Part:BBa_K1031300

HbpR-Terminator

Introduction

HbpR (For more details:http://2013.igem.org/Team:Peking/Project/BioSensors/HbpR) is a 63-kDa prokaryotic transcriptional activators from NtrC family. It shares a highly conserved homology to members of the XylR/DmpR subclass. HbpR was found in Pseudomonas azelaica^[1], which can use 2-hydroxybiphenyl (2-HBP) and 2, 2’-dihydroxybiphenyl as sole carbon and energy sources through enzymes encoded by hbpCAD operon in meta-cleavage pathway.

Metabolic Operon

Figure.1 (a) HbpR as the regulator to control the expression of hbp operon. Blue and green rectangles denote hbpCA and hbpD genes controled by P_C and P_D, respectively. The orange rectangle show the hbpR gene which encodes HbpR protein. When exposed to the effectors, such as 2-hydroxybiphenyl, HbpR will activate transcription at P_C and P_D. (b) Pathway for the primary metabolism of 2-hydroxybiphenyl and 2-propylphenol in P. azelaica HBP1. The enzymes for each step of the degradation are also indicated.

Protein Domains

Figure.2 Schematics for the domain organization of HbpR protein. N represents the N-terminal of HbpR and C represent the C-terminal. A, B, C and D denote 4 domains of HbpR, respectively, and the numbers below them denote domain boundaries at amino-acid-sequence resolution.C-domain contains an AAA+ ATPase motif^[2]. It has the ability to hydrolyze ATP and to interact with σ⁵⁴ to recruit RNA polymerase for transcription activation. D-domain binds to DNA via a typical helix-turn-helix motif. A-domain is necessary for the recognition of aromatic effector molecules to activate transcription.

Inducible Promoter Structure

Figure.3 The sequences preceding hbpC promoter contains the binding sites for HbpR (UAS,Upstream Activating Sequences^[3], boxed in red). Sequence numbers denote the locations of UASs relative to the transcriptional start site of hbpC and hbpD. HbpR binds to UAS C-1 and UAS C-2. The 32-bp space sequence between the centers of UASs C-1 and C-2 is critical for the cooperative multimerization of HbpR.The transcription output from the hbpC promoter is mainly mediated by the proximal UASs C-1/C-2. However, when the UASs C-1/C-2 are deleted, the UASs C-3/C-4 still could compensate the ability of the hbpC promoter to be induced by 2-HBP, albeit at a much lower level. The presence of UAS pair C-3/C-4 mediated a higher promoter activity for transcription of hbpR^[4].

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

Characterization of Biosensor

Construction and Tuning

Fig.4 Construction and optimization of the HbpR biosensor.
(a) Schematics for the HbpR biosensor circuit. A library of constitutive promoters preceding the coding sequence of HbpR and a library of RBS sequences preceding sfGFP, respectively were used to fine-tune the HbpR biosensor circuit. (b) Performance of HbpR biosensor using the constitutive promoters of different strength, described with induction ratios. The effectors 2-HBP and 2-ABP are plotted in color intensities. (c) Dose-response curves of HbpR when exposed to gradient concentrations of 2-ABP. Three curves represent different HbpR biosensors where sfGFP are controlled by RBS sequences of different strength. (d) As in (c), dose-response curves of HbpR biosensors when exposed to gradient concentrations of 2-HBP. The induction ratio was calculated by dividing the fluorescence intensity of biosensor exposed to object inducers by the basal fluorescence intensity of the biosensor itself.

On-Off Detection

Fig.5 ON/OFF test results for the improved HbpR biosensor.
(a) ON-OFF response of HbpR biosensor to overall 78 aromatic compounds. Click Here for the full names of aromatic compounds. The biosenor showed induction ratios higher than 10 folds when exposed to 2-HBP and 2-ABP. (b) The detection profile of HbpR biosensor is highlighted in yellow in the aromatics spectrum. The structure formula of typical inducer 2-HBP and 2-ABP is shown.

Dose-response Curve

Fig.6 Detailed dose-response curves of our best HbpR biosensor (BBa_J23114-HbpR and P_C-BBa_B0032-sfGFP), induced by 2-HBP and 2-ABP, respectively.

