Difference between revisions of "Part:BBa K4361100"

 
 
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<partinfo>BBa_K4361100 short</partinfo>
 
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BlcR, as described in BBa_K1758370, codon optimized for expression in E. coli.
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BlcR is a transcription factor originating from the bacterium <i>Agrobacterium tumefaciens</i>. A single BlcR monomer contains an effector binding domain near the C-terminus which recognizes the effectors, succinic semialdehyde (SSA) and gamma-Butyrolactone GBL, but also the rape drug gamma-hydroxybutyric acid (GHB). The N-terminal region allows for the dimerization of two BlcR monomers. Dimeric BlcR can bind to the <i> blc </i> operator sequence (<b> Figure 1 </b>). <br> <br>
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BlcR was originally added to the Parts Registry as [[Part:BBa_K1758370]] by the Bielefeld-CeBiTec iGEM 2015 team. Their sequence for BlcR has been codon optimized for expression in <i>E.coli</i> to improve the expression of the protein.
  
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<a href="https://static.igem.wiki/teams/4361/wiki/part-pages/blcr-housestyle-opaque-1.png"><img src="https://static.igem.wiki/teams/4361/wiki/part-pages/blcr-housestyle-opaque-1.png" style="width:500px;margin-left:175px"></a>
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<figcaption> <b>Figure 1.</b> BlcR crystal structure. DNA binding domain (DBD) in pink and the effector binding domain (EBD) in purple.</figcaption>
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<span class='h3bb'>Sequence and Features</span>
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<span class='h3bb'><h3>Sequence and Features</h3></span>
 
<partinfo>BBa_K4361100 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4361100 SequenceAndFeatures</partinfo>
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<h3>Usage and biology</h3>
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BlcR is an allosteric transcription factor BlcR from the bacterium <i>Agrobacterium tumefaciens</i>. This plant bacterium is able to use GBL, a precursor of GHB, as an energy source. In the absence of GBL, GHB or SSA, BlcR will bind to the <i>blc</i> operator sequence [[Part:BBa_K4361000]] and acts as a repressor for the transcription of the <i>blc</i> proteins. When GBL, GHB or SSA binds to BlcR, it is released from the DNA and the <i>blc</i>A, <i>blc</i>B and <i>blc</i>C proteins are transcribed and digest GBL to succinate (<b> Figure 2 </b>).
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<a href="https://static.igem.wiki/teams/4361/wiki/design/blcr-mechanism.png"><img src="https://static.igem.wiki/teams/4361/wiki/design/blcr-mechanism.png" style="width:500px;margin-left:175px"></a>
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<figcaption> <b>Figure 2.</b> The regulatory mechanism of BlcR on the <i>blc</i> operon and the pathway from gamma-butyrolactone to succinic acid of <i>Agrobacterium tumefaciens</i>.</figcaption>
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In our project we have designed a novel bioelectronic sensor for the detection of GHB in drinks by combining the specificity of the BlcR regulatory mechanism with the reliability of electronics. BlcR is tethered by dsDNA oligonucleotides carrying the <i>blc</i> operator sequence [[Part:BBa_K4361000]] to the surface of a gold interdigitated electrode (IDE). We can measure the capacitance of the IDE, which is influenced by the protein molecules around the fingers of the electrode. If GHB enters the system, it will bind to BlcR, which will then release from the electrode causing the capacitance to increase. Electronic hardware will interpret the change in capacitance and produce an output signal to warn the user that their drink has been spiked.
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This part is included in a composite part for the efficient production and purification of BlcR [[[[Part:BBa_K4361103]]]]
  
  

Latest revision as of 12:26, 12 October 2022


BlcR, codon optimized

BlcR is a transcription factor originating from the bacterium Agrobacterium tumefaciens. A single BlcR monomer contains an effector binding domain near the C-terminus which recognizes the effectors, succinic semialdehyde (SSA) and gamma-Butyrolactone GBL, but also the rape drug gamma-hydroxybutyric acid (GHB). The N-terminal region allows for the dimerization of two BlcR monomers. Dimeric BlcR can bind to the blc operator sequence ( Figure 1 ).

BlcR was originally added to the Parts Registry as Part:BBa_K1758370 by the Bielefeld-CeBiTec iGEM 2015 team. Their sequence for BlcR has been codon optimized for expression in E.coli to improve the expression of the protein.

Figure 1. BlcR crystal structure. DNA binding domain (DBD) in pink and the effector binding domain (EBD) in purple.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 694
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 78
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 589

Usage and biology

BlcR is an allosteric transcription factor BlcR from the bacterium Agrobacterium tumefaciens. This plant bacterium is able to use GBL, a precursor of GHB, as an energy source. In the absence of GBL, GHB or SSA, BlcR will bind to the blc operator sequence [[Part:BBa_K4361000]] and acts as a repressor for the transcription of the blc proteins. When GBL, GHB or SSA binds to BlcR, it is released from the DNA and the blcA, blcB and blcC proteins are transcribed and digest GBL to succinate ( Figure 2 ).
Figure 2. The regulatory mechanism of BlcR on the blc operon and the pathway from gamma-butyrolactone to succinic acid of Agrobacterium tumefaciens.

In our project we have designed a novel bioelectronic sensor for the detection of GHB in drinks by combining the specificity of the BlcR regulatory mechanism with the reliability of electronics. BlcR is tethered by dsDNA oligonucleotides carrying the blc operator sequence Part:BBa_K4361000 to the surface of a gold interdigitated electrode (IDE). We can measure the capacitance of the IDE, which is influenced by the protein molecules around the fingers of the electrode. If GHB enters the system, it will bind to BlcR, which will then release from the electrode causing the capacitance to increase. Electronic hardware will interpret the change in capacitance and produce an output signal to warn the user that their drink has been spiked.

This part is included in a composite part for the efficient production and purification of BlcR [[Part:BBa_K4361103]]