Difference between revisions of "Part:BBa K4361001"

Revision as of 17:25, 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).

Usage and Biology

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 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 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.

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 through Part:BBa_K4361022, shows our work on the second approach.

-Kd,app of 120 nM according to EMSA