Part:BBa_K4361021

BlcR-binding oligo, 57 bp, consensus sequence

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 their contribution to our partnership, the DTU iGEM 2022 team analyzed the BlcR binding operator with NCBI Blast. In total, they found 25 variants of the sequence originating from different strains of A. tumefaciens, varying in similarity to what we define as the wildtype oligo, Part:BBa_K4361001. Surprisingly, while they found that all sequences did have conserved nucleotides, these were not specifically for IR1 and IR2 but instead for alternative inverted repeats (see Sequence and Features and Usage and Biology below). From the multiple sequence alignment, two sequences were chosen to be further investigated by us: the consensus sequence formed by the most common nucleotide for each position in the alignment (this part), and the operator of A. tumefaciens strain Q15, because of its relatively low similarity with the wildtype sequence (Part:BBa_K4361022).

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.

Alternative inverted repeats
As shown above in Sequence and Features as IR1, IR2 and IR3, with > and < respectively indicating forward and reverse direction, this consensus sequence seems to contain three sets of matching IRs that are each reverse complements of each other. Especially IR2 and IR3 are highly conserved in all sequences analyzed by DTU whereas the IRs mentioned in previous oligos are not. This suggests not only that these alternative repeat pairs are highly important for the function of the Blc operator, but possibly that the 'original' IRs are not the actual true binding site of BlcR. Furthermore, the results below show that BlcR may be able to bind this sequence more strongly than the wildtype DNA. While it is not yet known how BlcR interacts with these new binding sequences, according to our results the binding of BlcR to DNA may not be as well understood as previously thought and should be investigated further in future research. For example, a crystallography model of BlcR bound to DNA would make a significant contribution to research on this protein and similar transcription factors.
Figure 2 shows the result of the NCBI Blast, with the consensus sequence shown at the bottom of the multiple sequence alignment on the right. The alternative IRs are clearly visible as the nucleotides most highly conserved throughout the sequences. As AE007872.2 corresponds to Part:BBa_K4361001, it can be seen that the sequence of the wildtype oligo is fully conserved within the sequence of this part. According to literature, binding of BlcR to DNA should be wholly dependent on the presence of the known, 'original' IRs. This does not explain why the addition of nucleotides at the 5' end, which is effectively the only difference between the wildtype and consensus sequences, results in increased binding of BlcR to the DNA. However, these added nucleotides coincide with the forward-facing alternative IR1, which supports the hypothesis that BlcR has a different mechanism of binding to DNA than previously described in literature.

Figure 2. 25 sequences similar to the BlcR operator from A. tumefaciens C58 (AE007872.2) were found with an nBLAST on NCBI (searching ‘somewhat similar sequences’ and word size of 7). Left: an unrooted tree of the sequences with the NCBI accession codes at the tips. Right: the multiple alignment of the sequences coloured by the percentage of identity with a normalized logo plot underneath and the consensus sequence. The new observed inverted repeats (IR1, IR2, IR3) are outlined. The sequences were aligned using MAFT and visualized with JalView. The tree was generated using TreeHugger 0.5 and visualized using FigTree v1.4.4.

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 3, 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.

Figure 3. 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, Part:BBa_K4361009, this part, 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 4 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 4. 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.