(599ca) Optimization of Protein Production in Microfluidic Reactors By Material Selection, Scale, and Flow Control

Shankles, P. G. - Presenter, University of Tennessee
Timm, A., Oak Ridge National Lab
Retterer, S., Oak Ridge National Laboratory
Foster, C. M., Oak Ridge National Lab
Caveney, P., University of Tennessee
Doktycz, M. J., Oak Ridge National Laboratory

Title: Optimization of protein production in microfluidic reactors by material selection, scale, and flow control

Cell-free protein synthesis (CFPS) systems have become a common tool used for the production of proteins. Extracts, sourced from a variety of cells such as E. coli, wheat- germ, rabbit reticulocyte lysate, insect cells, and others have been studied and optimized for high yield efficacy. The most common parameters to be altered during optimization include extract production methods, nutrient concentrations, and the amount of DNA plasmid used in reactions. Considerably less attention has been paid to optimizing the platform in which the reaction is carried out. Conventionally, reactions are done in static reactors or under flow conditions in which little consideration is given to the effect that flow or materials encountered by the components of the reaction might have on expression levels. To achieve the highest rate of production possible, the CFPS system as well as the reactor design must be taken into account when optimizing a system. Specifically, the material, channel dimensions, and flow parameters all play a role in determining the overall rate of protein expression and ultimately protein yield of a reaction.
The goal of this research is to better understand the impact of reactor specifications and reaction conditions on cell-free reactions in microfluidic devices. Using microfluidic reactors, the effects of mechanical stimuli on expression level have been analyzed. The reactor environment is controlled in three ways: bulk material of the reactor for surface interactions has been assessed using PDMS, silicon, and PMMA based reactors; the internal volume of the reactor varied from 15 µl to 250 µl in order to confirm scalability of the reaction; and the flow within the device was analyzed for its impact on expression due to pressure variation and shear stress on the reaction components. The reaction components are mixed on chip to have a clear starting point for the reaction. Because of this, mixing of reaction components was also analyzed. Here, a model fluorescent protein (sfGFP) was used to quantify the expression levels of the E. coli based cell-free system. We show that a simple flow reactor can achieve equivalent or even higher protein yields than a static reactor of equivalent size. Future opportunity lies in relating platform design parameters to cell-free system from other sources (yeast, mammalian, etc.) and other proteins to provide further insight into how reactors can be tailored for optimal production of target proteins.



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