Development and validation of corynebacterium DNA microarrays.
We have developed DNA microarray techniques for studying Corynebacterium glutamicum. A set of 52 C. glutamicum genes encoding enzymes from primary metabolism was amplified by PCR and printed in triplicate onto glass slides. Total RNA was extracted from cells harvested during the exponential-growth and lysine production phases of a C. glutamicum fermentation. Fluorescently labeled cDNAs were prepared by reverse transcription using random hexamer primers and hybridized to the microarrays. To establish a set of benchmark metrics for this technique, we compared the variability between replicate spots on the same slide, between slides hybridized with cDNAs from the same labeling reaction, and between slides hybridized with cDNAs prepared in separate labeling reactions. We found that the results were both robust and statistically reproducible. Spot-to-spot variability was 3.8% between replicate spots on a given slide, 5.0% between spots on separate slides (though hybridized with identical, labeled cDNA), and 8.1% between spots from separate slides hybridized with samples from separate reverse transcription reactions yielding an average spot to spot variability of 7.1% across all conditions. Furthermore, when we examined the changes in gene expression that occurred between the two phases of the fermentation, we found that results for the majority of the genes agreed with observations made using other methods. These procedures will be a valuable addition to the metabolic engineering toolbox for the improvement of C. glutamicum amino acid-producing strains.
Loos
A
A
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Glanemann
C
C
Willis
L B
LB
O'Brien
X M
XM
Lessard
P A
PA
Gerstmeir
R
R
Guillouet
S
S
Sinskey
A J
AJ
D003352:Corynebacterium / Q000235:genetics* / Q000254:growth & development; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results
D018076:DNA, Complementary; D012329:RNA, Bacterial