Difference between revisions of "Part:BBa K1031803"

Revision as of 19:44, 27 September 2013

Pu-B0031-sfGFP-Terminator (XylR)

For detailed information concerning XylR and Pu promoter, please visit 2013 Peking iGEM Biosensor XylR

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 167
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 219

Construction

Pc promoter J23114 is selected to initiate the transcription of XylR. Based on this circuit, we constructed a library of RBS (Ribosome Binding Site) including B0031[1], B0032[2], B0033[3] and B0034[4] for tunning for expression level of reporter gene sfGFP. K1031803 consists of Pu promoter, RBS B0031 and reporter gene sfGFP (Fig 5).

Fig.6 Construction of reporter circuit ''Pu''-B0031-sfGFP. The orange arrow represents ''Pu'' promoter for XylR. The green oval stands for RBS B0031. sfGFP coding sequence is shown with dark blue, while terminator B0015[https://parts.igem.org/Part:BBa_B0015] is in dark red.

Data shown

We obtained XylR from the TOL plasmid of Pseudomonas putida mt-2 [11][13]. It was then exploited to build the XylR biosensor. We used a library of constitutive promoters (Pc) to control the expression level of XylR. We expected this would fine-tuning the performance of XylR biosensor, because previous studies indicated that, the expression level of XylR is critical for its regulatory performance on Pu promoter. Results showed that the XylR biosensor with a medium strong Pc promoter, BBa_J23106, works the best (Fig. 7).

Fig.7 The induction ratios of all 78 aromatic compounds obtained from the ON/OFF Test using Test Protocol 1. ('''a''') XylR biosensor could respond to several aromatics whose induction ratios are not sufficiently high. This result was inconsistent with previous studies [5], which is probably due to the vaporization of hydrophobic aromatic compounds, as we discussed in the main text. ('''b''') The aromatics-sensing profile of XylR biosensor. The aromatic species that can elicit strong responses of XylR biosensor are highlighted in orange in the aromatics spectrum. The structure formula of the typical inducer(s) is also presented around the spectrum. 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. Click Here for the full names of aromatic compounds.

As shown in Fig. 8, the performance of XylR is quite different from the previous studies. The conventional inducers for XylR, for instance, TOL, m-Xyl and 3-CITOL, could not elicit any significant responses. It was probably because that these hydrophobic aromatic compounds can vaporize and permeate the sealing film used in our experiment. Regarding this problem, we performed the ON/OFF Test using centrifuge tubes, rather than the conventional 96-well microplate. Results indicated that the XylR biosensor indeed could give response to conventional hydrophobic compounds such as TOL and m-Xyl if the vaporization could be prevented (Fig. 8).

Fig.8 The induction ratios of hydrophobic aromatics compounds obtained from the ON/OFF Test using the new protocol (the same with Test Protocol 1 except that the experiments were performed in centrifuge tubes to avoid the vaporization).

