Part:BBa_K4361103

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 an effector binding domain near the C-terminus which recognizes the effectors, succinic semialdehyde (SSA) and gamma-Butyrolactone GBL gamma-hydroxybutyric acid (GHB) and related molecules. The N-terminal region allows for the dimerization of two BlcR monomers. Dimeric BlcR can bind to the blc <i/> operator sequence.
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 <i>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.

<figure> <a href=""><img src="" style="width:300px;margin-left:275px"></a> <figcaption> Figure 2. Figure 1.The regulatory mechanism of BlcR on the blc operon and the pathway from gamma-butyrolactone to succinic acid of Agrobacterium tumefaciens.</figcaption> </figure>

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 766
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 901
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 150
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 661
  

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

Wet Lab module 1 focused on optimizing the expression and purification of BlcR. After applying the engineering cycle multiple times, a protocol was developed which allowed for the expression and relatively high yield of the protein. After expression in E. coli BL21(DE3) cells, the protein was captured on a His column with Ni-NTA beads and subsequently washed off through TEV digestion. The washed off solution was then further purified with size exclusion.

The functionality of this part was later proven in experiments with the BlcR-binding DNA oligos, see Part:BBa_K4361000 through Part:BBa_K4361022 and our results for module 3.