Part:BBa_K4361000
BlcR-binding oligo, 51 bp, scrambled
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. This is further described in the wildtype oligo, Part:BBa_K4361001. To test the binding strength of BlcR to its operator sequence and variations thereof, one needs a negative control. To create the negative control, the 51 nt sequence of the wildtype oligo was scrambled using the Genscript Sequence Scramble tool, set to species 'Human'. This tool randomizes the order of the nucleotides of the input sequence, resulting in this part. As a scrambled variant of the original sequence, it has the same amount of each nucleotide, but lacks the specific binding sequence which is otherwise recognized by BlcR. As such, BlcR should not be able to specifically bind this molecule, allowing for its use as a negative control during measurements.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 37
- 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 37
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 37
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 37
- 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 this part 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, this part and Part:BBa_K4361001 were run on a gel after incubation with and without BlcR, the results of which are visible in Figure 2. 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.
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