Difference between revisions of "Part:BBa K4361001"
 
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<partinfo>BBa_K4361001 short</partinfo>
 
<partinfo>BBa_K4361001 short</partinfo>
  
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-Contains 2 inverted repeats, each binding 1 BlcR dimer
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BlcR is a transcription factor originating from the bacterium <i>Agrobacterium tumefaciens</i> ([[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, short DNA sequences whose ends are reverse complements of each other. For the Blc operator, these sequences are 'ACTCTAATgATTCAAGT' and 'ATTAGttgaactCTAAT', as highlighted under <b>Sequence and Features</b> below as Inverted repeat pair 1 (IR1) and Inverted repeat pair 2 (IR2), respectively. The capitalized nucleotides in each sequence form the inverted repeat pairs, although it should be noted that for the first pair the ends of the sequence are not perfect reverse complements (see [[Part:BBa_K4361004]] and [[Part:BBa_K4361005]]).
  
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-Kd,app of 120 nM according to EMSA
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<span class='h3bb'><h3>Sequence and Features</h3></span>
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<partinfo>BBa_K4361001 SequenceAndFeatures</partinfo>
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<h3>Usage and Biology</h3>
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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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<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.
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'''Oligo variants'''<br>
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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.
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<html>
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<h3>Results</h3>
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To qualitatively test the binding strength of BlcR to different oligos, electrophoresis experiments using a TapeStation were performed in which BlcR was combined with the varying sequences. Any DNA bound by BlcR is visible as a shifted band in the gel, and the ratio between the two bands can be used as an indication of the effectiveness with which BlcR binds to the given DNA sequence. As a first test, </html>[[Part:BBa_K4361000]]<html> and this part were run on a gel after incubation with and without BlcR, the results of which are visible in <b>Figure 2</b>. The graph clearly shows that addition of BlcR causes a shift of the DNA on the gel. Also, as expected, BlcR binds the wildtype sequence more strongly than a random, scrambled oligo. This first experiment shows that gel electrophoresis can indeed be used to, at least qualitatively, test the binding affinity of BlcR to different DNA oligos.
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<figure>
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<a href="https://static.igem.wiki/teams/4361/wiki/parts/add-blcr-wide.png"><img src="https://static.igem.wiki/teams/4361/wiki/parts/add-blcr-wide.png" style="width:300px;margin-left:275px"></a>
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<figcaption> <b>Figure 2.</b> Results of the first electrophoresis experiment, to measure the fraction of DNA bound to BlcR either in the
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<span style="color:#4CCAF1;">presence</span>
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or
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<span style="color:#4460EF">absence</span>
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of BlcR. First set of bars represent results with </html>[[Part:BBa_K4361000]]<html>, second set of bars correspond to those of 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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</figure>
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In addition to testing the effect of adding BlcR, another experiment was performed in which SSA was added as a substitute for GHB, the results of which are shown <b>Figure 3</b>. As stated above, addition of SSA is expected to decrease the amount of BlcR bound to DNA, which is indeed observed in this experiment. These results show that electrophoresis experiments can also be used to show the effect of SSA to the amount of DNA bound by BlcR.
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<figure>
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<a href="https://static.igem.wiki/teams/4361/wiki/parts/add-ssa-0-1.png"><img src="https://static.igem.wiki/teams/4361/wiki/parts/add-ssa-0-1.png" style="width:300px;margin-left:275px"></a>
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<figcaption> <b>Figure 3.</b> Results of the electrophoresis experiment in which the fraction of DNA bound to BlcR was determined for different types of oligos in the
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<span style="color:#4CCAF1;">presence</span>
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or
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<span style="color:#4460EF">absence</span>
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of 25 &mu;M SSA. First set of bars represent results with </html>[[Part:BBa_K4361000]]<html>, second set of bars correspond to those of 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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</figure>
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For further details on these experiments and the results, see <a href="https://2022.igem.wiki/tudelft/results">the Results page on our wiki</a>.
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</html>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4361001 SequenceAndFeatures</partinfo>
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Latest revision as of 15:15, 12 October 2022


BlcR-binding oligo, 51 bp, wild type

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, short DNA sequences whose ends are reverse complements of each other. For the Blc operator, these sequences are 'ACTCTAATgATTCAAGT' and 'ATTAGttgaactCTAAT', as highlighted under Sequence and Features below as Inverted repeat pair 1 (IR1) and Inverted repeat pair 2 (IR2), respectively. The capitalized nucleotides in each sequence form the inverted repeat pairs, although it should be noted that for the first pair the ends of the sequence are not perfect reverse complements (see Part:BBa_K4361004 and Part:BBa_K4361005).

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

To qualitatively test the binding strength of BlcR to different oligos, electrophoresis experiments using a TapeStation were performed in which BlcR was combined with the varying sequences. Any DNA bound by BlcR is visible as a shifted band in the gel, and the ratio between the two bands can be used as an indication of the effectiveness with which BlcR binds to the given DNA sequence. As a first test, Part:BBa_K4361000 and this part were run on a gel after incubation with and without BlcR, the results of which are visible in Figure 2. The graph clearly shows that addition of BlcR causes a shift of the DNA on the gel. Also, as expected, BlcR binds the wildtype sequence more strongly than a random, scrambled oligo. This first experiment shows that gel electrophoresis can indeed be used to, at least qualitatively, test the binding affinity of BlcR to different DNA oligos.
Figure 2. Results of the first electrophoresis experiment, to measure the fraction of DNA bound to BlcR either in the presence or absence of BlcR. First set of bars represent results with Part:BBa_K4361000, second set of bars correspond to those of 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.
In addition to testing the effect of adding BlcR, another experiment was performed in which SSA was added as a substitute for GHB, the results of which are shown Figure 3. As stated above, addition of SSA is expected to decrease the amount of BlcR bound to DNA, which is indeed observed in this experiment. These results show that electrophoresis experiments can also be used to show the effect of SSA to the amount of DNA bound by BlcR.
Figure 3. Results of the electrophoresis experiment in which the fraction of DNA bound to BlcR was determined for different types of oligos in the presence or absence of 25 μM SSA. First set of bars represent results with Part:BBa_K4361000, second set of bars correspond to those of 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 these experiments and the results, see the Results page on our wiki.