Enhancing Yield of Low Copy Number E. coli Plasmids by Optimization of Cell Growth Media

Undergraduate #7
Discipline: Biological Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)

Jennifer Ream - Texas State University
Co-Author(s): Kyle D. Smith and Kevin Lewis, Texas State University, San Marcos, TX

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Plasmid minipreps are commonly used to purify circular plasmid DNAs from small cultures of bacterial cells. High copy number plasmids produce high yields of DNA using this procedure. However, preps of low and single copy plasmids result in a low titer of desired DNA and are sometimes accompanied with undesired chromosomal DNA. Previous work performed in this lab has demonstrated that use of Terrific Broth (TB) to grow E. coli cells improved the yield of plasmid DNA relative to Lysogeny broth (LB) and several other commonly used broths. TB contains glycerol and potassium phosphate buffer that are not present in LB, and also contains five times as much yeast extract. Surprisingly, the higher plasmid yields with TB were not due to the presence of glycerol or buffering. Major goals of the current study are to determine the precise component(s) in TB that make it a superior broth and to develop new methods to improve yields of low copy and single copy plasmids isolated from minipreps. Initial experiments have attempted to determine if increasing the amount of yeast extract in LB could make it equivalent to or superior to TB. Plasmid preps were conducted with increasing supplemental concentrations of yeast extract in LB broth (LBX)e, and compared with the yields of preps performed with traditional LB and TB broths. Cell densities were monitored using OD550 measurements; agarose gels and DNA fluorometry were used to determine the efficiency of the prep protocols using LBX broths. It was found that higher yeast extract amounts increased both the cell densities and DNA yields in a dose-dependent manner. The results indicate that the superiority of TB is due to its yeast extract content and not the additional ingredients that distinguish the broth from LB. Additional experiments are being performed to try to enhance DNA yields further and to determine if use of this broth during transformation of DNA into E. coli cells could also generate higher efficiencies.

Funder Acknowledgement(s): NIH, Robert A. Welch Foundation, Houston-Louis Stokes Alliance for Minority Participants

Faculty Advisor: Kevin Lewis,