Comparison of plasmid library culture methods

DNA libraries are essential for many biological experiments: MPRAs, CRISPR screens, deep mutational scans, and directed evolution, among many others. They are typically constructed in plasmids and amplified in E. coli, but uneven amplification due to differences in clone growth rates can skew the representation of library elements, ultimately leading to suboptimal experimental outcomes. It is thought that the culture method used to amplify plasmid libraries can impact the uniformity of amplification but, to our knowledge, no one has directly compared library culture methods. To determine the impact of culture method on plasmid library amplification, we amplified two dissimilar plasmid libraries with five culture methods: liquid, semisolid agarose, cell spreader-spread plates with high or low colony density, and bead-spread plates with high colony density. We then sequenced the resulting plasmid libraries to determine library uniformity after amplification. We found that no method amplified libraries more uniformly than liquid culture, the simplest method. With high (~100X) transformation coverage, culture method had a minimal impact on library uniformity and the original library composition was always maintained. These results will allow the many users of plasmid libraries to make an informed choice of culture method when creating new plasmid libraries or amplifying existing ones.