Difference between revisions of "Part:BBa K1758370"
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<p> We performed <a data-toggle="tooltip" title="Electrophoretic Mobility Shift Assay">EMSA-shifts</a> and verified: BlcR binds to the operator site described in <a href="#Pan2013">Pan et al. 2013</a>. | <p> We performed <a data-toggle="tooltip" title="Electrophoretic Mobility Shift Assay">EMSA-shifts</a> and verified: BlcR binds to the operator site described in <a href="#Pan2013">Pan et al. 2013</a>. | ||
<figure> | <figure> | ||
− | <img src="https://static.igem.org/mediawiki/2015/3/38/Bielefeld-CeBiTec_CFPS_EMSA_BlcR.png" alt="EMSA BlcR and BlcR-sfGFP" | + | <img src="https://static.igem.org/mediawiki/2015/3/38/Bielefeld-CeBiTec_CFPS_EMSA_BlcR.png" alt="EMSA BlcR and BlcR-sfGFP" C> |
<figcaption> <i>EMSA shifts caused by addition of BlcR protein (see <a href="https://parts.igem.org/Part:BBa_K1758370" target="_blank">BBa_K1758370</a>) and BlcR-sfGFP fusion protein (see <a href="https://parts.igem.org/Part:BBa_K1758204" target="_blank">BBa_K1758204</a>), respectively, to Cy3-labeled <i>blc</i>-operator site. 5 pmol of following proteins were applied: 1: BlcR-sfGFP, 2: ArsR-sfGFP as negative control (see <a href="https://parts.igem.org/Part:BBa_K1758203" target="_blank">BBa_K1758203</a>), 3: BlcR, 4: none. </i></figcaption> | <figcaption> <i>EMSA shifts caused by addition of BlcR protein (see <a href="https://parts.igem.org/Part:BBa_K1758370" target="_blank">BBa_K1758370</a>) and BlcR-sfGFP fusion protein (see <a href="https://parts.igem.org/Part:BBa_K1758204" target="_blank">BBa_K1758204</a>), respectively, to Cy3-labeled <i>blc</i>-operator site. 5 pmol of following proteins were applied: 1: BlcR-sfGFP, 2: ArsR-sfGFP as negative control (see <a href="https://parts.igem.org/Part:BBa_K1758203" target="_blank">BBa_K1758203</a>), 3: BlcR, 4: none. </i></figcaption> | ||
</figure> | </figure> | ||
+ | |||
+ | <p> A BlcR-sfGFP fusion protein is also able to bind the <i>blc</i>-operator in <a href="https://parts.igem.org/Part:BBa_K1758376"> BBa_K1758376</a>, as depicted in the following figure. | ||
+ | |||
+ | <figure><a href="https://static.igem.org/mediawiki/2015/5/55/Bielefeld_Cebitec_EMSA_blcR_sfGFP_2.png"> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/5/55/Bielefeld_Cebitec_EMSA_blcR_sfGFP_2.png" alt="Sorry, cannot load this file at the moment" style="width:300px"></a> | ||
Revision as of 05:12, 19 September 2015
BlcR, repressor of blcABC operon, with promoter and RBS
Usage and biology
BlcR, a gene of Agrobacterium tumefaciens, represses the blcABC-operon. The latter enables the bacterium to grow on γ-butyrolactone as carbon and energy source as depicted in the following figure (Pan et al. 2013).
The Blc-Operon and the catabolism of GBL in A. tumefaciens. GHB: γ-hydroxybutyrate; GBL: γ-butyrolactone; SSA: Succinic semialdehyde; SA: Succinate.
Characterization – BlcR function
We performed EMSA-shifts and verified: BlcR binds to the operator site described in Pan et al. 2013.
A BlcR-sfGFP fusion protein is also able to bind the blc-operator in BBa_K1758376, as depicted in the following figure.
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 812 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 520
Illegal NgoMIV site found at 761 - 1000COMPATIBLE WITH RFC[1000]