Effects of growth medium selection on plasmid DNA production and initial processing steps.
Cultures of recombinant Escherichia coli containing the plasmid pSVbeta were grown in three medium formulations to assess their effects on the characteristics of supercoiled plasmid DNA production for plasmid-based gene therapy. A semi-defined medium containing casamino acids (SDCAS) was found to support higher cell densities and higher plasmid stability than a similar medium containing soya amino acids (SDSOY) or Luria-Bertani medium (LB). Differences were observed in the cell harvest characteristics, plasmid DNA primary recovery, plasmid DNA yield and quality between cells grown on LB and on SDCAS medium. Cells grown on SDCAS medium were more difficult to resuspend after harvest than those grown in LB medium and were less susceptible to alkaline lysis. The plasmid DNA content from SDCAS was predominantly supercoiled and was less contaminated by chromosomal DNA than plasmid DNA extracts derived from cells grown on LB medium. It was hypothesised that the different carbon:nitrogen ratio (C:N) of the medium may have been responsible for changing the cell wall polysaccharide composition resulting in the change in cell harvest and lysis characteristics. Results indicated that changing the C:N ratio of SDCAS medium between 1.21:1 and 12.08:1 resulted in no alteration in cell wall polysaccharide composition or in cell susceptibility to chemical lysis or physical breakage. Plasmid DNA yields increased ten-fold with ten-fold increase in the C:N ratio of SDCAS medium.
O'Kennedy
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RD
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, UK.
Baldwin
C
C
Keshavarz-Moore
E
E
D001709:Biotechnology / Q000379:methods; D002244:Carbon / Q000737:chemistry; D002455:Cell Division / Q000235:genetics; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development*; D008070:Lipopolysaccharides / Q000378:metabolism; D009584:Nitrogen / Q000737:chemistry; D010957:Plasmids / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D003470:Culture Media; D008070:Lipopolysaccharides; D002244:Carbon; D009584:Nitrogen