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QLK3-2002-01923
Biosensors for in situ evaluation of bioavailability of pollutants based on transcriptional regulators à la carte (BIOCARTE)
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Contacts
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| Type of Project |
Shared cost |
| Contract No |
QLK3-2002-01923 |
| Total Cost |
|
| EC Contribution |
1,025,764 EUR |
| Start Date |
01-11-2002 |
| Duration |
36 Months |
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:
- development of specialised genetic and molecular technologies for the generation
and isolation of transcriptional regulators responsive to predetermined chemical structures
(regulators à la carte).
- 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.
- 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.
- design and formatting of strain microarrays on glass slides for simultaneous monitoring
of multiple aromatic compounds.
- development of a high throughput station for on-line multi-parametric follow-up of the above
pollutants in the environment.
Activities
The work will be organized around five poles of activity:
- engineering the regulator à la carte. The A-domain of HbpR/XylR is both necessary and
sufficient for binding aromatic effectors. 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.
- 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.
- 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.
- 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.
- 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.
Contacts
Coordinator
Participant
