Difference between revisions of "Part:BBa K4361103"
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<partinfo>BBa_K4361103 short</partinfo>
 
<partinfo>BBa_K4361103 short</partinfo>
  
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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 gamma-hydroxybutyric acid (GHB) and related molecules. The N-terminal region allows for the dimerization of two BlcR monomers. Dimeric BlcR can bind to the <i> blc <i/> operator sequence. <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. <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> by us to improve expression of the protein ([[Part:BBa_K4361100]]). This composite part consists of codon optimized BlcR, a 6xHis-tag for purification, and a TEV cleavage site for removal of the tag from the protein ([[Part:BBa_K4361104]]). This combination allowed us to both make the expression as efficient as possible, as well as allowing for high-yield purification.
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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> by us to improve expression of the protein ([[Part:BBa_K4361100]]). This composite part consists of codon-optimized BlcR, a 6xHis-tag for purification [[Part:BBa_K4361102]], and a TEV cleavage site for removal of the tag from the protein ([[Part:BBa_K4361102]]). With this composite part, we were able to effectively produce and purify BlcR in <i> E. coli </i>.  
  
  
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<figure>
 
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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>
 
<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> Figure 1.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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<figcaption> <b>Figure 1.</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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<h3>Usage and biology</h3>
 
<h3>Usage and biology</h3>
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<i>In vivo</i> the Blc operator consists of two inverted repeat pairs (see [[Part:BBa_K4361001]]), which can each bind a single BlcR dimer, separated by a 3 nt linker. The specific length of the linker allows for the correct orientation relative to each other of two dimers bound to the DNA, such that they are able to tetramerize. If the spacer were of a different length, the dimers would have different orientations to each other, possibly inhibiting tetramerization (see [[Part:BBa_K4361014]]). With two BlcR dimers bound and forming a tetramer, RNA polymerases originating from an upstream promoter are sterically hindered from moving along the DNA past the Blc operator, inhibiting expression of downstream <i>blc</i> genes, creating a selfregulating system. Each BlcR monomer contains a binding site that recognizes <i>gamma</i>-hydroxybutyrate (GHB) and derivative molecules. When a BlcR tetramer binds GHB with one of its binding sites, it reverts back to two dimers and unbinds from the DNA, once more enabling downstream transcription.
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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 1 </b>.  
  
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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. One of the plates is covered in a BlcR-binding DNA oligo. The sensor also contains BlcR dimers, which bind to the DNA oligos. When the dimers displace water molecules by binding to the DNA, the permittivity and thereby the capacitance of the capacitor changes, which can be measured and set as a baseline after an equilibrium has been reached. 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 continuously measuring the capacitance, the solution contacting the biosensor can be monitored for changes in its GHB content.
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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 1.</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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</figure>
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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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<h3>Results</h3>
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<h3>Experimental results</h3>
 
<a href='https://2022.igem.wiki/tudelft/notebook#section2'>Wet Lab module 1</a> focused on optimizing the expression and purification of BlcR. After applying the engineering cycle multiple times, a protocol was developed which allowed for the expression and relatively high yield of the protein. After expression in <i>E. coli</i> BL21(DE3) cells, the protein was captured on a His column with Ni-NTA beads and subsequently washed off through TEV digestion. The washed off solution was then further purified with size exclusion.
 
<a href='https://2022.igem.wiki/tudelft/notebook#section2'>Wet Lab module 1</a> focused on optimizing the expression and purification of BlcR. After applying the engineering cycle multiple times, a protocol was developed which allowed for the expression and relatively high yield of the protein. After expression in <i>E. coli</i> BL21(DE3) cells, the protein was captured on a His column with Ni-NTA beads and subsequently washed off through TEV digestion. The washed off solution was then further purified with size exclusion.
  

Revision as of 10:36, 12 October 2022


BlcR with 6xHis-tag and TEV protease cleavage site

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 <i/> operator sequence.
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 by us to improve expression of the protein (Part:BBa_K4361100). This composite part consists of codon-optimized BlcR, a 6xHis-tag for purification Part:BBa_K4361102, and a TEV cleavage site for removal of the tag from the protein (Part:BBa_K4361102). With this composite part, we were able to effectively produce and purify BlcR in E. coli .


Figure 1. The regulatory mechanism of BlcR on the blc operon and the pathway from gamma-butyrolactone to succinic acid of Agrobacterium tumefaciens.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 766
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 901
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 150
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 661

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 1 .

Figure 1. 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.

Experimental results

Wet Lab module 1 focused on optimizing the expression and purification of BlcR. After applying the engineering cycle multiple times, a protocol was developed which allowed for the expression and relatively high yield of the protein. After expression in E. coli BL21(DE3) cells, the protein was captured on a His column with Ni-NTA beads and subsequently washed off through TEV digestion. The washed off solution was then further purified with size exclusion.

The functionality of this part was later proven in experiments with the BlcR-binding DNA oligos, see Part:BBa_K4361000 through Part:BBa_K4361022 and our results for module 3.