Part:BBa_K4361100

BlcR, codon optimized

BlcR is a transcription factor originating from the bacterium Agrobacterium tumefaciens (Part:BBa_K4361100). 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.
This part is based on 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 make expression of the protein as efficient as possible.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 694
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 78
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 589
  

Usage and Biology

In vivo, BlcR dimers recognize and bind a specific region in its host genome (denoted as the Blc operator, see [[Part:BBa_K4361001]]). Two operators lie close to each other on the DNA, separated by a 3 nt linker sequence. When two dimers each bind an operator sequence, their position relative to each other allows for the formation of a BlcR tetramer. This binding and tetramerization represses the expression of downstream genes of the blc family that allow A. tumefaciens to digest GHB-like molecules. When GHB is present in solution and occupies a GHB-binding domain in a BlcR tetramer, the tetramer reverts to two dimers and releases from the DNA it previously bound. This allows for the transcription of downstream genes and subsequent digestion of the molecules. 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 DNA oligos containing the BlcR-binding sequence (see [[Part:BBa_K4361001]]). The sensor also contains BlcR dimers, which bind to the DNA oligos. By doing so water molecules in the system 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.