Difference between revisions of "Part:BBa K4361014"

Revision as of 11:31, 12 October 2022

BlcR-binding oligo, 56 bp, IR1 + 5 bp linker + IR2

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 (IRs), short DNA sequences whose ends are reverse complements of each other. For the Blc operator, these sequences are 'ACTCTAATgATTCAAGT' (IR1) and 'ATTAGttgaactCTAAT' (IR2), as further explained in Part:BBa_K4361001.
As explained below under Usage and Biology, the length of the linker sequence between IRs (normally 3 nt) is crucial for BlcR's ability to correctly bind the DNA and tetramerize. This part has been designed to show whether or not a larger linker indeed inhibits this behaviour. To create this part, the final five nucleotides of the wildtype oligo (gcggg) have been added between the original linker and IR2. The BlcR-binding domain of this part thus consists of IR1 outer 5-tcagcggg-IR2. As a 3 nt linker is hypothesized to result in two BlcR dimers binding on the same side of a DNA strand, an 8 nt linker as found in this part should result in two dimers binding on opposite parts of the DNA, severely reducing the amount of BlcR bound to DNA strands.

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 (as seen with this part). 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 blcABC 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 reverts back to two dimers and unbinds from the DNA, enabling downstream transcription and subsequent digestion of the newly present substrate (see Figure 1).

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 the wildtype BlcR-binding 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.

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

Results