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Volume 2
     

Biosensors for in situ evaluation of bioavailability of pollutants based on transcriptional regulators à la carte (BIOCARTE)

   
Project

QLK3-2002-01923

Cell factory area

3.2.2

EU Contribution

1,025,764 Euro

Duration

36 months

Type

RS

Starting date

01 November 2002

Keywords
aromatic effectors
plate-based bioassays
prokaryotic regulators
ABSTRACT

BIOCARTE exploits the properties of prokaryotic transcriptional regulators of the XylR/HbpR family to design à la carte regulators that will be employed to determine the bioavailability of nitro-, bromo- and chloroaromatic chemicals and biphenylethers. The core of this proposal is the application of high-throughput combinatory genetic technologies to the protein module that sets the specificity of the response to distinct aromatic compounds. This will allow the generation and selection of regulator variants able to recognize and to trigger responses to a large variety of chemical species.

The selected proteins will be employed in various biosensor schemes through the coupling of the cognate promoters to genetically formatted lux and gfp reporters. This genetic standardisation will go in parallel with matching instrumentation, hardware and software suited for measuring the optical output of the system and its use in the field.

OBJECTIVES

BIOCARTE will pursue five objectives:

  1. development of specialised genetic and molecular technologies for the generation and isolation of transcriptional regulators responsive to predetermined chemical structures (regulators à la carte).
  2. standarisation of the procedures to fuse genetically the promoter/regulator pairs to reporter systems with an optical output (e.g. lux and gfp) and to optimise its expression in Pseudomonas putida.
  3. elaboration and validation of high-density microtiter plate-based bioassays for assessment of bioavailability of nitro- and chloroaromatic compounds and biphenylethers in soil and liquid environmental samples.
  4. design and formatting of strain microarrays on glass slides for simultaneous monitoring of multiple aromatic compounds.
  5. development of a high throughput station for on-line multi-parametric follow-up of the above pollutants in the environment.
DESCRIPTION OF THE WORK

The work will be organized around five poles of activity:

  1. engineering the regulator à la carte. The A-domain of HbpR/XylR is both necessary and sufficient for binding aromatic effectors. Hence, this activity will include the set up of general genetic utensils to generate large libraries of variants in the effector-binding pocket of the regulators, in vivo tests for selection of regulator variants responsive to the desired chemicals, affinity and specificity maturation of the preselected variants and molecular tools for the selection of novel DNA-binding specifities.
  2. engineering the reporter system into the chromosome of P. putida. Two reliable site-specific recombination vector systems will be developed to genetically assemble each of the novel regulators within a predetermined site of the bacterial chromosome.
  3. engineering the test strain/s. The prototype strains inserted with the lux or gfp fusions will be adapted to a predefined window of activity, to which the instrumental hardware will be matched.
  4. developing a microtiter plate test for luminescent/fluorescent P. putida cells. The resulting set-up will be applied to detect different chemicals in the environment.
  5. designing an automated remote control station adapted to luminescent/fluorescent P. putida strains. This will focus on the development of an analytical system applicable on-line to monitoring specific classes of environmental pollutants. Ultimately, the set-up will be expanded to the continuous culture of the test strains and to the design of automated biosensor stations.
DELIVERABLES

BIOCARTE will develop a set of genetic tools to evolve and select XylR/HbpR regulator variants specifically responsive to different pollutants. The regulators will be operative in robust Pseudomonas strains bearing stable chromosomal insertions with lux and gfp fusions responsive to the chemical species under scrutiny. The strains will be employed as the primary sensor component in a series of assays including microtiter plates, strain microarrays in microfluidic chambers and an automated station.

CONSORTIUM
COORDINATOR
 
Dr Juan L. Ramos
Dept. of Biochemistry and Molecular & Cell Biology of Plants
Estación Experimental del Zaidín
C/ Prof. Albareda, 1
18008 Granada, Spain
Tel: +34-958-121 011
Fax: +34-958-135 740
jlramos@eez.csic.es
 
PARTNERS
 
Dr Víctor de Lorenzo
CNB-CSIC / Centro Nacional de Biotecnología del CSIC
Campus Universidad Autónoma
8043 Madrid, Spain
Tel: +34-91-585 45 36
Fax: +34-91-585 45 06
vdlorenzo@cnb.uam.es

Dr Jan Roelof van der Meer
EAWAG-Dübbendorf
Dept. Microbiology
Inst. Env. Sci. Technol. (EAWAG)
Ueberlandstrasse 133
8600 Dübendorf, Switzerland
Tel: +41-1-823 54 38
Fax: +41-1-823 55 47
vdmeer@eawag.ch

Dr Janet K. Jansson
SH-Hüddinge
Section for Natural Sciences
Södertörns högskola
Box 4101
141 04 Huddinge, Sweden
Tel: +46-8-5858 8744
Fax: +46-8-5858 8510
janet.jansson@sh.se

Dr Lesley Anne Glover
UNI-Aberdeen
Dept. of Molecular & Cell Biology
University of Aberdeen
Inst. of Medical Sciences
Foresterhill,
Aberdeen AB25 2ZD, United Kingdom
Tel: +44-1224-273 099
Fax: +44-1224-273 144
l.a.glover@abdn.ac.uk

Teófilo Díez-Caballero
Biosensores SL
Ausias March, 1
12593 Moncófar (Castellón), Spain
Tel: +34-964-579 313
Fax: +34-964-579 397
biosensores@ctv.es