Difference between revisions of "Part:BBa K4361015"
 
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<partinfo>BBa_K4361015 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4361015 SequenceAndFeatures</partinfo>
  
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<h3>Usage and biology</h3>
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<h3>Usage and Biology</h3>
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<i>In vivo</i> the Blc operator consists of pair 1 and 2 linked together by a 3 nt spacer, and each pair can bind a single BlcR dimer (see [[Part:BBa_K4361100]]). With a spacer of specifically 3 nt, the centers of each pair are exactly 20 nt apart, which supports the hypothesis that the two dimers orient themselves at the same rotation angle to the DNA to form a tetramer. 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, ribosomes originating from an upstream RBS 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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The <i>blc</i> operator contains 2 pairs of inverted repeats, linked together by a 3 nt spacer, and each pair is assumed to bind one BlcR dimer (see [[Part:BBa_K4361100]]). With a spacer of specifically 3 nt, the centers of each pair are exactly 20 nt apart, which supports the hypothesis that the two dimers orient themselves at the same rotation angle to the DNA to form a tetramer. 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 hindered from transcribing past the <i>blc</i> operator, inhibiting expression of downstream <i>blcABC</i> genes. Each BlcR monomer contains a binding site that is specific to <i>gamma</i>-butyrolactone (GBL) <i>gamma</i>-hydroxybutyric acid (GHB) and succinic semialdehyde (SSA). When a BlcR tetramer binds GHB with one of its binding sites, tetramerization is inhibited and BlcR becomes dissociated from the DNA, enabling downstream transcription and subsequent digestion of the newly present substrate (see <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, with a solution containing BlcR in between. One of the plates is covered in the wildtype BlcR-binding oligo. The sensor also contains BlcR dimers, which bind to the DNA oligos. When the dimer 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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<html>
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<figure>
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<a href="https://static.igem.wiki/teams/4361/wiki/parts/blcr-ssa.png"><img src="https://static.igem.wiki/teams/4361/wiki/parts/blcr-ssa.png" style="width:400px;margin-left:225px"></a>
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<figcaption> <b>Figure 1.</b> General overview of the unbinding mechanism of BlcR from DNA in the presence of SSA. Left: two BlcR dimers bound to DNA as a tetramer. Middle: SSA is introduced into the system. Right: BlcR dimers bind SSA and unbind from the DNA.</figcaption>
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</figure>
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</html>
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In our project, these oligos are used to tether BlcR to the surface of a gold electrode, of which we measure the capacitance. When BlcR molecules dissociate from the DNA in response to the binding of GHB, water molecules are displaced towards the surface of the electrode, which causes an increase in capacitance. This is the signal we interpret to indicate the presence of GHB.
  
 
'''Oligo variants'''<br>
 
'''Oligo variants'''<br>
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The wildtype Blc operator has been theorized to not bind BlcR optimally, since BlcR regulates its own expression and that of proteins involved in the breakdown of GHB-like molecules. This means BlcR has to quickly unbind if said molecules are detected by <i>A. tumefaciens</i>, such that the bacterium can digest the molecules for nutrients. In our system, however, we would like BlcR to be more stably bound to DNA, such that it will only unbind in the presence of high GHB concentrations. This can be accomplished through two approaches: adjusting BlcR itself (see [[Part:BBa_K4361200]] through [[Part:BBa_K4361227]] and [[Part:BBa_K4361300]] through [[Part:BBa_K4361319]]), or changing the DNA molecule it binds to. This set of Parts, [[Part:BBa_K4361000]] up to [[Part:BBa_K4361022]], shows our work on the second approach.
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The wildtype <i>blc</i> operator has been theorized to not bind BlcR optimally, since BlcR regulates its own expression and that of proteins involved in the breakdown of GHB-like molecules. This means BlcR has to quickly unbind if these molecules taken up by <i>A. tumefaciens</i>, such that the bacterium can digest the molecules for nutrients. In our system, however, we would like BlcR to be more stably bound to DNA, such that it will only unbind in the presence of high GHB concentrations. This can be accomplished through two approaches: adjusting BlcR itself (see [[Part:BBa_K4361200]] through [[Part:BBa_K4361227]] and [[Part:BBa_K4361300]] through [[Part:BBa_K4361319]]), or changing the DNA molecule it binds to. This set of Parts, ranging from [[Part:BBa_K4361000]] up to [[Part:BBa_K4361022]], shows our work on the second approach.
  
