransciptional regulation of hokD. It consists of the fused sequences of a constitutive promoter (<html><a href="/Part:BBa_J23100">BBa_J23100</a></html>), a cis-repressing sequence (cr), hokD (<html><a href="/Part:BBa_K1497008">BBa_K1497008</a></html>) and a terminator (<html><a href="/Part:BBa_B0015">BBa_B0015</a></html>).
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<center>
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<figure >
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<img width=30%; src="https://static.igem.org/mediawiki/parts/a/af/RRlocked.png">
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<figcaption><b>Figure 1:</b> Design of RRlocked.</figcaption>
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</figure>
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</center>
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</html>
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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. If the corresponding trans-activating RNA-sequence (taRNA) (<html><a href="/Part:BBa_K1602049">RRKey-BBa_K1602049</a></html>) 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.
  
 
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<img width=30%; src="https://static.igem.org/mediawiki/parts/2/22/Schema_killswitch.png">
 
<img width=30%; src="https://static.igem.org/mediawiki/parts/2/22/Schema_killswitch.png">
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<figcaption><b>Figure 1:</b> Interaction of taRNA and crRNA leads to expression of hokD.</figcaption>
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<figcaption><b>Figure 2:</b> Interaction of taRNA and crRNA leads to expression of hokD.</figcaption>
 
</figure>
 
</figure>
 
</center>
 
</center>

Revision as of 06:22, 25 September 2015

RRlocked

This part is half of a two-part killswitch-system for E.coli utillizing a riboregulator for posttransciptional regulation of hokD. It consists of the fused sequences of a constitutive promoter (BBa_J23100), a cis-repressing sequence (cr), hokD (BBa_K1497008) and a terminator (BBa_B0015).

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

The sequence of the crRNA is based on an 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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]