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Electric DNA chips for bioprocess control

   
Project

QLK3-1999-00533

Cell factory area

3.3.2

EU Contribution

1 457 441 Euro

Duration

36 months

Type

Research project

Starting date

01-03-2000

Keywords
Bacillus subtilis
electric DNA chips
Escherichia coli
oligo-nucleotide
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ABSTRACT
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In this project an electric DNA chip array will be developed for the monitoring of the expression of selected genes under process conditions. The chips are based on micro-electrode arrays produced with silicone technology and activated with oligo-nucleotide probes that bind to the target sequence to be analysed. The hybridisation of enzyme labelled cDNAs of selected mRNAs with the corresponding oligo-nucleotide on the chip will be analysed by an electric signal generated by enzymatic production of an electroactive product. With this instrument a number of genes that are thought to be critical in selected bioprocesses, will be monitored at-line.

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OBJECTIVES
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Earlier work has shown that mRNA analysis may be useful for process monitoring. The over-all objective of this project is to develop a technique by which the electric chip technology is used for monitoring the expression of genes that are thought to be critical for production processes using microorganisms in bioreactors. Expression of strategically selected genes will be investigated in three types of processes:

  • Production of an enzyme in Bacillus subtilis.
  • Production of a recombinant protein in E. coli.
  • Production of metabolites of the aromatic amino acid biosynthesis (tryptophan and shikimic acid) in E. coli.

The technique will also be developed for early detection of phage infections in bioreactors.

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DESCRIPTION OF THE WORK
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A micro-array silicon chip with immobilised nucleotide probes will be used to bind the selected target mRNA. Enzyme labeled cDNAs will be used to measure the relative amount of process relevant mRNA. The electric signal is generated by the production of electro active paminophenol from the following enzymatic reaction. In the processes for production of enzyme and recombinant protein, the mRNA for genes of the product, chaperones, proteases, inclusion body binding proteins (in E. coli) and phage induction will be chosen as targets for analysis. In the process for metabolite production, the mRNA of genes involved in the biosynthetic pathway and selected stress genes will be monitored. These tasks are divided into:

i) instrument development,

ii) analysis development, and

iii) application of the methods in industrial processes. The silicon-based chips and the instrumentation will be developed by ISIT, while the analytic development and process applications are made by a co-operation of three industrial and four academic partners.

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DELIVERABLES
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An electric DNA/RNA chip-array instrument will be developed. This comprises the liquid sample handling system, the chips and the data processing unit. This instrument, together with the reports on methodology of transcriptional analysis in fermentation processes, are the main deliverables of the project.

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CONSORTIUM
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COORDINATOR
  Dr. Sven-Olof Enfors
Dept. of Biotechnology
Royal Institute of Technology
10044 Stockholm, Sweden
Tel: +46 87909164
Fax: +46 87231890
enfors@biotech.kth.se

PARTNERS
  Dr. Michael Hecker
Ernst-Moritz-Arndt-Universität
Inst f. Mikrobiol. und Molecularbiol.
Friedrich-Ludwig-Jahn-Straße 15a
17487 Greifswald, Germany
Tel: +49 3834864201
Fax: +49 3834864202
hecker@microbio7.biologie.uni-greifswald.de

Dr. Rainer Hintsche
Fraunhofer-Institu für Siliziumtechnologie
Fraunhoferstr. 1
25524 Itzehoe, Germany
Tel: +49 4821174221
Fax: +49 4821174251
hintsche@isit.fhg.de

Dr. Peter Neubauer
Martin-Luther-Universität
Dept. of Biochemistry and Biotechnology
Weinbergweg 16a
06120 Halle, Germany
Tel: +49 3455524938
Fax: +49 3455527013
neubauer@biochemtech.uni-halle.de

Dr. Grzegorz Wegrzyn
Dept. of Molecular Biology
University of Gdansk
Kladki 24
80-822 Gdansk, Poland
Tel: +48 583463014
Fax: +48 583010072
wegrzyn@biotech.univ.gda.pl

Dr. Mogens Wümpelmann
Novo Nordisk A/S
Novo Allé
2880 Bagsvaerd, Denmark
Tel: +45 44422251
Fax: +45 44426645
mwu@novo.dk

Dr. Harald Skogman
BioGaia Fermentation AB
Box 965
220 09 Lund, Sweden
Tel: +46 46181716
Fax: +46 46159291
harald.skogman@fermentation.biogaia.se

Dr. Åsa Manelius
Pharmacia & Upjohn AB
Mariefredsvägen 37
645 41 Strängnäs, Sweden
Tel: +46 15227415
Fax: +46 15218358
asa.manelius@eu.pnu.com
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