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| − | <span class='h3bb'><h3>Sequence and Features</h3></span> | + | <span class='h3bb'>Sequence and Features</span> |
| | <partinfo>BBa_K4361007 SequenceAndFeatures</partinfo> | | <partinfo>BBa_K4361007 SequenceAndFeatures</partinfo> |
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| | <h3>Usage and biology</h3> | | <h3>Usage and biology</h3> |
| − | <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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| − | 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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| − | '''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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| | <html> | | <html> |
| | <h3>Results</h3> | | <h3>Results</h3> |
| − | 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>, the amount of DNA bound by BlcR is increased significantly when compared to the results for the wildtype oligo. This suggests a higher binding affinity between BlcR and DNA, so this oligo was selected to be further analyzed, as described below.
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| | <figure> | | <figure> |
| | <a href="https://static.igem.wiki/teams/4361/wiki/parts/oligos-07.png"><img src="https://static.igem.wiki/teams/4361/wiki/parts/oligos-07.png" style="width:300px;margin-left:275px"></a> | | <a href="https://static.igem.wiki/teams/4361/wiki/parts/oligos-07.png"><img src="https://static.igem.wiki/teams/4361/wiki/parts/oligos-07.png" style="width:300px;margin-left:275px"></a> |
| − | <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> | + | <figcaption> <b>Figure 2.</b> Results of the second Tapestation 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.</figcaption> |
| | </figure> | | </figure> |
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| − | In a second electrophoresis experiment, two runs were done for the two controls and five selected oligos that showed binding similar to or increased from the wildtype </html>(this part, [[Part:BBa_K4361008]], [[Part:BBa_K4361009]], [[Part:BBa_K4361021]], and [[Part:BBa_K4361022]])<html>. One run was performed under similar conditions as those in the experiment described above, while SSA was added to the second run. As a substitute for GHB, it is expected that the addition results in the separation of BlcR tetramers into dimers, leading to them unbinding from the DNA. Indeed, <b>Figure 2</b> shows that this is the case for all samples. More strikingly for the selected oligos, the amount of bound DNA drops from increased levels when compared to the wildtype oligo to similar levels after addition of SSA. This means that there is a relatively larger change in the amount of bound DNA, which would be beneficial for application in an electronic biosensor as the bigger change in the signal would be easier to measure.
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| | <figure> | | <figure> |
| | <a href="https://static.igem.wiki/teams/4361/wiki/parts/add-ssa-07.png"><img src="https://static.igem.wiki/teams/4361/wiki/parts/add-ssa-07.png" style="width:300px;margin-left:275px"></a> | | <a href="https://static.igem.wiki/teams/4361/wiki/parts/add-ssa-07.png"><img src="https://static.igem.wiki/teams/4361/wiki/parts/add-ssa-07.png" style="width:300px;margin-left:275px"></a> |
| − | <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 | + | <figcaption> <b>Figure 3.</b> Results of the third Tapestation experiment, in which the fraction of DNA bound to BlcR was determined for different types of oligos in the |
| | <span style="color:#4CCAF1;">presence</span> | | <span style="color:#4CCAF1;">presence</span> |
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| | <span style="color:#4460EF">absence</span> | | <span style="color:#4460EF">absence</span> |
| − | of 25 μM SSA. The first set of bars represents the results with </html>[[Part:BBa_K4361000]]<html> (scrambled oligo, negative control), the second set of bars corresponds to those of </html>[[Part:BBa_K4361001]]<html> (wildtype oligo, positive control). The third set 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> | + | of 25 μM SSA. The first set of bars represents the results with </html>[[Part:BBa_K4361000]]<html> (scrambled oligo, negative control), the second set of bars corresponds to those of </html>[[Part:BBa_K4361001]]<html> (wildtype oligo, positive control). The third set depicts the measured fraction of bound DNA for this part.</figcaption> |
| | </figure> | | </figure> |
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| − | For further details on the experiments with our DNA oligos and the results, see <a href="https://2022.igem.wiki/tudelft/results">the Results page on our wiki</a>.
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| | </html> | | </html> |
