Abstract
Microorganisms used for various types of bioprocesses are subjected to
constantly changing environmental conditions to which they adapt by adjusting
their cellular physiology. Changes in the genomic expression program are the
first signs of adaptation to chancing conditions or to potential process
disturbances. However, tools suitable for high-throughput expression
monitoring of process-relevant genes are scarce. Moving to postgenomic era
with a growing number of organisms has increased the interest in functional
genomics, and the need for fast and reliable transcriptional profiling methods
is thus growing. We have developed a rapid method for transcriptional
profiling of microbial cultivations based on a novel technique called TRAC[1]
"transcriptional profiling with the aid of affinity capture". This method
allows fast gene expression analysis for sets of mRNAs by solution
hybridisation with a pool of target-specific oligonucleotide probes of
distinct sizes that are identified and quantified by capillary
electrophoresis. The assay procedure has been semi-automated for simultaneous
treatment of 96 samples using magnetic bead particle processor. To further
enhance the robustness of the method it was set up to work with crude cell
lysates. TRAC has been shown to produce results highly consistent with mRNA
quantification by Northern hybridisation. Computational methods have been
applied for design of target-specific oligonucleotide probes and to assign
them into minimal number of pools[2]. The whole assay procedure can be
performed in three hours, implying its usefulness in bioprocess monitoring and
control.
The developed TRAC method application has been used for monitoring the levels
of a set of mRNAs in the filamentous fungus Trichoderma reesei in fermentation
conditions. Chosen gene markers for bioprocess monitoring are involved in
various cellular pathways including unfolded protein response, protection
against various stress conditions, oxygen and nutrient limitation responses,
protein synthesis and growth. Data collected from different types of
fermentations shows the potential of the method for use in optimisation of
production processes and provides novel information about regulation of
various genes during different phases of long cultivations.
[1] Söderlund, H., Kataja, K., Paloheimo, M., Ilmen, M. and Takkinen, K.
(2003) Method and test kit for quantitative and/or coparative assessment of
variations in polynucleotide amounts in cell or tissue samples. Finland
FI20010041 / Int.pat.appl. No. PCT/FI02/00023
[2] Kivioja, T., Arvas, M., Kataja, K., Penttilä, M., Söderlund, H. and
Ukkonen, E. (2002) Assigning probes into a small number of pools separable by
electrophoresis. Bioinformatics, 18, S199-206.
Original language | English |
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Publication status | Published - 2004 |
Event | 3rd Recombinant Protein Production Meeting: A comparative view on host physiology - Tavira, Portugal Duration: 11 Nov 2004 → 14 Nov 2004 |
Conference
Conference | 3rd Recombinant Protein Production Meeting |
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Country/Territory | Portugal |
City | Tavira |
Period | 11/11/04 → 14/11/04 |