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Improving DNA Plasmid Production in Escherichia Coli

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Improving DNA Plasmid Production in Escherichia Coli Improving DNA Plasmid Production in Escherichia Coli by ADAM SINGER (Under the Direction of Mark A. Eiteman) ABSTRACT The ability to produce large quantities of plasmid DNA is imperative for wide scale availability of DNA vaccines. Large scale, high yield production relies on the synergy between host strain, plasmid, medium and production scheme. Screening as many variables as quickly and cost effectively as possible is the goal. In this study, Escherichia coli strains were transformed with two plasmids and screened for plasmid yield in shake flasks in chemically defined medium supplemented with either glucose or glycerol. High yield candidates were grown in feed batch fermentations at two specific growth rates, µ = 0.14 h-1 and µ = 0.24 h-1. As predicted, high production in shake flasks was predictive of high production in fermentations. Using our media and process, we were able to reach volumetric yields of approximately 600 mg/L and specific yields of approximately 17.82 mg/g, regardless of growth rate. We were also able to increase productivity (mg/Lh) over 30%. INDEX WORDS: E. coli, fed-batch, gene therapy, plasmid production Improving DNA Plasmid Production in Escherichia Coli by ADAM SINGER B.S., Biological Engineering, University of Georgia, 1998 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2007 © 2007 Adam Singer All Rights Reserved Improving DNA Plasmid Production in Escherichia Coli by ADAM SINGER Major Professor: Mark A. Eiteman Committee: Elliot Altman Sidney Kushner Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2007 iv DEDICATION To my wife Dana and my daughter Sydney-Rose. Thank you for standing by me. v ACKNOWLEDGEMENTS I would like to thank my major professor Dr. Mark A. Eiteman and Dr. Elliot Altman for their guidance and belief that I could accomplish this task. They have had an instrumental part in my engineering education. Thanks to my committee member Dr. Sidney Kushner for his support during the initial stages of this project. Thank you Merial Ltd for providing me with the financial, technical, and personal support to finish this project. I would especially like to thank Frank Milward, Lauri Kreimeyer and Xuan Guo for their continued support. Without their continuing support, I would never have taken the first steps. Special thanks to Amanda Randall, Mike Vincent, Ernie Veal, Thasan Murugesu, Jayanta Sinha, Fabien Lux and Nicolas Carboulec for your technical support and help. Lastly, I would like to thank my family and friends for picking me up when I fell. Dana, Sydney-Rose, Rory, Mom, Burt, Phyllis and Paul. I love you all. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.............................................................................................................v LIST OF TABLES....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix CHAPTER 1 INTRODUCTION & LITERATURE REVIEW...........................................................1 INTRODUCTION.....................................................................................................2 DNA VACCINES .....................................................................................................3 PLASMID VECTOR OVERVIEW ..........................................................................6 HOSTS ......................................................................................................................9 PLASMID PRODUCTION.....................................................................................10 REFERENCES........................................................................................................18 2 OPTIMIZATION OF DNA PLASMID PRODUCTION BY USE OF DIFFERENT ESCHERICHIA COLI HOST STRAINS.....................................................................25 Introduction .............................................................................................................26 Materials and Methods ............................................................................................27 Results .....................................................................................................................31 Discussion ...............................................................................................................41 References ...............................................................................................................54 3 CONCLUSION............................................................................................................58 vii APPENDICES ...............................................................................................................................60 A Raw Data from Clonal Selection .................................................................................60 B Raw Data from Shake Flasks.......................................................................................65 C Raw Data from Fermentations.....................................................................................68 D Raw Data From Dry Cell Weight Data........................................................................99 viii LIST OF TABLES Page Table 1: A sample of current DNA vaccine research efforts..........................................................4 Table 2: A comparison of vaccine methods....................................................................................5 Table 3: Advantages and disadvantages of DNA vaccines. ...........................................................5 Table 4: Major genotypes used in plasmid DNA host strains ......................................................11 Table 5: Comparison between batch and fed-batch fermentations...............................................14 Table 6: Summary of DNA yield results.. ....................................................................................17 Table 7: Strains used in this study.. ..............................................................................................28 Table 8: DNA yield, maximum specific growth rate and acetate concentration for strains containing plasmid pLL14. Strains were grown in defined medium in shake flasks to an OD of 1.......................................................................................................................33 Table 9: DNA yield, maximum specific growth rate and acetate concentration for strains containing plasmid pUC19. Strains were grown in defined medium in shake flasks to an OD of 1.......................................................................................................................34 Table 10: Maximum DNA yields for feed batch fermentations at µ= 0.14 and µ = 0.28. ............42 Table 11: The effect of heat treatments on DNA yields during a fed-batch process.....................48 Table 12: Affect of growth rate on specific DNA yield and volumetric productivity...................48 Table 13: DNA yields obtained in published fermentation studies.. .............................................50 ix LIST OF FIGURES Page Figure 1: Schematic of the major components shared by most DNA vaccine plasmid vectors. Important features include the origin of replication, the antibiotic marker, the eukaryotic promoter, and the gene of interest. ................................................................7 Figure 2: Basic schematic of inputs and outputs for a fed-batch fermentation. This graph is a representation of the growth rates in a fed-batch fermentation. The fermentation starts with growth at µ=µmax during the batch phase and transitions growth to µ<µmax according to the feed rates.............................................................................................15 Figure 3: Growth rates and volumetric DNA yields for various E. coli strains containing pLL14 strains grown in defined medium containing either glycerol (●) or glucose (○).Strains were grown in shake flask to an OD of 1. .....................................................................35 Figure 4: Growth rates and volumetric DNA yields for various E. coli strains containing pUC19 strains grown in defined medium containing either glycerol (●) or glucose (○).Strains were grown in shake flask to an OD of 1. .....................................................................36 Figure 5: Comparison of DNA yield ratio and specific growth ratio for strains containing either pLL14 (●) or pUC plasmids (○). Strains were grown in defined medium in shake flask to an OD of 1.................................................................................................................37 Figure 6: Acetate concentrations and volumetric DNA yields for various E. coli strains containing either pLL14 (●) or pUC plasmids (○). Strains were grown in defined medium in shake flask to an OD of 1............................................................................38 x Figure 7: Production of DNA in SCS1-L during a glucose-limited fed-batch process with µ=0.12 h-1: Volumetric DNA yield (▲), specific DNA yield (■) and OD ({). Heat treatment effects are shown to the right of the dashed line. ..........................................43 Figure 8: Production of DNA in BL21 during a glucose-limited fed-batch process
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