Biomedical Engineering Reference
In-Depth Information
The protein pattern and expression levels of cellular proteins during
recombinant gene expression were monitored by 2D electrophoresis [
47
,
48
,
50
].
To monitor transcription profiles in the course of experiments, custom-made
DNA microarrays for E. coli K-12 or E. coli BL21 were designed (Eurofins/MWG/
Operon, Ebersberg, Germany). Based on sequence data of both strains and infor-
mation on genes of recombinant compounds (DE3 lysogen, T7 RNA polymerase,
plasmid-related genes), oligo probe sets were assembled on the microarray [
53
].
The array design (accession numbers A-MARS-11 for the BL21 array and A-MARS-
12 for the K-12 array) is accessible via ArrayExpress (
http://www.ebi.ac.uk/
microarray-as/ae/
)[
54
]. The workflow for analysis of microarray data is described in
Scharl et al. [
55
], and the gcExplorer is available at the Comprehensive R Archive
Network (CRAN,
http://cran.R-project.org/package=gcExplorer
).
2.4.3 GFP Online Stress Marker
To obtain real-time information on the cellular stress level during production of
recombinant proteins, an approach based on GFP fused to a stress-responsive pro-
moter, introduced into the production host either on a plasmid or integrated into the
genome, has been established. In a first step, the GFP signal and its capability for
online stress monitoring of recombinant fermentation processes were evaluated
[
44
]. The fluorescence signal of expressed GFP was measured by 2D multiwave-
length fluorescence spectroscopy, thereby allowing noninvasive in vivo stress
monitoring online (Fig.
3
). Results of these stress monitoring approaches are
detailed by Nemecek et al. [
56
].
Fig. 3 Difference in online fluorescence signals of two fed-batch cultivations of E. coli HMS174(DE3)
pET30ahSOD with and without the stress-monitoring plasmid pMMB67HE:dnaKp:GFPmut3.1.
Selected wavelength combinations: ex450/em490/510/530/550/570/590; ex470/em510/530/550/570/
590; ex490/em530/550/570/590. Sample number indicates measurement intervals of 5 min; sample 0
represents the first measurement after feed start
Search WWH ::
Custom Search