Difference between revisions of "Part:BBa K4361001"

Revision as of 16:55, 6 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 and Inverted repeat pair 2, 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). In vivo the Blc operator consists of pair 1 and 2 linked together by a 3 nt spacer. As mentioned before, each pair can bind a single BlcR dimer. With a spacer of specifically 3 nt, the centers of each pair are exactly 20 nt apart, which may support 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 genes. 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 reverses back into 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. By doing so water molecules in are displaced, which changes the permittivity and thereby the capacitance of the capacitor, which can be measured to be set as a baseline. 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 measuring the capacitance, the solution contacting the biosensor can be continuously monitored for changes in its GHB content.

This original sequence (designated BBa_K4361001) contains two sets of different ‘inverted repeat pairs’, short DNA sequences whose ends are complementary to their own inverse sequence, separated by a 3 nt spacer. Each inverted repeat pair is able to bind a BlcR dimer, with the spacing between pairs allowing for tetramerization of the protein.

This is further described in the wildtype oligo, Part:BBa_K4361001.

-Contains 2 inverted repeats, each binding 1 BlcR dimer

-Kd,app of 120 nM according to EMSA

Sequence and Features