Reference

[1] Abril, M. A., Michan, C., Timmis, K. N., & Ramos, J. (1989). Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway.Journal of bacteriology, 171(12), 6782-6790.
[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.
[3] Valls, M., Silva‐Rocha, R., Cases, I., Muñoz, A., & de Lorenzo, V. (2011). Functional analysis of the integration host factor site of the σ54Pu promoter of Pseudomonas putida by in vivo UV imprinting. Molecular microbiology, 82(3), 591-601.
[4] Devos, D., Garmendia, J., Lorenzo, V. D., & Valencia, A. (2002). Deciphering the action of aromatic effectors on the prokaryotic enhancer‐binding protein XylR: a structural model of its N‐ terminal domain. Environmental microbiology, 4(1), 29-41.
[5] Salto, R., Delgado, A., Michán, C., Marqués, S., & Ramos, J. L. (1998). Modulation of the function of the signal receptor domain of XylR, a member of a family of prokaryotic enhancer-like positive regulators. Journal of bacteriology,180(3), 600-604.
[6] Garmendia, J., & De Lorenzo, V. (2000). The role of the interdomain B linker in the activation of the XylR protein of Pseudomonas putida. Molecular microbiology, 38(2), 401-410.
[7] Tropel, D., & Van Der Meer, J. R. (2004). Bacterial transcriptional regulators for degradation pathways of aromatic compounds. Microbiology and Molecular Biology Reviews, 68(3), 474-500.
[8] Pérez-Martín, J., & de Lorenzo, V. (1996). ATP Binding to the σ< sup> 54-Dependent Activator XylRTriggers a Protein Multimerization Cycle Catalyzed by UAS DNA. Cell, 86(2), 331-339.
[9] de las Heras, A., Chavarría, M., & de Lorenzo, V. (2011). Association of dnt genes of Burkholderia sp. DNT with the substrate‐blind regulator DntR draws the evolutionary itinerary of 2, 4‐ dinitrotoluene biodegradation. Molecular microbiology, 82(2), 287-299.
[10] de las Heras, A., & de Lorenzo, V. (2011). Cooperative amino acid changes shift the response of the σ54‐dependent regulator XylR from natural m‐xylene towards xenobiotic 2, 4‐ dinitrotoluene. Molecular microbiology, 79(5), 1248-1259.
[11] Garmendia, J., De Las Heras, A., Galvão, T. C., & De Lorenzo, V. (2008). Tracing explosives in soil with transcriptional regulators of Pseudomonas putida evolved for responding to nitrotoluenes. Microbial Biotechnology, 1(3), 236-246.
[12] Kim, M. N., Park, H. H., Lim, W. K., & Shin, H. J. (2005). Construction and comparison of< i> Escherichia coli whole-cell biosensors capable of detecting aromatic compounds. Journal of microbiological methods, 60(2), 235-245.
[13] Garmendia, J., Devos, D., Valencia, A., & De Lorenzo, V. (2001). À la carte transcriptional regulators: unlocking responses of the prokaryotic enhancer‐binding protein XylR to non‐natural effectors. Molecular microbiology, 42(1), 47-59.

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+</html>
+<html>
+<style type="text/css">
+#ReferenceXylR{line-height:20px; text-align:justify; position:absolute; top:4850px ; left:100px; width:800px; border-bottom:0px; color:#1b1b1b; font-size:14px;font-family:arial,calibri,helvetica,sans-serif;}
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 '''Gene cluster and chemical pathway'''
-XylR is an intensively studied regulatory protein mined from Pseudomonas putida[1]. It responds strongly to toluene, xylene and 4-chlro-toluene, while weakly to 3-methyl benzyl alcohol[1]. XylR activates the Pu promoter to express the "upper pathway" (XylMABC) when exposed to m-xylene ('''Fig. 1'''). It also activates the Ps1 promoter, thus to produce another transcriptional activator, called XylS, to turn on the expression of the downstream pathway (XylXYZLTEGFJGKIH, the meta-cleavage operon)[1][2]('''Fig. 1, Fig. 2'''). Notably, the entire regulatory network is also controlled by several global regulatory elements, such as IHF. This provides an explanation for the genetic-context-dependent performance of XylR-Pu pair; namely, when expressed in different bacterial species, the regulatory performance of XylR/Pu pair often fails[3]. Therefore, fine-tuning is probably necessary.
+XylR is an intensively studied regulatory protein mined from Pseudomonas putida<html><sup><a href="#ReferenceXylR">[1]</a></sup></html>. It responds strongly to toluene, xylene and 4-chlro-toluene, while weakly to 3-methyl benzyl alcohol[1]. XylR activates the Pu promoter to express the "upper pathway" (XylMABC) when exposed to m-xylene ('''Fig. 1'''). It also activates the Ps1 promoter, thus to produce another transcriptional activator, called XylS, to turn on the expression of the downstream pathway (XylXYZLTEGFJGKIH, the meta-cleavage operon)[1][2]('''Fig. 1, Fig. 2'''). Notably, the entire regulatory network is also controlled by several global regulatory elements, such as IHF. This provides an explanation for the genetic-context-dependent performance of XylR-Pu pair; namely, when expressed in different bacterial species, the regulatory performance of XylR/Pu pair often fails[3]. Therefore, fine-tuning is probably necessary.
 <html>