Difference between revisions of "Part:BBa K1602053"
 
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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's design is very simmilar to <html><a href="/Part:BBa_K1602050">RRlocked</a></html> as 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. In addition to <html><a href="/Part:BBa_K1602050">RRlocked</a></html> the sequence of <html><a href="/Part:BBa_K1497008">hokD</a></html> is flanked bei two restriction sites: BamHI and HindIII. These additional restriction sites allow for an easy exchange of the hokD-sequence with any gene of interest flanked by BamHI and HindIII via restriction cloning without the need to assemble the whole regulatory part from the beginning.
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<figcaption><b>Figure 1:</b> Design of RRlocked_site with the two additional restriction sites BamHI and HindIII.</figcaption>
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        <h1>RRhok_site</h1>
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                <b>RRhok_site</b> is a composit of the RRkey (BBa_K1602049), the RRlocked (BBa_K1602050) part and a restriktion site for BamI and HindIII. There is the possibility to change the GOI.
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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">
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<figcaption><b>Figure 2:</b> Interaction of taRNA and crRNA leads to expression of hokD.</figcaption>
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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 <html><a href="http://rsdnerf.com/">Riboswitch Designer</a></html> 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.
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===Functional Parameters===
 
===Functional Parameters===
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===References===
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1. Isaacs, F.J., et al., Engineered riboregulators enable post-transcriptional control of gene expression. Nat Biotechnol, 2004. 22(7): p. 841-7.
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1602053 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K1602050 parameters</partinfo>
 
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Latest revision as of 05:26, 25 September 2015

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. These additional restriction sites allow for an easy exchange of the hokD-sequence with any gene of interest flanked by BamHI and HindIII via restriction cloning without the need to assemble the whole regulatory part from the beginning.

Figure 1: Design of RRlocked_site with the two additional 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 2: 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]