Team:Peking/Project/BioSensors/HbpR

From 2013.igem.org

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<a href="https://2013.igem.org/Team:Peking">home</a>
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<a href="https://2013.igem.org/Team:Peking">Home</a>
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<li><a href="https://2013.igem.org/Team:Peking/Team/Members">Members</a></li>
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                                <li><a href="https://2013.igem.org/Team:Peking/Project/SensorMining">Biosensor Mining</a></li>
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</ul>
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<div id="ProjectTitle">
<div id="ProjectTitle">
<h1 id="ProjectName">Biosensors</h1>
<h1 id="ProjectName">Biosensors</h1>
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                <h1 id="ProjectSubname">A FAST, EASY AND ACCURATE METHOD TO DETECT TOXIC AROMATIC COMPOUNDS</h1>
 
                 <img src="https://static.igem.org/mediawiki/igem.org/9/96/Peking2013-biosensortitile-zyh.jpg"/>
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                 <h1 id="SensorsListTitle">Biosensors</h1>
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                 <h1 id="SensorsListTitle"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors">Biosensors</a></h1>
                 <ul id="SensorsList">
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                <li><a href="#BottomNavigation">CapR</a><li>
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                    <li class="SensorsListItem" style="font-size:18px; height:15px; width:180px; position:relative; left:-10px;">Individual Biosensors<li>
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                <li><a>DmpR</a><li>
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                    <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/XylS">XylS</a><li>
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                <li><a>HbpR</a><li>
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                    <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/XylR">XylR</a><li>
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                <li><a>HcaR</a><li>
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                    <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/HbpR">HbpR</a><li>
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                <li><a>HpaR</a><li>
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                    <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/HcaR">HcaR</a><li>
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                <li><a>NahR</a><li>
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                    <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/HpaR">HpaR</a><li>
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                    <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/HpaR#ContentHpaR4">PaaX</a><li>
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                <li><a>XylR</a><li>
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                    <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/DmpR">DmpR</a><li>
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                <li><a>XylS</a><li>
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                    <li class="SensorsListItem"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/NahR">NahR</a><li>
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                    <li class="SensorsListItem" style="font-size:18px; height:40px; width:180px; position:relative; left:-10px; top:25px;"><a href="https://2013.igem.org/Team:Peking/Project/BioSensors/MulticomponentAnalysis">Multi-component Analysis</a></li>
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             <h1 id="PageTitle">HbpR</h1>
             <h1 id="PageTitle">HbpR</h1>
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             <h2 id="OverviewHbpR1"> Overview </h2>
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             <h2 id="OverviewHbpR1"> Mechanism </h2>
             <h3 id="OverviewHbpR2"> Build Our Own Sensor! </h2>
             <h3 id="OverviewHbpR2"> Build Our Own Sensor! </h2>
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<img id="NahRFigure1" scr="https://static.igem.org/mediawiki/igem.org/e/e2/Peking2013-NahRFigure1-zyh.jpg"/>
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<img id="FigurePic2" src=" https://static.igem.org/mediawiki/igem.org/e/e3/HbpR_Figure2_2013Peking_WH1.png "/>
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<p id="ContentHbpR1">
<p id="ContentHbpR1">
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HbpR is a 63-kDa protein belonging to NtrC family of prokaryotic transcriptional activators. It shares a high homology to members of the XylR/DmpR subclass.  
