Massively parallel gene expression variation measurement of a synonymous codon library

Cell-to-cell variation in gene expression strongly affects population behavior and is key to multiple biological processes. While codon usage is known to affect ensemble gene expression, how codon usage influences variation in gene expression between single cells is not well understood.

Here, we used a Sort-seq based massively parallel strategy to quantify gene expression variation from a green fluorescent protein (GFP) library containing synonymous codons in Escherichia coli. We found that sequences containing codons with higher tRNA Adaptation Index (TAI) scores, and higher codon adaptation index (CAI) scores, have higher GFP variance. This trend is not observed for codons with high Normalized Translation Efficiency Index (nTE) scores nor from the free energy of folding of the mRNA secondary structure. GFP noise, or squared coefficient of variance (CV²), scales with mean protein abundance for low-abundant proteins but does not change at high mean protein abundance.

Our results suggest that the main source of noise for high-abundance proteins is likely not originating at translation elongation. Additionally, the drastic change in mean protein abundance with small changes in protein noise seen from our library implies that codon optimization can be performed without concerning gene expression noise for biotechnology applications.