Figure 1
From: Improving membrane protein expression and function using genomic edits
Workflow to determine genetic mutants detrimental to membrane protein expression. (A) The pooled, bar-coded E. coli transposon insertion library (TnLib) containing ~150k strains with single gene disruptions was transformed with a plasmid encoding an inner membrane protein GFP fusion (IMP-GFP), resulting in the transposon library TnLib/IMP. (BC = bar code) (B) Growth and expression of five IMP-GFP fusions were tested under a range of inducer concentration to find toxic growth conditions. (C) Fluorescence-activated cell sorting (FACS) isolated the unique bar-coded mutants grown in toxic membrane protein expression conditions into gates of no, low, or high GFP signal. (D) Bar codes of mutants enriched in sorted populations were amplified using BarSeq, and the abundance of each bar code was determined using next generation sequencing. (E) Enrichment ratios were used to determine preferential IMP-GFP expression associated with each gene disruption.