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HbpR is a 63 kDa prokaryotic transcriptional activators from NtrC family. It shares a highly conserved homology to members of the XylR/DmpR subclass. In our project, HbpR was <a href="https://2013.igem.org/Team:Peking/Project/SensorMining">bioinformatically mined</a> from <i>Pseudomonas azelaica</i> <a href="#ReferenceHbpR"><sup>[1]</sup></a>. <i>Pseudomonas azelaica</i> can use 2-hydroxybiphenyl (2-HBP) and 2, 2’-dihydroxybiphenyl as sole carbon and energy sources through enzymes encoded by <i>hbpCAD</i> operon in meta-cleavage pathway.
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HbpR was found in Pseudomonas azelaica [ ]. Pseudomonas azelaica 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.
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<p id="ContentHbpR2">
<p id="ContentHbpR2">
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The regulatory gene hbpR encodes a transcriptional activator HbpR, which activates <i>P<sub>C</sub></i> and <i>P<sub>D</sub></i> promoters when exposed to inducers (<B>Fig. 1</B>). Thus three enzyme-coding genes, <i>hbpCA</i> and <i>hbpD</i>, are expressed to degrade 2-HBP (<B>Fig. 2</B>).  
-
The regulatory gene hbpR encodes a transcriptional activator HbpR, which activates PC and PD promoters when exposed to inducers (FIG. 1). Thus three enzyme HbpCA, HbpD are expressed, degrading 2-HBP via meta-cleavage pathway (FIG. 2).  
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</p>
</p>
<p id="ContentHbpR3">
<p id="ContentHbpR3">
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The HbpR protein is composed of four domains, namely Domain A, Domain B, Domain C and Domain D (<B>Fig. 3</B>) . Domain C contains an AAA+ ATPase motif <a href="#ReferenceHbpR"><sup>[2]</sup></a>. It has the ability to hydrolyze ATP and to interact with &sigma;<sup>54</sup> to recruit RNA polymerase for transcription activation. Domain D binds to DNA via a typical helix-turn-helix motif. Domain A is necessary for the recognition of aromatic effector molecules to activate transcription.
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The HbpR protein is composed of four domains, namely A-domain, B-domain C-domain and D-domain (FIG. 3). C-domain contains a triple-AAA ATPase motif [ ]. It has the capacity to hydrolyze ATP and interact with 54 RNA polymerase. D domain binds to DNA via a typical helix-turn-helix motif. A-domain is necessary for recognition of chemical effector molecules that unleashes activity of the transcription activator.
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</p>
</p>
<p id="ContentHbpR4">
<p id="ContentHbpR4">
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Generally, these transcriptional activators act at a distance of 100 to 200 bp from the actual promoter, binding to the sequence, which are called enhancer-like elements or upstream activating sequences (UAS) [3]. 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 (FIG. 4).  
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Generally, AAA+ ATPase-dependent transcriptional activators act at a distance of 100 to 200 bp from the promoter core component, which are called enhancer-like elements or upstream activating sequences (UAS) <a href="#ReferenceHbpR"><sup>[3]</sup></a>. 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 (<B>Fig. 4</B>).
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Most if not all of the transcriptional outputs from the hbpC promoter are mediated by the proximal UASs C-1/C-2. However, 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 [4].  
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The transcription output from the <i>hbpC</i> 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 <i>hbpC</i> promoter to be induced by 2-HBP, albeit at a much lower level. The presence of UAS pair C-1/C-2 mediated a higher promoter activity for transcription of hbpR <a href="#ReferenceHbpR"><sup>[4]</sup></a>.  
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<p id="ContentHbpR5">
<p id="ContentHbpR5">
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HbpR detects a limited range of effectors. Previous studies have revealed that HbpR detects 2-hydroxybiphenyl, 2, 2-dihydroxybiphenyl and structural analogs including 2-aminobiphenl and 2-hydroxybiphenylmethane (TABLE 1) [5].  
+
The aromatics-sensing of HbpR regulator is quite specific. Previous studies have revealed that HbpR detects 2-hydroxybiphenyl, 2, 2-dihydroxybiphenyl and structural analogs including 2-aminobiphenl and 2-hydroxybiphenylmethane (Table 1) <a href="#ReferenceHbpR"><sup>[5]</sup></a>.
</p>
</p>
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<p id="ContentHbpR6">
 
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A single mutation in the hbpR gene (T613C) causes a Trp-Arg substitution at amino acid residue 205. This residue is conserved among all members of XylR/DmpR subclass and significant for inter-domain repression [6].
 
