ScienceDirect - Journal of Biotechnology : Ef...

R. D. O’Kennedy^a, C. Baldwin^b and E. Keshavarz-Moore^{, , a}

^a The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK

^b Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK

Abstract

Cultures of recombinant Escherichia coli containing the plasmid pSVβ 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.

Author Keywords: Gene therapy; Plasmid DNA; Growth medium; Downstream processing

Article Outline

1. Introduction

2. Materials and methods

2.1. Plasmids and bacterial strains

2.2. Growth media

2.3. Culture conditions and dry cell weight determination

2.4. Total viable counts and plasmid stability

2.5. Plasmid DNA preparation and analysis

2.6. Absorbance measurements

2.7. Electrophoresis

2.8. Cell lysis susceptibility and cell wall strength

2.9. Polysaccharide assay

3. Results and discussion

3.1. Cell growth and plasmid stability

3.2. DNA characteristics

3.3. Cell harvest and lysis characteristics

3.4. Effect of C:N ratio on total polysaccharide production, cell breakage and plasmid yield

4. Conclusions

References