Fig. 2

From a random sequence to an active promoter by stepwise mutations (example from three random sequences). a Evolved expression levels of the lac genes are plotted for three strains that carry different random sequences (blue, orange, and purple) as a function of the number of acquired mutations. Expression level of 1 is defined as the expression measured from the WT lac promoter, and 0 is defined as the background read of the control strain, ΔLacOperon (in which the lac operon is deleted and no YFP gene was integrated). After the accumulation of mutations, de novo expression is observed (as well as the ability to utilize lactose). The number of generations is indicated near each mutation. Mutations shown were verified by reinsertion into their non-evolved ancestors. Error bars represent s.d. of YFP measurements of three biological replicates. b Sequences of the evolving promoters. For each strain, the top sequence is the random sequence before evolution, second, and third lines are the random sequence with the evolved mutations (first and second mutations, respectively). Increasing similarity to the canonical E. coli promoter motifs can be observed by different mutations. For each evolving promoter, the canonical promoter is shown as the bottom line where capital bases indicate a match. For RandSeq3, the evolved promoter is an extended −10 promoter, thus the −35 motif is not indicated