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<p id="ContentHbpR7">
<p id="ContentHbpR7">
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Using <i>hbpR</i> gene coding sequence, a gene circuit working as a biosensor for 2-HBP and 2-ABP was constructed, as described in the <a href="https://2013.igem.org/Team:Peking/Project/BioSensors#BiosensorContent1">Biosensor Introduction</a> section (<B>Fig. 5a</B>).  
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We used PCR to get hbpR gene from bacterial strain and Pc was synthesized by Genscript Company. We construct a gene circuit working as biosensors for 2-HBP and 2-ABP. The hbpR was controlled by a constitutive promoter on plasmid pSB4K5. Another plasmid pUC57 containing Pc-RBS-sfGFP was double transformed with pSB4K5 to construct a biosensor for substituted biphenyls (FIG. 5a). Cells were cultured to appropriate OD600 and stored at -80℃ in 20% glycerol for induction test.
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We first performed <a href="https://2013.igem.org/Team:Peking/Team/Notebook/Protocols#Content3">ON/OFF test</a> on primary HbpR biosensor (BBa_J23106-HbpR and Pc-BBa_B0034-sfGFP) when it was exposed to various aromatic compounds following <a href="https://2013.igem.org/Team:Peking/Team/Notebook/Protocols#Content1">Test Protocol 1</a>. Unfortunately, the basal expression level is considerably high and only 2-HBP and 2-ABP showed slightly induction effect.  
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We firstly tested the on/off response of sensor cell HbpR (BBa_J23106-HbpR and Pc-BBa_B0034-sfGFP) when it was exposed to other aromatic compounds following test protocol 1. Unfortunately, the basal expression level is relatively high and only 2-HBP and 2-ABP showed slightly induction effect (Data not shown) possibly because the HbpR protein originally exists in Pseudomonas azelaica instead of E.coli.  
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Therefore, we used a promoter library to fine-tune the expression level of HbpR to see whether this genetic tailoring could correct its performance. The expression levels of these constitutive promoters, J23109, J23113, J23114, and J23117, are 106, 21, 256, and 162, respectively, according to the <a href="http://parts.igem.org/Part:BBa_J23119">Partsregistry</a>. Results of <a href="https://2013.igem.org/Team:Peking/Team/Notebook/Protocols#Content">ON/OFF test</a> showed that, the HbpR biosensor using a weak constitutive promoter (BBa_J23114) performed best (<B>Fig. 5b</B>).  
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Therefore, we then constructed a promoter library to fine-tune the expression intensity. Results of the sensor strains with different initiation strength showed that BBa_J23114 performed better in this construction (FIG. 5b). We chose BBa_J23114 rather than BBa_J23113 because of the relatively higher fluorescence intensity of cells containing the former promoter. We also created a library for the RBS of sfGFP to figure out which kind of Pc-RBS-sfGFP is optimal for our biosensor system. Experiment results showed that for inducers 2-ABP and 2-HBP, RBS BBa_B0032 had a higher induction ratio compared with RBS BBa_B0031 and RBS BBa_B0034. (FIG. 6c,d)
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On the basis of this improvement, we further exploited a library of RBS sequences to tune the expression level of sfGFP, because a strong RBS sequence might cause high leakage expression at <i>P<sub>C</sub></i> promoter. Experiment results showed that, for inducers 2-ABP and 2-HBP, RBS BBa_B0032 brought a higher induction ratio, compared with RBS BBa_B0031 or RBS BBa_B0034 (<B>Fig. 5c, d</B>).
</p>
</p>
<p id="ContentHbpR8">
<p id="ContentHbpR8">
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After the HbpR biosensor with the best configuration was determined (using constitutive promoter BBa_J23114 and RBS BBa_B0032), subsequently it was subjected to <a href="https://2013.igem.org/Team:Peking/Team/Notebook/Protocols#Content3">ON/OFF test</a> via <a href="https://2013.igem.org/Team:Peking/Team/Notebook/Protocols#Content1">Test Protocol 1</a>. Results showed that, as expected, 2-HBP and 2-ABP elicited significant induction ratios compared with the other tested aromatic compounds (<B>Fig. 6a, b</B>).
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After we had determined the most appropriate expression level of HbpR regulator, the best HbpR sensor cell was subjected to on/off test via protocol 1. Response result showed that 2-HBP and 2-ABP had significant induction ratios compared with the other tested aromatic compounds (FIG. 6a,b).
 
