Extended Data Fig. 9: Kinetic Luminescence Activity Assays of −3, −5, and TP6 Promoter Variants.
From: Systematic molecular evolution enables robust biomolecule discovery
To quantify the relative activities of each variant (independent of possible phage backbone mutations), phage from each replicate at time point t = 10 days were isolated and each T7 RNAP variant was cloned into an IPTG-inducible reporter construct. Subcloned variants were transformed into S2060 cells containing LuxAB driven by either the (a) −3 variant, (b) −5 variant, or (c) TP6 promoter and grown overnight to an OD > 1. Bacteria were then diluted 1:100 and grown to an OD of exactly 1.2 in DRM using a high-throughput robotic turbidostat method as described previously22. Once the bacteria reached an OD of 1.2 (approximately 2 hours), cells were induced with either 1 mM IPTG or [-] IPTG controls (n = 3 for each condition). Bacteria were autonomously maintained at an OD of 1.2 for the duration of the experiment and luminescence readings were taken once every 45 minutes for 10 hours. Fold change in luminescence (as shown in Fig. 6E), was calculated by averaging the luminescence in each turbidostat once the luminescence reached equilibrium (t > 8 hours) and then normalized to the average luminescence of the [-] IPTG controls within the same time window. WT T7 RNAP was used as a control for each respective promoter reporter construct (n = 6 for each WT control).
