Fig. 3

A typical random sequence of ~100 bases requires only one mutation to evolve into a promoter. a A summary of 40 different random sequences and the different type (outer circle) and number (inner circle) of mutations needed to transform a random sequence to a functional promoter. Zero represents random sequences that were active already before any adaptation and ≥2 represent random sequences that require two or more mutation, in which we include all the library strains that could not evolve the expression via mutations in the random sequences. ~10% of random sequences require no mutation for expression of the lac genes (red segment). For 57.5% of random sequences, a single mutation found within the random sequence enabled expression of the lac genes (similar to RandSeq 1, 2, and 3 shown earlier) (blue segment). Other strains either relocated an existing promoter from another locus in the genome to be upstream to the lac promoter (15%, purple) or found point mutations in the intergenic region upstream to the random sequence (12.5%, orange). b Expression of the lac genes before evolution and after the first mutation that was associated with the ability to utilize lactose (upper and lower panel, respectively). YFP reads normalized to OD600 are shown. Expression level of 1 is defined as the expression measured from the WT lac promoter (right vertical dashed line), 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 (left vertical dashed line). The ~10% of random sequences that conferred the ability to utilize lactose even before evolution is found to have significant expression of the lac genes (upper panel)