         </p>
         </p>
<p id="ContentHbpR9">
<p id="ContentHbpR9">
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Then we carefully examined the dose-respone curves of HbpR when it was exposed to effectors 2-HBP and 2-ABP according to the <a href="https://2013.igem.org/Team:Peking/Team/Notebook/Protocols#Content1">Test Protocol 1</a> (<B>Fig. 7</B>).
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We then tested a dose-response curve of HbpR when it was exposed to effectors 2-HBP and 2-ABP according to test protocol 1(Fig. 7).
 
         </p>
         </p>
<p id="ContentHbpR10">
<p id="ContentHbpR10">
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In summary, we have constructed and fine-tuned the biosensor for 2-HBP and 2-ABP using HbpR protein, which add materials to practical biosensors for synthetic biology. We also want to acknowledge Professor van der Meer for his material assistance.
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In summary, we have constructed and fine-tuned the biosensing circuit for 2-HBP and 2-ABP using HbpR protein. The aromatic-sensing profile of HbpR biosensor is quite narrow, making it an effective sensor for 2-HBP and 2-ABP, the chemicals that need to be tightly monitored due to their extremely toxicity for human health.
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</p>
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<p id="FigureHbpR1">
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<B>Figure 1.</B> HbpR as the regulator to control the expression of <i>hbp</i> operon. Blue and green rectangles denote <i>hbpCA</i> and <i>hbpD</i> genes controled by <i>P<sub>C</sub></i> and <i>P<sub>D</sub></i>, respectively. The orange rectangle show the <i>hbpR</i> gene which encodes HbpR protein. When exposed to the effectors, such as 2-hydroxybiphenyl, HbpR will activate transcription at <i>P<sub>C</sub></i> and <i>P<sub>D</sub></i>.
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FIG. 1 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.
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<p id="FigureHbpR2">
<p id="FigureHbpR2">
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FIG 2. Pathway for the initial metabolism of 2-hydroxybiphenyl and 2-propylphenol in P. azelaica HBP1. The responsible enzymes are indicated below each arrow.
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<B>Figure 2.</B> Pathway for the primary metabolism of 2-hydroxybiphenyl and 2-propylphenol in <i>P. azelaica</i> HBP1. The enzymes for each step of the degradation are also indicated .
</p>
</p>
<p id="FigureHbpR3">
<p id="FigureHbpR3">
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FIG. 3 Sketch map of protein HbpR. N represents the N terminal of HbpR and C represent the C terminal of HbpR. A, B, C and D show 4 domains of HbpR and the numbers below them show domain boundaries at amino acid level.
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<B>Figure 3.</B> 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.
</p>
</p>
<p id="FigureHbpR4">
<p id="FigureHbpR4">
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FIG. 4 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.
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<B>Figure 4.</B> The sequences preceding <i>hbpC</i> 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 (See <b>Fig. 1</b> as reference).
</p>
</p>
<p id="FigureHbpR5">
<p id="FigureHbpR5">
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FIG. 5 Construction of the HbpR strain 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.  
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<B>Figure 5.</B> Construction and optimization of the HbpR biosensor. (<b>a</b>) 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>b</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. (<b>c</b>) Dose-response curves of HbpR when exposed to gradient concentrations of 2-ABP. Three curves represent HbpR biosensors with sfGFP controlled by RBS sequences of different strength. (<b>d</b>) As in (<b>c</b>), 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.
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<p id="FigureHbpR6">
<p id="FigureHbpR6">
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FIG. 6 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.
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<B>Figure 6.</B> <a href="https://2013.igem.org/Team:Peking/Team/Notebook/Protocols#Content3">ON/OFF test</a> results for the improved HbpR biosensor. (<b>a</b>) ON-OFF response of HbpR biosensor to overall 78 aromatic compounds. <a href="https://static.igem.org/mediawiki/igem.org/2/24/Peking2013_Chemicals_V3%2B.pdf">Click Here</a> 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>b</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.
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FIG. 7 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).
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<B>Figure 7.</B> Detailed dose-response curves of our best HbpR biosensor (BBa_J23114-HbpR and <i>P<sub>C</sub></i>-BBa_B0032-sfGFP), induced by 2-HBP and 2-ABP, respectively.
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<p id="FigureHbpR8">
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<B>Table 1</B> <b>Summary of HbpR aromatics-sensing characteristics</b>
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[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.
[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.
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[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.
 
