Part:BBa_K4361022
BlcR-binding oligo, 49 bp, strain variant
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 (Part:BBa_K4361021), and the operator of A. tumefaciens strain Q15, because of its relatively low similarity with the wildtype sequence (this part).
Sequence and Features
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- 12COMPATIBLE WITH RFC[12]
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- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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).
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 this part, 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 and CP049218.1 to this part, it can be seen that outside of the conserved nucleotides the two sequences do not resemble each other. Still, as shown in the results further below, BlcR has a higher binding affinity to this sequence than to the wildtype. Since the Q15 operator does not have the full original IR1 and IR2 sequences but it does contain the alternative IR2 and IR3, this sequence supports the hypothesis that BlcR has a different mechanism of binding to DNA than previously described in literature.
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 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.
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