Abstract
This protocol is used to assay the effect of protein over-expression on fitness of E. coli. It is based on a plasmid expression of a protein of interest from an arabinose-regulated pBAD promoter followed by the measurement of the intracellular protein abundance by Western blot along with the measurement of growth parameters of E. coli cell expressing this protein.
Keywords: Gene-dosage toxicity, Arabinose, Protein abundance, Western-blot, Fitness, Over-expression, Plasmid
Background
Gene dosage experiments are crucial for understanding the effects of protein over-expression on fitness and determining the optimal levels of protein abundance. Several genes are toxic even when expressed at very low levels. It is therefore important to express the protein from a tightly regulated promoter to minimize leaky expression. Here we have elucidated conditions for expression of proteins under the well-characterized arabinose induced pBAD promoter, and designed protocols to measure the intracellular abundance and fitness of E. coli cells harboring the overexpression plasmid.
Materials and Reagents
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Acknowledgments
This work was funded by NIH RO1 GM111955.
References
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