Fig. 2.
Improved signal peptide performance following directed evolution of the TIR a A directed evolution approach was used to convert a TIRUNEVOLVED to a TIREVOLVED. The TIR is defined as the region from the Shine-Dalgarno (half-dome) to codon 5 of the signal peptide. b mRNA has a high propensity to form structures, thus a TIRUNEVOLVED can be sequestered into short- (top) or long-range structures (middle). Directed evolution should select a TIREVOLVED that is less structured and more accessible to the ribosome during translation initiation (bottom). c An overview of the directed evolution process. A TIRLIBRARY was constructed by completely randomising the six nucleotides immediately upstream of the AUG start codon, and partially randomising the six nucleotides immediately downstream of the AUG start codon (allowing synonymous codons changes only). The TIRLIBRARY was transformed into E. coli BL21(DE3) pLysS and plated on increasing concentrations of ampicillin. A TIREVOLVED was identified on the plate containing the highest concentration of ampicillin relative to the TIRUNEVOLVED variant. d β-lactamase production levels from TIRUNEVOLVED/TIREVOLVED pairs was assessed by immuno-blotting. In this experiment, β-lactamase production was induced for 2 h, then a volume of cells corresponding to 0.2 OD600 units of cells were harvested, separated by a 12% SDS-PAGE and protein levels were determined by immuno-blotting with antisera to β-lactamase. To ensure that protein loading was consistent between the samples, the membrane was stained with Amido black after immuno-detection. ‘Pre’ denotes the precursor form of the protein, which contains the signal peptide fused version of β-lactamase, which we presume to be in the cytoplasm as the signal peptide is still present. ‘Mat’ precursor denotes the mature version of β-lactamase, which presumably is in the periplasm as the signal peptide has been cleaved. (E) β-lactamase activity from TIRUNEVOLVED/TIREVOLVED pairs was assessed using the disc diffusion assay. Here a filter disc containing 2 mg of ampicillin was placed on top of an LB-agar plate containing a lawn of bacteria expressing β-lactamase from either a TIRUNEVOLVED or a TIREVOLVED. The diameter of the growth-inhibition zone was measured for each experiment. In all cases, a TIREVOLVED conferred more resistant to ampicillin than TIRUNEVOLVED