Title:

Optimizing of Bioreactor-Based Plasmid DNA Production in E.coli

Double stranded DNA (dsDNA) exhibits many sequence independent properties that are often overlooked in nucleic acid materials. To exploit these properties, gram-scale quantities of dsDNA are needed. Plasmid DNA (pDNA) is circular dsDNA that is easily isolated from bacteria, but current methods are not efficient and produce low yields. To increase yields, we reverse engineered current industrial processes and adapted them to an academic lab setting to create a simple, replicable, scalable method for pDNA production and purification. Now with an established base protocol in place, optimization of the culture conditions to further increase yields is the next step. This will be accomplished through testing alternate culturing protocols such as sulfate limitation and temperature shifts. The productivity of the bioreactor run is measured using commercially available mini prep kits to assess how much pDNA was produced per volume of culture. Current work focuses on learning proper operation of the bioreactor using the simple fed-batch fermentation protocol already used in our group before testing more complex procedures. To date, we have produced cultures yielding 136 mg of pDNA per liter of culture which is comparable to yields usually obtained from our protocol.

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