Difference between revisions of "Part:BBa K4361020"

Latest revision as of 23:46, 11 October 2022

BlcR-binding oligo, 51 bp, Cro repressor variant

BlcR is a transcription factor originating from the bacterium Agrobacterium tumefaciens (Part:BBa_K4361100). Cro is a repressor protein found in bacteriophage λ. Both proteins form homodimers in vivo to create a dimer with a single DNA-binding domain, though the DNA sequences they each bind differ greatly. As described in Usage and Biology below, one of the core principles of our project was to increase the binding strength between BlcR and DNA. One approach to achieve this was to create a chimera protein which combines the DNA-binding and dimerization domain of Cro with the GHB-recognition and tetramerization domain of BlcR. While its application would be similar to those of the previous parts (Part:BBa_K4361000 through Part:BBa_K4361019), it would require a different binding oligo due to its differing DNA-binding domain.
This part has been designed to test the binding strength between Cro-BlcR and DNA. To create this part, the nucleotides originally designated as belonging to IR1 and IR2 in Part:BBa_K4361001 have been exchanged for the consensus sequence which the Cro repressor binds ('TATCACCgcgGGTGATA', Cro IR1) and its reverse complement ('TATCACCcgcGGTGATA', Cro IR2). As the other nucleotides are identical to those in the wildtype oligo, the BlcR-binding domain of this part thus consists of Cro IR1-tca-Cro IR2, where tca is the original 3 nt linker sequence between IRs. The layout of this oligo presumes that other than the DNA-binding domain, the overall structure of Cro-BlcR is identical to that of wildtype BlcR, including its orientation to the DNA, and it retains its ability to form tetramers when bound to DNA.

Usage and biology

In vivo the Blc operator consists of pair 1 and 2 linked together by a 3 nt spacer, and each pair can bind a single 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 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 reverts back to 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. When the dimer displace water molecules by binding to the DNA, the permittivity and thereby the capacitance of the capacitor changes, which can be measured and set as a baseline after an equilibrium has been reached. 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 continuously measuring the capacitance, the solution contacting the biosensor can be 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 up to this part, shows our work on the second approach.