Library characterization and workflow. (A) We synthesized all combinations of 114 promoters and 111 RBS sites to create a library containing 12,653 constructs. The library was then cloned into an expression plasmid to express superfolder GFP, and mCherry was also independently expressed from a constitutive promoter to act as an intracellular control. The cell library was harvested for DNASeq, RNASeq, and FlowSeq to quantify DNA, RNA, and protein levels, respectively, for each construct. In FlowSeq, cells were sorted into bins of varying GFP-to-mCherry ratios, barcoded, and sequenced to reconstruct protein levels for each individual construct. (B) GFP expression levels for the library varied over approximately four orders of magnitude compared with relatively constant red fluorescence (Inset). (C) One hundred forty-four sequence-verified clones were individually subjected to flow cytometry analysis to act as controls. Displayed are GFP levels of two representative clones, P007-R065 (Left) and P081-R062 (Right), which show that individual constructs generally fall into 2 to 3 bins. (D, Upper) Library is split into 12 log-spaced bins based on the GFP-to-RFP ratio. (D, Lower) Individual bins have large differences in the number of cells that fall into each one.
