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

Fig.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

Fig.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

Fig.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

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

we have confirmed the orthogonality among inducers of different biosensors, which is one of the main features we expect for our aromatics-sensing toolkit. Our sensors are well suited to multicomponent analysis.

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

Orthoganaility between inducer A (originally detected by biosensor I) and B (originally detected by biosensor II) were tested in the following manner (Fig.7). To test the effect of inducer B upon the dose-response curve of inducer A obtained by biosensor I:

(1) Fluorescence intensity of biosensor I elicited by inducer A of concentration gradient was measured as standard results (Fig.7a, Lane 1);

(2) And fluorescence intensity of biosensor I induced by inducer A of concentration gradient in the presence of a certain concentration of inducer B was measured (Fig.7a, Lane 2 and 3) and compared with the standard results.

The effect of inducer A upon the dose-response curve of inducer B obtained by biosensor II was tested vice versa (Fig.7b).

Fig.7 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.

We managed to demonstrate the orthogonality among inducers of different biosensors in a more quantitative and visible way. If inducer A and B were orthogonal, the fluorescence intensity should be identical no matter with or without the irrelevant inducer B. That is to say, the ideal experimental points should be aligned in a line whose slope is one.

The orithogonality of inducers of XylS, NahR, HbpR and DmpR biosensors have been carefully confirmed using the test assay introduced above (Fig.8). The experimental points were processed by linear fitting and the slopes of the fitting curves were compared with 1. The closer the slope was to 1, the more orthogonal the inducers were. The results showed that inducers of biosensor XylS and NahR (Fig.8a, b), XylS and HbpR( Fig.8c, d), NahR and HbpR (Fig.8e, f), XylS and DmpR (Fig.8g, h), NahR and DmpR (Fig.8i, j), and HbpR and DmpR (Fig.8k, l) are all highly orthogonal, which is summarized in Fig.8

Fig.8 Experimental points and the linear fitting curves of the orthogonality test. The black dashed lines are with the slopes of 1, showing as the reference line. The slopes of the experimental fitting curves were showed in the upside portion of the figure, all of them were around 1. These data showed the orthogonality among inducers of 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 experimental points and linear fitting curves of biosensor and its inducers are marked in different colors: XylS in red, NahR in green, HbpR in orange and DmpR in dark cyan.

Reference

[1] Kaldalu, N., Toots, U., de Lorenzo, V., & Ustav, M. (2000). Functional domains of the TOL plasmid transcription factor XylS. Journal of bacteriology, 182(4), 1118-1126.
[2] Ramos, J. L., Michan, C., Rojo, F., Dwyer, D., & Timmis, K. (1990). Signal-regulator interactions, genetic analysis of the effector binding site of xyls, the benzoate-activated positive regulator of Pseudomonas TOL plasmid meta-cleavage pathway operon. Journal of molecular biology, 211(2), 373-382.
[3] Ramos, J. L., Marqués, S., & Timmis, K. N. (1997). Transcriptional control of the Pseudomonas TOL plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators. Annual Reviews in Microbiology, 51(1), 341-373.
[4] Domínguez-Cuevas, P., Marín, P., Busby, S., Ramos, J. L., & Marqués, S. (2008). Roles of effectors in XylS-dependent transcription activation: intramolecular domain derepression and DNA binding. Journal of bacteriology, 190(9), 3118-3128.
[5] Kaldalu, N., Mandel, T., & Ustav, M. (1996). TOL plasmid transcription factor XylS binds specifically to the Pm operator sequence. Molecular microbiology,20(3), 569-579.