Part:BBa_K4361009

BlcR-binding oligo, 51 bp, IR1 perfect RV 2 outer 5 + IR2

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.
As described in Part:BBa_K4361004 and Part:BBa_K4361005, the original sequence of IR1 is not a perfect reverse complement of itself. Furthermore, as described in Part:BBa_K4361007, only the outer 5 nucleotides of IR2 are reverse complementary instead of the outer 8 in IR1, meaning IR1 can be changed to also only have its outer 5 nucleotides be reverse complementary. This part has been designed to combine the changes in Part:BBa_K4361005 and Part:BBa_K4361007, meaning nucleotides 6-12 of IR1 RV 2 have been replaced by those of IR2, resulting in 'ACTCTttgaactAGAGT' (IR1 RV 2 outer 5). The BlcR-binding domain of this part thus consists of IR1 RV 2 outer 5-tca-IR2, where tca is the original 3 nt linker sequence between IRs.

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

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

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 1, the amount of DNA bound by BlcR is comparable to that measured for the wildtype oligo. This suggests a similar binding affinity between BlcR and DNA, so this oligo was selected to be further analyzed, as described below.

Figure 1. 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.

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 ([[Part:BBa_K4361007]], [[Part:BBa_K4361008]], this part, [[Part:BBa_K4361021]], and [[Part:BBa_K4361022]]). 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, Figure 2 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.

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. The first set of bars represents the results with Part:BBa_K4361000 (scrambled oligo, negative control), the second set of bars corresponds to those of Part:BBa_K4361001 (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.

For further details on the experiments with our DNA oligos and the results, see the Results page on our wiki.