title: D-xylose detection in Escherichia coli by a xylose binding protein-dependent response.
abstract: A gene circuit for the controlled expression of a marker gene and for the assay of xylose concentration in Escherichia coli has been designed and tested. The xylF coding sequence for the xylose binding protein (XBP) was cloned in pT7T318U downstream from the promoter for xylanase A from B. subtilis (Pbsu), together with the GFP coding sequence (gfp) under the control of the xylF promoter, forming the pT7T3-GFP-XBP construct. GFP fluorescence in Escherichia coli JW3538-1 xylF-transformed with pT7T3-GFP-XBP was approximately 1.4 × higher after 520 min growth in the presence of 5mM xylose than in cells transformed with pT7T3-GFP. Under saturating xylose concentration, flow cytometry analysis showed that all cells resulted in homogeneous populations, and the population with XBP showed a fluorescence greater than that without XBP. Activity of the xylF promoter in cells transformed with pT7T3-GFP-XBP was ≈ 40% higher than with the pT7T3-GFP. No response was observed with arabinose and ribose, showing that the expression effects were specific for xylose, demonstrating the potential use of the gene circuit as a biosensor.
authors.affiliation: Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP 14049-900, Brazil.
authors.forename: Lucas F
authors.initials: LF
authors.lastname: Ribeiro
authors.forename: Fabiana
authors.initials: F
authors.lastname: Bressan
authors.forename: Gilvan P
authors.initials: GP
authors.lastname: Furtado
authors.forename: Flavio
authors.initials: F
authors.lastname: Meireles
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Ward
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics*; D014994:Xylose / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D011506:Proteins; D049452:Green Fluorescent Proteins; D014994:Xylose; D043364:Endo-1,4-beta Xylanases; C092541:2-hydroxymuconate-semialdehyde hydrolase
keywords: Biological circuit; Flow cytometry; GFPuv; Green ﬂuorescent protein (GFP); PxylF; PxylF promotor; XBP; green ﬂuorescent protein (excitation: 395nm); promoter xylose inducible from Escherichia coli (control gene expression of xylFGH); xylose binding protein from Escherichia coli