Part:BBa_K1031300

HbpR-Terminator

Introduction HbpR (For more details:[http://2013.igem.org/Team:Peking/Project/BioSensors/HbpR]) is 54-dependent transcriptional factor belonging to NtrC family of prokaryotic transcriptional activators. It shares a high homology to members of the XylR/DmpR subclass. HbpR was found in Pseudomonas azelaica. which can use 2-hydroxybiphenyl (2-HBP) and 2, 2’-dihydroxybiphenyl as sole carbon and energy sources through enzymes encoded by hbpCAD functioning in meta-cleavage pathway.

Metabolic Operon

Fig. 1a. Operon of hbpR. Orange rectangle shows hbpR gene. Blue and green rectangles show hbpCA and hbpD genes controled by PC and PD respectively. The orange rectangle show hbpR gene which encodes HbpR protein. When exposed to effector 2-hydroxybiphenyl, HbpR will activate PC and PD.
Fig. 1b. Pathway for the primary metabolism of 2-hydroxybiphenyl and 2-propylphenol in P. azelaica HBP1. The enzymes for each step are also indicated .

Protein Domains

Fig. 2 Four domains of HbpR protein. A domain is sensing domain, conformation change may happen with inducer's binding. B domain is a linker. C domain contains an AAA ATPase motif. It has the capacity to hydrolyze ATP and to interact with sigma54 RNA polymerase. D domain binds to DNA via a typical helix-turn-helix motif.

Fig. 3. Regions containing the binding sites for HbpR (UAS) are shown within boxes in the sequence. Sequence numbers refer to the locations of the transcriptional start sites of hbpC and hbpD. HbpR binds to UAS C-1 and UAS C-2. The 32-bp space between the centers of UASs C-1 and C-2 is critical for cooperative interactions. when the UASs C-1/C-2 are deleted and UASs C-3/C-4 are placed in an appropriate position with respect to the promoter region, the hbpC promoter is still inducible with 2-HBP, albeit at a lower level. The presence of UAS pair C-3/C-4 mediated a higher promoter activity for transcription of hbpR.

Inducible Promoter Structure

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1673
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 387
  

Construction and Tuning

We used PCR to get hbpR gene from bacterial strain and inducible promoter Pc' was synthesized by Genscript Company. The gene hbpR was controlled by a constitutive promoter Pc on plasmid pSB4K5. Another plasmid pUC57 containing Pc'-RBS-sfGFP was double transformed with pSB4K5 to construct HbpR biosensor. To tune its performance, Pc constitutive promoter library and RBS library for reporter were constructed.(Fig. 4)

Fig. 4. Construction of the HbpR biosensor and improvements of its performance. (a) Structure of plasmids for hbpR gene and the reporter gene sfGFP. There is a library for the constitutive promoter before HbpR and the RBS before sfGFP respectively, both of which function to fine-tune the expression level of HbpR. (b) Induction ratio of HbpR controlled by promoters with different expression intensity. The effectors 2-HBP and 2-ABP are plotted in different colors. Data were collected via ELIASA. (c) Induction ratio of HbpR when exposed to a series of concentration of 2-ABP. The reporter system includes Pc-RBS-sfGFP. Three lines represent sfGFP controlled by different RBS. Fluorescence intensity of sfGFP is detected and calculated to plot induction ratio. (d) Induction ratio of HbpR when exposed to a series of concentration of 2-HBP.

On-Off Detection

Fig. 5. On-Off test results for sensor strain 114-32 HbpR. (a) On/off response of strain HbpR to 78 aromatic compounds. (For the full name of the compounds, CLICK HERE(hyperlink is needed here)). The strain showed induction ratio more than 10 folds when exposed to 2-HBP and 2-ABP. (B) The detection range of sensor strain HbpR is profiled in yellow at the aromatics spectrum. The structure formula of typical inducer 2-HBP and 2-ABP is showed near its chemical formula.

Dose-response Curve

Fig. 6. Dose response curves for the induction effect of 2-HBP and 2-ABP to the best-performed HbpR sensor strain (BBa_J23114-HbpR and Pc-BBa_B0032-sfGFP).

Reference:
[1] Jaspers, M. C., Suske, W. A., Schmid, A., Goslings, D. A., Kohler, H. P. E., & van der Meer, J. R. HbpR, a new member of the XylR/DmpR subclass within the NtrC family of bacterial transcriptional activators, regulates expression of 2-hydroxybiphenyl metabolism in Pseudomonas azelaica HBP1. Journal of bacteriology, (2000).182(2), 405-417.
[2] Neuwald AF, Aravind L, Spouge JL, Koonin EV AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res (1999)9: 27–43
[3] Pe´rez-Martı´n J, de Lorenzo. VATP binding to the s54-dependent activator XylR triggers a protein multimerization cycle catalyzed by UAS DNA. Cell (1996) 86: 331–339
[4] Jaspers, M. C., Sturme, M., & van der Meer, J. R. Unusual location of two nearby pairs of upstream activating sequences for HbpR, the main regulatory protein for the 2-hydroxybiphenyl degradation pathway of ‘Pseudomonas azelaica’HBP1. Microbiology, (2001).147(8), 2183-2194.
[5] Jaspers, M. C., Suske, W. A., Schmid, A., Goslings, D. A., Kohler, H. P. E., & van der Meer, J. R.. HbpR, a new member of the XylR/DmpR subclass within the NtrC family of bacterial transcriptional activators, regulates expression of 2-hydroxybiphenyl metabolism in Pseudomonas azelaica HBP1. Journal of bacteriology, (2000)182(2), 405-417.
[6] Jaspers, M. C., Sturme, M., & van der Meer, J. R. Unusual location of two nearby pairs of upstream activating sequences for HbpR, the main regulatory protein for the 2-hydroxybiphenyl degradation pathway of ‘Pseudomonas azelaica’HBP1. Microbiology, (2001). 147(8), 2183-2194.