Difference between revisions of "Part:BBa K4361001"
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<h3>Results</h3> | <h3>Results</h3> | ||
| − | + | 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 both with and without BlcR were run on a gel, the results of which are visible in <b>Figure 1.</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. | |
<figure> | <figure> | ||
<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> | <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> | ||
| − | <figcaption> <b>Figure 1.</b> Results of the first | + | <figcaption> <b>Figure 1.</b> Results of the first electrophoresis experiment, to measure the fraction of DNA bound to BlcR either in the |
<span style="color:#4CCAF1;">presence</span> | <span style="color:#4CCAF1;">presence</span> | ||
or | or | ||
<span style="color:#4460EF">absence</span> | <span style="color:#4460EF">absence</span> | ||
| − | of BlcR. First set of bars represent results with </html>[[Part:BBa_K4361000]]<html>, second set of bars correspond to those of this part. </figcaption> | + | 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> |
</figure> | </figure> | ||
| − | + | 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 2.</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. | |
| − | + | ||
| − | + | ||
| − | + | ||
<figure> | <figure> | ||
<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> | <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> | ||
| − | <figcaption> <b>Figure | + | <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 in the |
<span style="color:#4CCAF1;">presence</span> | <span style="color:#4CCAF1;">presence</span> | ||
or | or | ||
<span style="color:#4460EF">absence</span> | <span style="color:#4460EF">absence</span> | ||
| − | of 25 μM SSA. First set of bars represent results with </html>[[Part:BBa_K4361000]]<html>, second set of bars correspond to those of this part. </figcaption> | + | of 25 μM 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> |
</figure> | </figure> | ||
| + | |||
| + | 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>. | ||
</html> | </html> | ||
Revision as of 15:52, 11 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
In vivo 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 blc genes, creating a selfregulating system. Each BlcR monomer contains a binding site that recognizes gamma-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.
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.
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 said molecules are detected 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, 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 both with and without BlcR were run on a gel, the results of which are visible in Figure 1.. 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.