 
<html>
 
<html>
 
<h3>Results</h3>
 
<h3>Results</h3>
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As described in the Results section of </html>[[Part:BBa_K4361000]] and [[Part:BBa_K4361001]]<html>, an electrophoresis experiment was performed with the majority of our designed oligos, wherein the aforementioned parts act respectively as the negative and positive control. By incubating them with BlcR and running them on a gel, the binding strength of BlcR to each sequence can be estimated by looking at the bands of free DNA and DNA bound by the protein. As can be seen in <b>Figure 1</b>, less DNA is bound by BlcR than with the wildtype sequence. This suggests a lower binding affinity between BlcR and DNA, so this oligo was not selected for further analysis.
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As described in the Results section of </html>[[Part:BBa_K4361000]] and [[Part:BBa_K4361001]]<html>, an electrophoresis experiment was performed with the majority of our designed oligos, wherein the aforementioned parts act respectively as the negative and positive control. By incubating them with BlcR and running them on a gel, the binding strength of BlcR to each sequence can be estimated by looking at the bands of free DNA and DNA bound by the protein. As can be seen in <b>Figure 2</b>, less DNA is bound by BlcR than with the wildtype sequence. This suggests a lower binding affinity between BlcR and DNA, so this oligo was not selected for further analysis.
  
 
<figure>
 
<figure>
 
<a href="https://static.igem.wiki/teams/4361/wiki/parts/oligos-15.png"><img src="https://static.igem.wiki/teams/4361/wiki/parts/oligos-15.png" style="width:300px;margin-left:275px"></a>
 
<a href="https://static.igem.wiki/teams/4361/wiki/parts/oligos-15.png"><img src="https://static.igem.wiki/teams/4361/wiki/parts/oligos-15.png" style="width:300px;margin-left:275px"></a>
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<figcaption> <b>Figure 1.</b> Results of the electrophoresis experiment in which the fraction of DNA bound to BlcR was determined for different types of oligos. The first bar and bottom dashed line represent the results with </html>[[Part:BBa_K4361000]]<html> (scrambled oligo, negative control), the second bar and top dashed line correspond to those with </html>[[Part:BBa_K4361001]]<html> (wildtype oligo, positive control). The third bar depicts the measured fraction of bound DNA for this part. Values represent the ratio between the intensity of the band corresponding to protein-bound DNA, and the sum of the protein-bound and protein-free bands.</figcaption>
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<figcaption> <b>Figure 2.</b> Results of the electrophoresis experiment in which the fraction of DNA bound to BlcR was determined for different types of oligos. The first bar and bottom dashed line represent the results with </html>[[Part:BBa_K4361000]]<html> (scrambled oligo, negative control), the second bar and top dashed line correspond to those with </html>[[Part:BBa_K4361001]]<html> (wildtype oligo, positive control). The third bar depicts the measured fraction of bound DNA for this part. Values represent the ratio between the intensity of the band corresponding to protein-bound DNA, and the sum of the protein-bound and protein-free bands.</figcaption>
 