Orthogonality of Different Sensor

If the presence of an inducer of biosensor A (not an inducer of biosensor B) doesn’t interfere with the dose response of biosensor B to any of its inducers, and vice versa, we call the B and A biosensors are "orthogonal"; namely, no synergistic/antagonistic effects happen between the inducers of A and B biosensors.(for more details, Chick Here)

Sensor	Host	Main Inducers
XylS	Pseudomonas putida	BzO 2-MeBzO 3-MeBzO 2,3-MeBzO 3,4-MeBzO
NahR	Pseudomonas putida	4-MeSaA 4-C1SaA 5-C1SaA SaA Aspirin
DmpR	Pseudomonas sp.600	Phl 2-MePhl 3-MePhl 4-MePhl 2-ClPhl
HbpR	Pseudomonas azelaica	o-Phenylphenol 2,6'-DiHydroxybiphenol

Figure.7 Summary of the orthogonality assay to evaluate the synergistic/antagonistic effects between the inducers of 4 representative biosensors.
No synergistic or antagonistic effects between the inducers of 4 representative biosensors (XylS, NahR, HbpR, and DmpR) were observed. For instance, although the sensing profiles of NahR and XylS overlap to some extent, the NahR-specific and XylS-specific inducers proved to be really orthogonal.

We have confirmed the orthogonality among inducers of different biosensors, which is one of the main features we expect for our aromatics-sensing toolkit; this allowed the combination of these biosensors to profile aromatics for the ease of practical applications.

Related Parts:

XylS: https://parts.igem.org/Part:BBa_K1031911 Wiki: http://2013.igem.org/Team:Peking/Project/BioSensors/XylS

NahR: https://parts.igem.org/Part:BBa_K1031610 Wiki: http://2013.igem.org/Team:Peking/Project/BioSensors/NahR

HbpR: https://parts.igem.org/Part:BBa_K1031300 Wiki: http://2013.igem.org/Team:Peking/Project/BioSensors/HbpR

DmpR: http://2013.igem.org/Team:Peking/Project/BioSensors/DmpR

We examined the orthogonality between 4 representative biosensors (Fig.8). The orthogonality test between two biosensors, biosensor I and biosensor II, was performed in the following procedure:

1. A typical inducer A for biosensor I and a typical inducer B for biosensor II were selected.
2. The dose response of biosensor I to inducer A was measured, under the perturbation of inducer B.
3. The dose-response of biosensor II to inducer B was measured, under the perturbation of inducer A.

If biosensor I and biosensor II are orthogonal, the dose response of biosensor I to inducer A should be constant, regardless of the concentrations of inducer B; and the dose response of biosensor II to inducer B should be constant, regardless of the concentrations of inducer A. Namely, for two "orthogonal" biosensors, the perturbation of an unrelated inducer has negligible effect on the dose response of a biosensor to its related inducer (Fig.9).

Figure.8 Orthogonality test assay for inducer A (detected by biosensor I) and inducer B (detected by biosensor II). (a) Biosensor I was added into the test assay. Different mixtures of inducers were added into lane 1, 2, and 3 respectively as listed above. Effect of inducer B upon the dose-response curve of inducer A was tested by comparing the fluorescence intensity of biosensor I among lane 1 ,2, and 3. (b) Biosensor II was added into the test assay. Different mixtures of inducers were added into lane 1, 2, and 3 respectively as listed above. Effect of inducer A upon the dose-response curve of inducer B was tested by comparing the fluorescence intensity of biosensor II among lane 1 ,2, and 3.

Figure.9 Correlation of the inducer B and the dose-response of biosensor I to its inducer A. Each point on the right plot represents a concentration of inducer A. It's x coordinate represents the fluorescence when inducer B is 0 and the y coordinate represents the fluorescence when the cell is exposed to a none-zero concentration of inducer B. If the dose-response of biosensor I is invariant to the concentration of inducer B, the x coordinate of a experimental point should be equal to its y coordinate and the experimental points are supposed to be aligned in a line whose slope is one.

The orthogonality between XylS, NahR, HbpR and DmpR biosensors have been carefully evaluated using the assay discussed above (Fig.8). The data were processed by linear fitting and the slopes of the fitting curves were compared with 1 (Fig.7, Fig.8). The closer the slope was to 1, the more orthogonal the two biosensors were. Results showed that the biosensor pairs, XylS and NahR (Fig.10a, b), XylS and HbpR (Fig.10c, d), NahR and HbpR (Fig.10e, f), XylS and DmpR (Fig.10g, h), NahR and DmpR (Fig.10i, j), and HbpR and DmpR (Fig.10k, l) are all orthogonal, as summarized in Fig.7.

Figure.10 Linear fitting of the data obtained from the orthogonality assay showing that the orthogonality between the 4 representative biosensors. The experiments and data processing were performed as described in Fig. 8 and Fig. 9.The black dashed line denotes slope=1 as the reference line. These fittings showed the orthogonality between biosensors, (a, b) XylS and NahR; (c, d) XylS and HbpR; (e, f) NahR and HbpR, (g, h) XylS and DmpR, (i, j) NahR and DmpR, and (k, l) HbpR and DmpR. The experiment data, linear fitting curves of biosensor, and cognate inducers are in different colors: XylS in red, NahR in green, HbpR in orange and DmpR in dark cyan.

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.