growth media selection

growth media selection - JOURNAL OF BIOSCIENCE AND...

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490 J OURNAL OF B IOSCIENCE AND B IOENGINEERING ©2007, The Society for Biotechnology, Japan Vol. 104, No. 6, 490–497. 2007 DOI: 10.1263/jbb.104.490 Growth Medium Selection and Its Economic Impact on Plasmid DNA Production Michael K. Danquah 1 * and Gareth M. Forde 1 Bioengineering Laboratory, Department of Chemical Engineering, Monash University, Clayton campus, Wellington Rd., Victoria 3800, Australia 1 Received 22 January 2007/Accepted 27 September 2007 Current developments in gene medicine and vaccination studies are utilizing plasmid DNA (pDNA) as the vector. For this reason, there has been an increasing trend towards larger and larger doses of pDNA utilized in human trials: from 100–1000 µ g in 2002 to 500–5000 µ g in 2005. The increasing demand of pDNA has created the need to revolutionalize current production levels under optimum economy. In this work, different standard media (LB, TB and SOC) for culturing recombinant Escherichia coli DH5 α harbouring pUC19 were compared to a medium optimised for pDNA production. Lab scale fermentations using the standard media showed that the highest pDNA volumetric and specific yields were for TB (11.4 µ g/ml and 6.3 µ g/mg dry cell mass respec- tively) and the lowest was for LB (2.8 µ g/ml and 3.3 µ g/mg dry cell mass respectively). A fourth medium, PDMR, designed by modifying a stoichiometrically-formulated medium with an optimised carbon source concentration and carbon to nitrogen ratio displayed pDNA volumetric and spe- cific yields of 23.8 µ g/ml and 11.2 µ g/mg dry cell mass respectively. However, it is the economic ad- vantages of the optimised medium that makes it so attractive. Keeping all variables constant except medium and using LB as a base scenario (100 medium cost [MC] units/mg pDNA), the optimised PDMR medium yielded pDNA at a cost of only 27 MC units/mg pDNA. These results show that greater amounts of pDNA can be obtained more economically with minimal extra effort simply by using a medium optimised for pDNA production. [ Key words: plasmid DNA, cultivation medium, fermentation] Recombinant DNA technology and the sequencing of the human genome have led to revolutionary discoveries espe- cially in the fields of gene therapy and nucleic acid vaccines ( 1 , 2). Plasmid DNA has acquired appreciable interest due to its attractive potential application in gene therapy and DNA vaccines applications ( 3 ). Gene therapy processes in- volve the introduction of one or more functional and spe- cific genes into a human recipient to repair certain genetic defects and aberrations. A pDNA vaccine can be developed from a pathogen’s genes to provide immunity against dis- eases ( 4 , 5). Plasmid DNA vaccines allow the foreign genes to be expressed transiently in transfected cells, mimicking intracellular pathogenic infection and inducing humoral and cellular immune responses ( 6 , 7). Plasmid DNA vaccines offer a new opportunity to immunize with materials that are mainly gene-based and are expressed by the cells of the re- cipient. This gives greater control over the entire immuniza- tion process ( 2 ).
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growth media selection - JOURNAL OF BIOSCIENCE AND...

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