Difference between revisions of "Part:BBa K1031300"

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== '''Orthogonality''' ==
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== '''Orthogonality of Different Sensor''' ==
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If the existence of each inducer A, which is not an inducer of B, doesn’t interfere the B’s dose-response to any of its inducers, vice versa, we call the biosensor A and B are orthogonal, and we are secured to apply the multi-component analysis with A and B.(for more details, <a href="http://2013.igem.org/Team:Peking/Project/BioSensors/MulticomponentAnalysis">Chick Here</a>)
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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.
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<B>Figure.6</B> Summary of the orthogonality among four sensors.
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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 <a href="http://2013.igem.org/Team:Peking/Project/BioSensors/MulticomponentAnalysis">multi-component analysis</a>.
 
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DmpR: http://2013.igem.org/Team:Peking/Project/BioSensors/DmpR
 
DmpR: http://2013.igem.org/Team:Peking/Project/BioSensors/DmpR
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<b>Fig.7</b> 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.
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<b>Figure.7</b> 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.
 
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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.
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If biosensor I and biosensor II are orthogonal, the dose-response of biosensor I is invariant to the concentration of inducer B and the dose-response of biosensor II is invariant to the concentration of inducer A. This invariance is showed in the form of <b>Fig.8</b>.</p>
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<B>Figure.8 </B>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.
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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'''
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The orithogonality of XylS, NahR, HbpR and DmpR biosensors have been carefully confirmed using the test assay introduced above (<b>Fig.9</b>). 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 biosensors were. The results showed that biosensor XylS and NahR (<b>Fig.9a,b</b>), XylS and HbpR (<b>Fig.9c,d</b>), NahR and HbpR (<b>Fig.9e,f</b>), XylS and DmpR (<b>Fig.9g,h</b>), NahR and DmpR (<b>Fig.9i,j</b>), and HbpR and DmpR (<b>Fig.9k,l</b>) are all investigated to be orthogonal, which is summarized in <b>Fig.6</b>
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<b>Fig.8</b> 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.
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<B>Figure.9</B> 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 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.
 
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Revision as of 00:57, 28 September 2013

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 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. (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 PC-BBa_B0032-sfGFP), induced by 2-HBP and 2-ABP, respectively.

Orthogonality of Different Sensor

If the existence of each inducer A, which is not an inducer of B, doesn’t interfere the B’s dose-response to any of its inducers, vice versa, we call the biosensor A and B are orthogonal, and we are secured to apply the multi-component analysis with A and B.(for more details, Chick Here)

SensorHostMain Inducers
XylSPseudomonas putidaBzO 2-MeBzO 3-MeBzO 2,3-MeBzO 3,4-MeBzO
NahRPseudomonas putida4-MeSaA 4-C1SaA 5-C1SaA SaA Aspirin
DmpRPseudomonas sp.600Phl 2-MePhl 3-MePhl 4-MePhl 2-ClPhl
HbpRPseudomonas azelaicao-Phenylphenol 2,6'-DiHydroxybiphenol

Figure.6 Summary of the orthogonality among four sensors.

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 multi-component 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).

Figure.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.


If biosensor I and biosensor II are orthogonal, the dose-response of biosensor I is invariant to the concentration of inducer B and the dose-response of biosensor II is invariant to the concentration of inducer A. This invariance is showed in the form of Fig.8.

Figure.8 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 orithogonality of XylS, NahR, HbpR and DmpR biosensors have been carefully confirmed using the test assay introduced above (Fig.9). 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 biosensors were. The results showed that biosensor XylS and NahR (Fig.9a,b), XylS and HbpR (Fig.9c,d), NahR and HbpR (Fig.9e,f), XylS and DmpR (Fig.9g,h), NahR and DmpR (Fig.9i,j), and HbpR and DmpR (Fig.9k,l) are all investigated to be orthogonal, which is summarized in Fig.6

Figure.9 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 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] 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.