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Latest revision as of 18:14, 28 October 2013

Biosensors

HbpR

Mechanism

Build Our Own Sensor!

HbpR is a 63 kDa prokaryotic transcriptional activators from NtrC family. It shares a highly conserved homology to members of the XylR/DmpR subclass. In our project, HbpR was bioinformatically mined from Pseudomonas azelaica [1]. Pseudomonas azelaica 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.

The regulatory gene hbpR encodes a transcriptional activator HbpR, which activates PC and PD promoters when exposed to inducers (Fig. 1). Thus three enzyme-coding genes, hbpCA and hbpD, are expressed to degrade 2-HBP (Fig. 2).

The HbpR protein is composed of four domains, namely Domain A, Domain B, Domain C and Domain D (Fig. 3) . Domain C 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. Domain D binds to DNA via a typical helix-turn-helix motif. Domain A is necessary for the recognition of aromatic effector molecules to activate transcription.

Generally, AAA+ ATPase-dependent transcriptional activators act at a distance of 100 to 200 bp from the promoter core component, which are called enhancer-like elements or upstream activating sequences (UAS) [3]. 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 (Fig. 4).

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-1/C-2 mediated a higher promoter activity for transcription of hbpR [4].

The aromatics-sensing of HbpR regulator is quite specific. Previous studies have revealed that HbpR detects 2-hydroxybiphenyl, 2, 2-dihydroxybiphenyl and structural analogs including 2-aminobiphenl and 2-hydroxybiphenylmethane (Table 1) [5].

Using hbpR gene coding sequence, a gene circuit working as a biosensor for 2-HBP and 2-ABP was constructed, as described in the Biosensor Introduction section (Fig. 5a).

We first performed ON/OFF test on primary HbpR biosensor (BBa_J23106-HbpR and Pc-BBa_B0034-sfGFP) when it was exposed to various aromatic compounds following Test Protocol 1. Unfortunately, the basal expression level is considerably high and only 2-HBP and 2-ABP showed slightly induction effect.

Therefore, we used a promoter library to fine-tune the expression level of HbpR to see whether this genetic tailoring could correct its performance. The expression levels of these constitutive promoters, J23109, J23113, J23114, and J23117, are 106, 21, 256, and 162, respectively, according to the Partsregistry. Results of ON/OFF test showed that, the HbpR biosensor using a weak constitutive promoter (BBa_J23114) performed best (Fig. 5b).

On the basis of this improvement, we further exploited a library of RBS sequences to tune the expression level of sfGFP, because a strong RBS sequence might cause high leakage expression at PC promoter. Experiment results showed that, for inducers 2-ABP and 2-HBP, RBS BBa_B0032 brought a higher induction ratio, compared with RBS BBa_B0031 or RBS BBa_B0034 (Fig. 5c, d).

After the HbpR biosensor with the best configuration was determined (using constitutive promoter BBa_J23114 and RBS BBa_B0032), subsequently it was subjected to ON/OFF test via Test Protocol 1. Results showed that, as expected, 2-HBP and 2-ABP elicited significant induction ratios compared with the other tested aromatic compounds (Fig. 6a, b).

Then we carefully examined the dose-respone curves of HbpR when it was exposed to effectors 2-HBP and 2-ABP according to the Test Protocol 1 (Fig. 7).

In summary, we have constructed and fine-tuned the biosensing circuit for 2-HBP and 2-ABP using HbpR protein. The aromatic-sensing profile of HbpR biosensor is quite narrow, making it an effective sensor for 2-HBP and 2-ABP, the chemicals that need to be tightly monitored due to their extremely toxicity for human health.

Figure 1. HbpR as the regulator to control the expression of hbp operon. Blue and green rectangles denote hbpCA and hbpD genes controled by PC and PD, 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 PC and PD.

Figure 2. 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 .

Figure 3. 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.

Figure 4. 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 (See Fig. 1 as reference).

Figure 5. 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 HbpR biosensors with sfGFP 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.

Figure 6. 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.

Figure 7. Detailed dose-response curves of our best HbpR biosensor (BBa_J23114-HbpR and PC-BBa_B0032-sfGFP), induced by 2-HBP and 2-ABP, respectively.

Table 1 Summary of HbpR aromatics-sensing characteristics

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.