Abstract

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.

Objectives

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.

Description of the work

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:

1. instrument development,
2. analysis development, and
3. 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.

Deliverables

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.

Contacts

Coordinator

• Sven-Olof ENFORS / Royal Institute of Technology Dept. of Biotechnology / SWEDEN

Participant

• Harald SKOGMAN / BioGaia Fermentation AB SWEDEN
• Michael HECKER / Ernst-Moritz-Arndt-University Inst. Microbiol. Molecular biol. / GERMANY
• Rainer HINTSCHE / Fraunhofer-Institu für Siliziumtechnologie GERMANY
• Peter NEUBAUER / Martin-Luther-Universität Dept. of Biochemistry and Biotechnology / GERMANY
• Novo Nordisk A/S DENMARK
• Åsa MANELIUS / Pharmacia & Upjohn AB SWEDEN
• Grzegorz WEGRZYN / University of Gdansk Dept. of Molecular Biology / POLAND