Difference between revisions of "Part:BBa K1602053"
 
(2 intermediate revisions by the same user not shown)
Line 2: Line 2:
 
<partinfo>BBa_K1602053 short</partinfo>
 
<partinfo>BBa_K1602053 short</partinfo>
  
−
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 assembly the whole regulatory part from the beginning.
+
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.
  
 
<html>
 
<html>
 
<center>
 
<center>
 
<figure >
 
<figure >
−
<img width=40%; src="https://static.igem.org/mediawiki/2015/c/cc/Killswitch_Abbildung_3_neu.png">
+
<img width=40%; src="https://static.igem.org/mediawiki/parts/a/aa/RRlocked_site.png">
−
<figcaption><b>Figure 1:</b> Design of RRlocked_site with the two addition restriction site BamHI and HindIII.</figcaption>
+
<figcaption><b>Figure 1:</b> Design of RRlocked_site with the two additional restriction sites BamHI and HindIII.</figcaption>
 
</figure>
 
</figure>
 
</center>
 
</center>

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]