Figure 9From: Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coliFitness effects of first five mutations fixed in G3 lineage. The first five mutations fixed in the G3 lineage (marC, miaA-hfq, rph, mdh, and groL) were constructed singly and in various combinations in E. coli EcHW24 (EcNR1 ΔmutS) using ssDNA mediated recombination; the marC::IS1 mutation was approximated by knocking out marC. The constructed mutants and E. coli EcHW24 were phenotyped by measuring the maximum specific growth rate in 0%, 0.5%, and 1% (w/v) isobutanol spiked minimal glucose media; relative fitness was calculated as mutant maximum specific growth rate (h-1) divided by E. coli EcHW24 maximum specific growth rate (h-1). Mutation combinations corresponding to the order of appearance in G3 are highlighted. Epitasis is assumed to follow a simple multiplicative fitness model , where w = relative fitness of a particular mutation combination, ε = total epistatic interaction parameter, and wi = relative fitness of single mutants; log epistasis is calculated as . Inset shows log(ε) at 1% (w/v) isobutanol for selected mutation combinations with notable epistasis.Back to article page