Design, manufacture, and application of DNA microarrays to study gene expression phenotypes of lysine-producing Corynebacterium glutamicum

Corynebacterium glutamicum partial genome DNA microarrays were constructed that were capable of assaying the transcriptional profile of the genes of pathways involved in central carbon metabolism and lysine biosynthesis. It was found that to ensure arrays of high quality, protocols applying the arrays should include DNase treatment of RNA samples. additional RNA filtration purification steps, and the use of gene specific primers in the formation of labeled cDNA through reverse transcription. After implementing these procedures, the accuracy and reproducibility of the array data were validated. The microarrays were used to explore the effects of the over-expression of the key anaplerotic enzyme pyruvate carboxylase and the use of different medium carbon source compositions, both of which have been shown to influence the yields of biomass on carbon and of lysine on biomass. Three different strains of C. glutamicum that were grown on six different minimal medium formulations that varied in their balance of glucose and lactate were assayed by isolating total mRNA samples from cultures in three different phases of growth and lysine production. Genes associated with glycolysis and the pentose phosphate pathway showed decreased transcript concentrations as the available carbon source was shifted from glucose to lactate, while those associated with the TCA cycle and the glyoxylate bypass demonstrated increased transcription. As the cultures stopped generating biomass and began generating lysine, mRNA of genes associated with lysine synthesis and export was measured at elevated concentrations.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemical Engineering.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2005

Author dc:creator

• Roberge, Christopher (Christopher M.)

Advisor dc:contributor.advisor

• Gregory Stephanopoulos.

• Chemical Engineering.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/32322

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/32322