Difference between revisions of "Part:BBa K1031911"

Revision as of 17:36, 23 September 2013

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[7]. 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.

Promoter Structure

The cognate promoter regulated by XylS, Pm, is σ70-dependent in E.coli, while in Pseudomonas putida, it is σ32/38-dependent[6]. It acts as the master regulator to control the ON/OFF expression of meta-operon on TOL plasmid pWW0[2]. 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 (a hyper link is needed here). Vertical axis represents the ON-OFF induction ratio. The Pm/XylS biosensor circuit which adopted Pc promoter J23114 clearly

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 5)

Fig 5. 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 4).

'''Fig 4''' 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.

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+<img id="XylSCurve2" src="https://static.igem.org/mediawiki/igem.org/6/67/Peking2013_Xyls_figur7.png" style="position:relative; top:-350px; left: 420px; ", width=400px; />
 <p style="position: relative; top:-315px;" >
 '''Fig 4''' 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.