IS3 Sort-Seq

In this study, we created a library of five mutagenized Insertion Seqeunce backbones using error-prone polymerase chain reaction (PCR). The library was cloned into a dual-reporter plasmid to observe how the sequences evolve gene regulatory "promoter" activity on both strands of the DNA (GFP top strand, RFP bottom strand). We sorted the bacteria in a Fluorescence Activated Cell Sorter into different bins of fluorescence (GFP and RFP: none, low, medium, and high expression). We then amplified inserts from the sorted pools using PCR and multiplexed the 5'-end of each sequence with a unique barcode to specify the different bins and replicates. We sequenced these PCRs using Illumina amplicon sequencing.