Difference between revisions of "Part:BBa K1031911"

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	<b>Dose-response curve to benzoate, salicylic acid and derivatives</b>		<b>Dose-response curve to benzoate, salicylic acid and derivatives</b>
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−	Pm/J23114-XylS biosensor circuit was subjected to induction experiments with concentration of inducer ranging at 10 µM, 30µM,100µM, 300µM and 1000µM according to protocol 1. (<b>Fig 5</b>).	+	Pm/J23114-XylS biosensor circuit was subjected to induction experiments with concentration of inducer ranging at 10 µM, 30µM,100µM, 300µM and 1000µM according to protocol 1. (<b>Fig.5</b>).
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	<img src="https://static.igem.org/mediawiki/2013/f/fd/Peking2013_XylS_Induce.png" style="width:800px;margin-left:60px"  ></a>		<img src="https://static.igem.org/mediawiki/2013/f/fd/Peking2013_XylS_Induce.png" style="width:800px;margin-left:60px"  ></a>

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Revision as of 17:08, 25 September 2013

XylS-Terminator

Introduction

XylS is an archetype transcriptional activator of AraC/XylS family, mined from the TOL plasmid pWW0 of the bacterium Pseudomonas putida. It is composed of a C-terminal domain (CTD) involved in DNA binding, and an N-terminal domain required for effector binding and protein dimerization^[1]. XylS detect benzoate and its’ derivatives, mainly methyl and chlorine substitution at 2-, 3- carbon^[2].

Fig.1 Regulatory circuits controlled by XylS and XylR on the TOL plasmid pWW0^[3]. Squares, XylS; circles, XylR; open symbol,s.transcriptional regulator without aromatic effector binding; closed symbols, effector-bound transcription factors that is in active form. See the main text for the detailed explanation for the regulatory loops^[4].

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Promoter Structure

The cognate promoter regulated by XylS, Pm, is σ⁵⁴-dependent in Pseudomonas putida; meanwhile in E.coli, it is σ⁷⁰-dependent^[5]. It acts as the master regulator to control the ON/OFF expression of meta-operon on TOL plasmid pWW0^[3]. In this meta-operon, XylXYZLTEGFJQKIH genes encode enzymes for the degradation of benzoate and its derivatives, producing intermediate metabolites as carbon sources (Fig.1).

Mechanism

Fig.2 Mechanism of XylS binding to Pm promoter.

Mechanism of transcription activation by XylS at Pm promoter. Step 1: Free DNA. The -10/-35 consensus boxes of σ⁷⁰-dependent promoter and the two XylS binding sites (D: distal; and P: proximal) are highlighted. The bending angle is supposed to be 35°, centered at XylS proximal binding site. Step 2: A first XylS monomer binds to Pm at the proximal site, shifts the bent center to the DNA sequence between the two XylS binding sites, and increases the bending angle to 50°. Step 3: This conformational change facilitates the binding of a second XylS monomer to the distal site, further increasing the DNA curvature to an overall value of 98°. Step 4: Contacts with RNAP, also probably with the σ-subunit, are established through the α-CTD of RNA polymerase, which dramatically facilitates the open complex formation and transcription initiation.

Tuning of Biosensor

Fig.3(a) Construction of biosensor circuit.
(b) Using a library of constitutive promoters (Pc) to fine-tune the induction ratio of XylS biosensor. Horizontal axis stands for different XylS biosensor circuits with different Pc promoters. These Pc promoters are of different strength to control the expression of XylS. The relative expression strength of these constitutive promoters, J23113, J23109, J23114, J23105, J23106 are 21, 106, 256, 623, and 1185, respectively, according to the Partsregistry. Four kinds of aromatic compounds, namely BzO, 2-MxeBzO, 3-MeBzO and 4-MeBzO, shown with different color intensities, were tested following Test Protocol 1. Vertical axis represents the ON-OFF induction ratio. The Pm/XylS biosensor circuit adopting a weak Pc promoter J23114 performed best throughout the four inducers.

Related Part:
105-XylS-Terminator: https://parts.igem.org/Part:BBa_K1031912
109-XylS-Terminator: https://parts.igem.org/Part:BBa_K1031913
114-XylS-Terminator: https://parts.igem.org/Part:BBa_K1031916

Sequence and Features

Characterization of Biosensor

ON/OFF test to 78 aromatic compounds

20 compounds showed apparent activation effect with the induction ratios over 20, they are listed as follows: BzO, 2-MeBzO, 3-MeBzO, 4-MeBzO, 2-FBzO, 4-FBzO, 2-ClBzO, 3-ClBzO, 4-ClBzO, 2-BrBzO, 4-BrBzO, 3-IBzO, 3-MeOBzO, SaA, 3-MeOSaA, 4-ClSaA, 5-ClSaA, 3-MeBAD, 3-ClTOL (Fig.4)

Fig.4 (a) The induction ratios of all 78 typical aromatic compounds in the ON/OFF test following Test Protocol 1. XylS biosensor could respond to 24 out of 78 aromatics with the induction ratio higher than 20, mainly benzoate, salicylic and their derivatives.
(b) The aromatics-sensing profile of XylS biosensor is summarized from (a), highlighted in red in the aromatics spectrum. The structure formula of typical inducers are listed around the central spectrum, near their chemical formula.

Dose-response curve to benzoate, salicylic acid and derivatives
Pm/J23114-XylS biosensor circuit was subjected to induction experiments with concentration of inducer ranging at 10 µM, 30µM,100µM, 300µM and 1000µM according to protocol 1. (Fig.5).

Fig.5 (a) Dose-response curves of XylS biosensor induced by benzoate and its derivatives. X-axis stands for concentration gradient of inducers at 10µM, 30µM, 100µM, 300µM and 1000µM. Different colors represent different kinds of inducers. Y-axis shows induction ratios. (b) Dose-response curves of XylS biosensor induced by salicylate and its derivatives. X-axis stands for concentration gradient of inducers at 10µM, 30µM, 100µM, 300µM and 1000µM. Y-axis denotes induction ratios. Different colors denote different kinds of inducers.

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.6). 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.6a, 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.6a, 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.6b).

Fig.6 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.7). 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.7a, b), XylS and HbpR( Fig.7c, d), NahR and HbpR (Fig.7e, f), XylS and DmpR (Fig.7g, h), NahR and DmpR ( Fig.7i, j ), and HbpR and DmpR (Fig.7k, l) are all highly orthogonal, which is summarized in Fig.7

Fig.7 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.
[2] Gerischer, U. (2002). Specific and global regulation of genes associated with the degradation of aromatic compounds in bacteria. Journal of molecular microbiology and biotechnology, 4(2), 111-122.