Difference between revisions of "Part:BBa K1758370"
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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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Revision as of 05:14, 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).

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.

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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]