</figure>
 
</figure>
  

Latest revision as of 15:24, 12 October 2022


BlcR-binding oligo, 71 bp, IR1 + IR2 + IR1

BlcR is a transcription factor originating from the bacterium Agrobacterium tumefaciens (Part:BBa_K4361100). In a homodimer state it contains a single DNA-binding domain that specifically binds one of two DNA sequences. Both sequences are so-called inverted repeat pairs (IRs), short DNA sequences whose ends are reverse complements of each other. For the Blc operator, these sequences are 'ACTCTAATgATTCAAGT' (IR1) and 'ATTAGttgaactCTAAT' (IR2), as further explained in Part:BBa_K4361001.
To our understanding, one BlcR dimer contains two domains that allow for tetramerization, only one of which is used during tetramerization in vivo. This part, Part:BBa_K4361016, and Part:BBa_K4361018 have been designed to show whether or not BlcR dimers are able to form multimers larger than tetramers when bound to DNA. To create this part, the original 3 nt linker sequence (tca) and a copy of IR1 have been added to the 3' end of IR2. The BlcR-binding domain of this part thus consists of IR1-tca-IR2-tca-IR1. As the distance between the centers of all IRs is still 20 nt, see also Usage and Biology below, this oligo theoretically allows for the correct orientation of three sequential BlcR dimers to bind to each other, resulting in a BlcR hexamer.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

The blc operator contains 2 pairs of inverted repeats, linked together by a 3 nt spacer, and each pair is assumed to bind one BlcR dimer (see Part:BBa_K4361100). With a spacer of specifically 3 nt, the centers of each pair are exactly 20 nt apart, which supports the hypothesis that the two dimers orient themselves at the same rotation angle to the DNA to form a tetramer. 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 hindered from transcribing past the blc operator, inhibiting expression of downstream blcABC genes. Each BlcR monomer contains a binding site that is specific to gamma-butyrolactone (GBL) gamma-hydroxybutyric acid (GHB) and succinic semialdehyde (SSA). When a BlcR tetramer binds GHB with one of its binding sites, tetramerization is inhibited and BlcR becomes dissociated from the DNA, enabling downstream transcription and subsequent digestion of the newly present substrate (see Figure 1).

Figure 1. General overview of the unbinding mechanism of BlcR from DNA in the presence of SSA. Left: two BlcR dimers bound to DNA as a tetramer. Middle: SSA is introduced into the system. Right: BlcR dimers bind SSA and unbind from the DNA.

In our project, these oligos are used to tether BlcR to the surface of a gold electrode, of which we measure the capacitance. When BlcR molecules dissociate from the DNA in response to the binding of GHB, water molecules are displaced towards the surface of the electrode, which causes an increase in capacitance. This is the signal we interpret to indicate the presence of GHB.

Oligo variants
The wildtype blc operator has been theorized to not bind BlcR optimally, since BlcR regulates its own expression and that of proteins involved in the breakdown of GHB-like molecules. This means BlcR has to quickly unbind if these molecules taken up by A. tumefaciens, such that the bacterium can digest the molecules for nutrients. In our system, however, we would like BlcR to be more stably bound to DNA, such that it will only unbind in the presence of high GHB concentrations. This can be accomplished through two approaches: adjusting BlcR itself (see Part:BBa_K4361200 through Part:BBa_K4361227 and Part:BBa_K4361300 through Part:BBa_K4361319), or changing the DNA molecule it binds to. This set of Parts, ranging from Part:BBa_K4361000 up to Part:BBa_K4361022, shows our work on the second approach.

Results

As described in the Results section of Part:BBa_K4361000 and Part:BBa_K4361001, an electrophoresis experiment was performed with the majority of our designed oligos, wherein the aforementioned parts act respectively as the negative and positive control. By incubating them with BlcR and running them on a gel, the binding strength of BlcR to each sequence can be estimated by looking at the bands of free DNA and DNA bound by the protein. As can be seen in Figure 2, less DNA is bound by BlcR than with the wildtype sequence. This suggests a lower binding affinity between BlcR and DNA, so this oligo was not selected for further analysis.
Figure 2. Results of the electrophoresis experiment in which the fraction of DNA bound to BlcR was determined for different types of oligos. The first bar and bottom dashed line represent the results with Part:BBa_K4361000 (scrambled oligo, negative control), the second bar and top dashed line correspond to those with Part:BBa_K4361001 (wildtype oligo, positive control). The third bar depicts the measured fraction of bound DNA for this part. Values represent the ratio between the intensity of the band corresponding to protein-bound DNA, and the sum of the protein-bound and protein-free bands.
For further details on the experiments with our DNA oligos and the results, see the Results page on our wiki.