Part:BBa_K1031911

XylS-Terminator

Overview

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 containing two helix-turn-helix motifs and an N-terminal domain required for effector binding and protein dimerization. XylS detects benzoate and its’ derivatives, mainly methyl and chlorine substitutes at 2-, 3- carbon.

Fig.1 Regulatory circuits controlling the expression from the TOL plasmid pWW0^[1].
Squares, XylS; circles, XylR; open symbol,s.transcriptional regulator without aromatics effector binding; closed symbols, effector-bound transcription factors that is in active form. See the main text for the detailed explanation for regulatory loops.</p>
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Promoter Structure

The cognate promoter regulated by XylS, Pm, is σ70-dependent in E.coli, while in Pseudomonas putida, it is σ32/38-dependent^[2]. It acts as the master regulator to control the ON/OFF expression of meta-operon on TOL plasmid pWW0<a href="#ReferenceXylS">[2]</a></sup></html>. In this meta-operon, XylXYZLTEGFJQKIH genes encode enzymes for the degradation of benzoate and its derivatives, generating intermediate products in TCA cycle. (Fig.1)

Mechanism

Fig.2 Mechanism of XylS binding to Pm promoter.

Mechanism transcription activation by XylS at Pm promoter. Step 1: Free DNA. The -10/-35 consensus sequence motifs of σ70-dependent promoter and the two XylS binding sites (D: distal; and P: proximal) are depicted. The bending angle is supposed to be 35°, centered at the 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 change favors the binding of a second monomer to the distal site, further increasing the DNA curvature to an overall value of 98° (here schematized as a right angle). Contacts with RNAP, also probably with the σ-subunit, are established through the α-CTD, which dramatically facilitates the open complex formation and transcription initiation as shown in Step 4.

Tuning of Biosensor

Fig.3(a) Construction of biosensor circuit.
(b) Induction ratio of XylS biosensor library that utilizes different constitutive Pc promoters to control the expression of XylS. Horizontal axis stands for different XylS biosensor with Pc promoters of different strength. The expression strength of these constitutive promoters, J23113, J23109, J23114, J23105, J23106 is 21, 106, 256, 623, and 1185, respectively, according to the Partsregistry. Four kinds of aromatics, 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 which adopted Pc promoter J23114 clearly

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

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 32
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 754
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 217
  

Summary: 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 The detective range of Pm/J23114-XylS biosensor circuit.
(a) The induction ratio column in the On-Off test following protocol 1. XylS could respond to 24 out of 78 aromatics with the induction ratio over 20, mainly benzoate, salicylic and their derivatives. (b) The detection range of sensor strain XylS is profiled in red at the aromatics spectrum. The structure formula of typical inducer is listed around the cycle spectrum, near its 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 curve of Pm/J23114-XylS biosensor circuit in response to 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 ratio reflected via fluorescence intensity. (b) Dose-response curve of Pm/J23114-XylS expression system in response to salicylate 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 ratio reflected via fluorescence intensity.

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] 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.
[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.