Optimization of Escherichia Coli Growth Conditions to Maximize Plasmid Yield

Open Access
Author:
McAnulty, Joshua Thomas
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Song Tan, Thesis Supervisor
  • Chen Pei David Tu, Honors Advisor
  • Wendy Hanna Rose, Faculty Reader
Keywords:
  • plasmid
  • plasmid preparation
  • bacterial growth
  • nucleosome
Abstract:
Plasmids are circular double-stranded DNA that replicate independently of chromosomal DNA. Additional genes encoded by the plasmid provide phenotypical traits that increase versatility and adaptability of the host organism. In molecular biology, plasmids are often used as vectors for producing recombinant DNA and proteins. The Tan laboratory uses bacterial plasmids to produce nucleosome DNA fragments for reconstituting nucleosomes used in biochemical and structural studies of chromatin enzymes. However, the protocol used to grow E. coli containing such plasmids has not been examined systematically to optimize plasmid yield. The aim of this thesis is to determine the optimal E. coli growth conditions to maximize plasmid yield. Quantitative Polymerase Chain Reaction (qPCR) of culture samples were used to determine the concentration of plasmids grown under different growth conditions. The effect of three different growth media (2xTY, PDMR, and TB) on plasmid yield was examined using both ampicillin and kanamycin resistant plasmids. The effect of growth temperature was also examined. My results suggest the use of kanamycin resistant plasmid grown at 37º C in TB media produced the largest plasmid yield 3.7x the yield using currently used growth conditions in our laboratory.