Difference between revisions of "Part:BBa K1602053"
| 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. | + | 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. |
| + | |||
| + | <html> | ||
| + | <center> | ||
| + | <figure > | ||
| + | <img width=40%; src="https://static.igem.org/mediawiki/2015/c/cc/Killswitch_Abbildung_3_neu.png"> | ||
| + | <figcaption><b>Figure 1:</b> Design of RRlocked_site with the two addition restriction site BamHI and HindIII.</figcaption> | ||
| + | </figure> | ||
| + | </center> | ||
| + | </html> | ||
| + | |||
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. | 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. | ||
| Line 9: | Line 19: | ||
<figure > | <figure > | ||
<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"> | ||
| − | <figcaption><b>Figure | + | <figcaption><b>Figure 2:</b> Interaction of taRNA and crRNA leads to expression of hokD.</figcaption> |
</figure> | </figure> | ||
</center> | </center> | ||
Revision as of 04:13, 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 assembly the whole regulatory part from the beginning.
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 91
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]