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, but also the rape drug gamma-hydroxybutyric acid (GHB). The N-terminal region allows for the dimerization of two BlcR monomers. Dimeric BlcR can bind to the blc operator sequence ( Figure 1 ).

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 (Part:BBa_K4361102), and a TEV cleavage site for removal of the tag from the protein (Part:BBa_K4361101). With this composite part, we were able to effectively produce and purify BlcR in E. coli .

Figure 1. BlcR crystal structure. DNA binding domain (DBD) in pink and the effector binding domain (EBD) in purple.

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

BlcR is an allosteric transcription factor BlcR from the bacterium Agrobacterium tumefaciens. This plant bacterium is able to use GBL, a precursor of GHB, as an energy source. In the absence of GBL, GHB or SSA, BlcR will bind to the blc operator sequence Part:BBa_K4361000 and acts as a repressor for the transcription of the blc proteins. When GBL, GHB or SSA binds to BlcR, it is released from the DNA and the blcA, blcB and blcC proteins are transcribed and digest GBL to succinate ( Figure 2 ).

Figure 2. The regulatory mechanism of BlcR on the blc operon and the pathway from gamma-butyrolactone to succinic acid of Agrobacterium tumefaciens.

In our project we have designed a novel bioelectronic sensor for the detection of GHB in drinks by combining the specificity of the BlcR regulatory mechanism with the reliability of electronics. BlcR is tethered by dsDNA oligonucleotides carrying the blc operator sequence Part:BBa_K4361000 to the surface of a gold interdigitated electrode (IDE). We can measure the capacitance of the IDE, which is influenced by the protein molecules around the fingers of the electrode. If GHB enters the system, it will bind to BlcR, which will then release from the electrode causing the capacitance to increase. Electronic hardware will interpret the change in capacitance and produce an output signal to warn the user that their drink has been spiked.

Experimental results

Wet Lab module 1focused on optimizing the expression and purification of BlcR. After applying the engineering cycle multiple times, a protocol was developed which allowed for effective protein production and purification. After expression in E. coli BL21(DE3) cells, the protein was purified with Ni-NTA column purification and gel filtration. We were able to obtain a highly pure and concentrated (36.1 µM) BlcR sample after executing our optimized production and purification protocol(Figure 3).

Figure 3. SDS PAGE of different elution fractions after size exclusion with BlcR protein sample. Protein bands corresponding to the monomer (~35 kDa) and dimer (~70 kDa) of BlcR are visible.

To characterize the binding of BlcR to its cognate DNA binding sequence Part:BBa_K4361000, we used electrophoresis mobility assay (EMSA). The concentration of the DNA was maintained constant while the concentration of BlcR was increased gradually from 0 to 1 µM (Figure 4).

Figure 4. EMSA study for the characterization of BlcR binding to the 51 bp Blc operator sequence. The concentration of Cy3 labeled DNA was maintained at 25 nM. Titration of dimeric BlcR from lane 1 to 10 . 1: 0μM, 2: 0.1μM, 3: 0.25μM, 4: 0.4μM, 5: 0.5μM, 6: 0.6μM, 7: 0.7μM, 8: 0.8μM, 9: 0.9μM, 10: 1μM.

We successfully validated binding of BlcR to the Blc operator sequence. From the results of EMSA we determined the binding affinity of BlcR to the Blc operator sequence, and established the degree of cooperative binding, expressed in the hill coefficient. We found a binding affinity of 390 nM, together with a hill coefficient of 1.79. These values were not far from what is currently reported in literature. Earlier research of Pan et al [1]. reported a binding affinity of 490 nM [1]. The 1.79 hill coefficient was further on used in modeling experiments. Read more about how we calculated the Kd and the hill coefficient in our model section.

Another EMSA study was performed to characterize the dissociation of BlcR from the blc operator in presence of SSA. We performed an EMSA study where we kept the concentration of 6xHis-BlcR and DNA constant at 1.6 µM and 25 nM respectively, and titrated the concentration of SSA in a range from 0 to 1 mM (Figure 5). With this study we successfully established the full dissociation of BlcR in presence of > 40 µM SSA.

Figure 5. EMSA study for the characterization of BlcR dissociating from to the 51 bp Blc operator sequence in presence of SSA. The concentration of Cy3 labeled DNA and 6xHis-BlcR was maintained at 25 nM and 1.6 μM respectively. Titration of SSA from lane 1 to 9 . 1: 0, 2: 64 nM, 3: 320 nM, 4: 1.6 μM , 5: 08 μM, 6: 40 μM, 7: 0.2 mM, 8: 1mM, 9: 25 nM DNA only.

From the EMSA experiments mentioned above, we established the successful production and purification of active BlcR. We also confirmed the activity of BlcR with the attached His-Tag. Suggesting that digestion with TEV protease is not necessary for the activity of BlcR, thus making the production and purification easier to carry out.

References

[1] Pan, Y., Fiscus, V., Meng, W., Zheng, Z., Zhang, L.-H., Fuqua, C. and Chen, L. (2011). The Agrobacterium tumefaciens Transcription Factor BlcR Is Regulated via Oligomerization. The Journal of Biological Chemistry, [online] 286(23), pp.20431–20440. doi:10.1074/jbc.M110.196154