Difference between revisions of "Part:BBa K4361100"
 
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<partinfo>BBa_K4361100 short</partinfo>
 
<partinfo>BBa_K4361100 short</partinfo>
  
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BlcR is a transcription factor originating from the bacterium <i>Agrobacterium tumefaciens</i> ([[Part:BBa_K4361100]]). A single BlcR monomer contains a domain near the C-terminus which recognizes <i>gamma</i>-hydroxybutyric acid (GHB) and related molecules. The N-terminal region allows for dimerization of two BlcR monomers, as well as forming a DNA-binding domain when in a dimer state. <br>
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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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This part is based on [[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> by us to make expression of the protein as efficient as possible.
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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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<h3>Usage and Biology</h3>
 
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<i>In vivo</i>, BlcR dimers recognize and bind a specific region in its host genome (denoted as the Blc operator, see [[Part:BBa_K4361001]]). Two operators lie close to each other on the DNA, separated by a 3 nt linker sequence. When two dimers each bind an operator sequence, their position relative to each other allows for the formation of a BlcR tetramer. This binding and tetramerization represses the expression of downstream genes of the <i>blc</i> family that allow <i>A. tumefaciens</i> to digest GHB-like molecules. When GHB is present in solution and occupies a GHB-binding domain in a BlcR tetramer, the tetramer reverts to two dimers and releases from the DNA it previously bound. This allows for the transcription of downstream genes and subsequent digestion of the molecules.
 
  
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In our project, we make use of BlcR's abilities to bind a specific DNA sequence and to react to the presence of GHB by incorporating it into a capacitive biosensor. This biosensor contains two parallel metal plates that act as a capacitor, with a solution containing BlcR in between. One of the plates is covered in DNA oligos containing the BlcR-binding sequence (see </html>[[Part:BBa_K4361001]]<html>). The sensor also contains BlcR dimers, which bind to the DNA oligos. By doing so water molecules in the system are displaced, which changes the permittivity and thereby the capacitance of the capacitor, which can be measured to be set as a baseline. When the sensor then comes into contact with GHB or a derivative molecule (succinic semialdehyde (SSA) for the majority of our experiments), BlcR unbinds which once again leads to a capacitance change. By measuring the capacitance, the solution contacting the biosensor can be continuously monitored for changes in its GHB content.
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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]]