Difference between revisions of "Part:BBa K1758370"

 
 
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BlcR, a gene of ''Agrobacterium tumefaciens'', represses the ''blcABC''-Operon which enables the bacterium to grow on gamma-butyrolactone as carbon and energy source ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083662/ Pan et al. 2013]).  
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<h2>Usage and biology</h2>
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<p> BlcR, a protein from <i>Agrobacterium tumefaciens</i>, represses the <i>blcABC</i>-operon. The latter enables the bacterium to grow on &gamma;-butyrolactone as carbon and energy source as depicted in the following figure (<a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083662">Pan et al. 2013</a>). </p>
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  <a href="https://static.igem.org/mediawiki/parts/8/82/Bielefeld-CeBiTec_blc_operon_and_reactions_image_reg.png"><img src="https://static.igem.org/mediawiki/parts/8/82/Bielefeld-CeBiTec_blc_operon_and_reactions_image_reg.png" alt="catabolism of GBL in A. tumefaciens" style="width:300px"></a>
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<p style="font-size:90%"> <i>The Blc-Operon and the catabolism of GBL in A. tumefaciens. GHB: &#947;-hydroxybutyrate; GBL: &#947;-butyrolactone; SSA: Succinic semialdehyde; SA: Succinate. </i></p>
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<h2> Characterization – BlcR function </h2>   
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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>.
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    <img src="https://static.igem.org/mediawiki/2015/3/38/Bielefeld-CeBiTec_CFPS_EMSA_BlcR.png" alt="EMSA BlcR and BlcR-sfGFP" C>
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    <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>
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<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.
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<figure><a href="https://static.igem.org/mediawiki/2015/5/55/Bielefeld_Cebitec_EMSA_blcR_sfGFP_2.png">
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    <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>
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<figcaption> <i> EMSA shift caused by addition of different amounts of the indicated protein to 0.05 pmol Cy3-labeled operator site. +/- refers to the presence of dithiothreitol in the reaction. </i>
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<h3>References</h3>
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<p id="Pan2013">
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Pan, Yi; Wang, Yi; Fuqua, Clay; Chen, Lingling (2013): In vivo analysis of DNA binding and ligand interaction of BlcR, an IclR-type repressor from Agrobacterium tumefaciens. In: Microbiology (Reading, England) 159 (Pt 4), S. 814–822. DOI: 10.1099/mic.0.065680-0.
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<!-- Add more about the biology of this part here
 
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Latest revision as of 05:15, 19 September 2015

BlcR, repressor of blcABC operon, with promoter and RBS

Usage and biology

BlcR, a protein from 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).

catabolism of GBL in A. tumefaciens

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.

EMSA BlcR and BlcR-sfGFP
EMSA shifts caused by addition of BlcR protein (see BBa_K1758370) and BlcR-sfGFP fusion protein (see BBa_K1758204), respectively, to Cy3-labeled blc-operator site. 5 pmol of following proteins were applied: 1: BlcR-sfGFP, 2: ArsR-sfGFP as negative control (see BBa_K1758203), 3: BlcR, 4: none.

A BlcR-sfGFP fusion protein is also able to bind the blc-operator in BBa_K1758376, as depicted in the following figure.

Sorry, cannot load this file at the moment
EMSA shift caused by addition of different amounts of the indicated protein to 0.05 pmol Cy3-labeled operator site. +/- refers to the presence of dithiothreitol in the reaction.

References

Pan, Yi; Wang, Yi; Fuqua, Clay; Chen, Lingling (2013): In vivo analysis of DNA binding and ligand interaction of BlcR, an IclR-type repressor from Agrobacterium tumefaciens. In: Microbiology (Reading, England) 159 (Pt 4), S. 814–822. DOI: 10.1099/mic.0.065680-0.


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
    Illegal NheI site found at 812
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 520
    Illegal NgoMIV site found at 761
  • 1000
    COMPATIBLE WITH RFC[1000]