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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 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, 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

Characterization of Biosensor

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 Microplate Reader. (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 (J23114-HbpR and Pc-B0032-sfGFP).

Orthogonality

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.