Difference between revisions of "Part:BBa K4361103"

Revision as of 07:06, 12 October 2022

BlcR with 6xHis-tag and TEV protease cleavage site

BlcR is a transcription factor originating from the bacterium Agrobacterium tumefaciens. A single BlcR monomer contains a domain near the C-terminus which recognizes gamma-hydroxybutyric acid (GHB) and related molecules. The N-terminal region allows for dimerization of two BlcR monomers, as well as forming a DNA-binding domain when in a dimer state.
BlcR was originally added to the Parts Registry as Part:BBa_K1758370 by the Bielefeld-CeBiTec iGEM 2015 team. Their sequence for BlcR has been codon optimized for expression in E.coli by us to improve expression of the protein (Part:BBa_K4361100). This composite part consists of codon optimized BlcR, a 6xHis-tag for purification, and a TEV cleavage site for removal of the tag from the protein (Part:BBa_K4361104). This combination allowed us to both make the expression as efficient as possible, as well as allowing for high-yield purification.

Usage and biology

In vivo the Blc operator consists of two inverted repeat pairs (see Part:BBa_K4361001), which can each bind a single BlcR dimer, separated by a 3 nt linker. The specific length of the linker allows for the correct orientation relative to each other of two dimers bound to the DNA, such that they are able to tetramerize. 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. One of the plates is covered in a BlcR-binding DNA oligo. The sensor also contains BlcR dimers, which bind to the DNA oligos. When the dimers 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.

Results